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W4242887310 abstract "Laboratory Automation and High-Throughput ChemistryDevelopment of a Semiautomated High-Throughput Transient Transfection SystemTransient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high-throughput protein production platform that could be used to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies, including proof of concept, antibody development, and biological structure and function.Bos et al. describe such a platform: a semiautomated process for polyethylenimine (PEI)-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid-handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods are developed for the liquid-handling system to automate the different processes associated with transient transfections, such as initial cell seeding, DNA–PEI complex activation, and DNA–PEI complex addition to the cells. Increasing the DNA–PEI complex incubation time results in lower protein expression. To minimize protein production variability, the methods are further optimized to achieve consistent cell seeding, control the DNA–PEI incubation time, and prevent cross-contamination among different tubespins. This semiautomated transfection process is applied to express 520 variants of a human IgG1 (hu IgG1) antibody (Bos, A. B., et al., J. Biotechnol. 2014, 180, 10–16).Automated Analysis of Dynamic Behavior of Single Cells in Picoliter DropletsKhorshidi et al. present a droplet-based microfluidic platform to automatically track and characterize the behavior of single cells throughout time. This high-throughput assay allows encapsulation of single cells in microdroplets and traps intact droplets in arrays of miniature wells on a polydimethylsiloxane (PDMS)–glass chip. Automated time-lapse fluorescence imaging and image analysis of the incubated droplets on the chip allow the determination of the viability of individual cells throughout time. To automatically track the droplets containing cells, the authors present a simple method based on circular Hough transform to identify droplets in images and quantify the number of live and dead cells in each droplet. Here, the authors study the viability of several hundred single isolated HEK293T cells throughout time and demonstrate a high survival rate of the encapsulated cells for up to 11 hours. The presented platform has a wide range of potential applications for single-cell analysis (e.g., monitoring heterogeneity of drug action throughout time and rapidly assessing the transient behavior of single cells under various conditions and treatments in vitro) (Khorshidi, M. A., et al., Lab Chip. 2014, 14 (5), 931–937).A Fully Automated, Dual-Online, Multifunctional, Ultra-High-Pressure Liquid Chromatography System for High-Throughput Proteomics AnalysisA fully automated, dual-online, multifunctional, ultra-high-pressure liquid chromatography (DO-MULTI-UPLC) system has been developed for high-throughput proteome analyses of complex peptide mixtures. The system uses two online solid phase extraction (SPE) columns (150 µm inner diameter × 3 cm), two capillary reverse phase (RP) columns (75 µm × 100 cm), and a strong cation exchange (SCX) column (150 µm × 15 cm) on a single system using one binary pump and one isocratic pump.With the automated operation of six switching valves, the selection of LC experiments between single-dimensional reverse phase liquid chromatography (RPLC) and online two-dimensional SCX/RPLC is achieved automatically, without manual intervention, while two RPLC columns are used independently and alternatively. By essentially removing the dead time for column equilibration between experiments, in either one-dimensional (1D) mode or 2D experimental mode, the current system demonstrates how it can increase the experimental throughput about twofold while keeping the intercolumn reproducibility of peptide elution time to less than 1% of the gradient time. The advantageous features of the proposed system are demonstrated by its application to proteome samples of varying complexities (Lee, H., et al., J. Chromatogr. A. 2014, 1329, 83–89).Large-Scale Cell Production of Stem Cells for Clinical Application Using the Automated Cell-Processing MachineCell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process.To solve this problem, Kami et al. focus on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported to date. The AutoCulture system used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. The authors further examine the feasibility of this system in a clinical setting.The authors observe similar characteristics by both the culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture is a feasible method for the cultivation of human cells for regenerative medicine.An automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products (Kami, D., et al., BMC Biotechnol. 2013, 13, 102).Microplates in Liquid Chromatography: A New Solution in Clinical Research? A ReviewMicroplates are routinely used in radio- or immuno-assays. Recently, microplates have found use not only in the analytical phase but also in the pre-analytical phase in bioanalyses (e.g., sample storage and preparation). This new connection of this technology to LC could be an economical, fast, and simple solution for many routine laboratories that handle large sequences of biological samples. This review summarizes the application of microplates in bioanalytical laboratories. The different types of sorbents, materials, and shapes of microplates are discussed, and the main advantages and disadvantages of the microplates used in clinical research are presented (Krcmova, L., et al., Talanta. 2013, 115, 973–979).Small-Scale, High-Throughput, Sequencing-Based Identification of New Therapeutic Tools in Cystic FibrosisAlthough 97–99% of cystic fibrosis transmembrane conductance regulator (CFTR) mutations have been identified, great efforts must be made to detect as-yet-unidentified mutations. Bonini et al. share a small-scale, next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. The authors apply this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had been identified.Using an in-house bioinformatics pipeline, Bonini et al. rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G is found in three unrelated CF patients. Analysis of minigenes and patients’ transcripts shows that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. The authors next test the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection results in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF.The data from the study confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches (Bonini, J., et al., Genet. Med. 2015).Microfluidic Chip Technology and Microreactor TechnologyA High-Throughput Cellulase Screening System Based on Droplet MicrofluidicsA new ultra-high-throughput screening assay for the detection of cellulase activity is based on microfluidic sorting. Cellulase activity is detected using a series of coupled enzymes leading to the formation of a fluorescent product that can be detected on a chip. Using this method, researchers Ostafe et al. achieve up to 300-fold enrichments of the active population of cells and greater than 90% purity after just one sorting round. In addition, the authors prove they can sort the cellulase-expressing cells from mixtures containing less than 1% active cells (Ostafe, R., et al., Biomicrofluidics. 2014, 8 (4), 041102).Accessing New Chemical Entities through Microfluidic SystemsFlow systems have been 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. Rodrigues et al. mention that they 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 minireview, 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, T., et al., Angew Chem. Int. Ed. Engl. 2014, 53 (23), 5750–5758).Microfluidic-Controlled Manufacture of Liposomes for the Solubilization of a Poorly Water-Soluble DrugBesides their well-described use as delivery systems for water-soluble drugs, liposomes have the ability to act as a solubilizing agent for drugs with low aqueous solubility. However, a key limitation in exploiting liposome technology is the availability of scalable, low-cost production methods for the preparation of liposomes.Kastner et al. describe a new method, using microfluidics, to prepare liposomal solubilizing systems that can incorporate low-solubility drugs (in this case, propofol). The setup, based on a chaotic advection micromixer, shows high drug loading (41 mol.%) of propofol as well as the ability to manufacture vesicles at prescribed sizes (between 50 and 450 nm) in a high-throughput setting. Results from the studies demonstrate the ability to merge liposome manufacturing and drug encapsulation in a single process step, leading to an overall reduced process time. These studies emphasize the flexibility and ease of applying lab-on-a-chip microfluidics for the solubilization of poorly water-soluble drugs (Kastner, E., et al., Int. J. Pharm. 2015).Paper-Based Microreactor Integrating Cell Culture and Subsequent Immunoassay for the Investigation of Cellular PhosphorylationInvestigations of cellular phosphorylation and signaling pathways have recently gained much attention for the study of the pathogenesis of cancer. Related conventional bioanalytical operations for this study, including cell culture and Western blot, are time-consuming and labor-intensive. In this work, a paper-based microreactor integrates cell culture and subsequent immunoassay on a single paper.The paper-based microreactor is a filter paper with an array of circular zones for running multiple cell cultures and subsequent immunoassays. Cancer cells are directly seeded in the circular zones without hydrogel encapsulation, and they are cultured for 1 day. Subsequently, protein expressions, including structural, functional, and phosphorylated proteins, of the cells could be detected by their specific antibodies. Study of the activation level of phosphorylated Stat3 of liver cancer cells stimulated by interleukin (IL)-6 cytokine is demonstrated by the paper-based microreactor.This technique can highly reduce tedious bioanalytical operation and sample and reagent consumption. Also, the time required by the entire process can be shortened. This work provides a simple and rapid screening tool for the investigation of cellular phosphorylation and signaling pathways for understanding the pathogenesis of cancer. In addition, the operation of the paper-based microreactor is compatible to the molecular biological training, and therefore it has the potential to be developed for routine protocols for various research areas in conventional bioanalytical laboratories (Lei, K. F., and Huang, C. H., ACS Appl. Mater. Interfaces. 2014, 6 (24), 22423–22429).Automation SystemsAutomated Flow Cytometric Analysis across Large Numbers of Samples and Cell TypesMultiparametric flow cytometry is a key technology for the characterization of immune cell phenotypes. However, robust high-dimensional postanalytic strategies for automated data analysis in large numbers of donors are still lacking. Here, Chen et al. report a computational pipeline, called FlowGM, which minimizes operator input, is insensitive to compensation settings, and can be adapted to different analytic panels.A Gaussian mixture model (GMM)-based approach is used for initial clustering, with the number of clusters determined using Bayesian information criterion. Metaclustering in a reference donor permits automated identification of 24 cell types among four panels. Cluster labels are integrated into Flow Cytometry Standard (FCS) files, thus permitting comparisons to manual gating. Cell numbers and the coefficient of variation (CV) are similar between FlowGM and conventional gating for lymphocyte populations, but notably FlowGM provides improved discrimination of “hard-to-gate” monocyte and dendritic cell (DC) subsets. FlowGM thus provides rapid high-dimensional analysis of cell phenotypes and is amenable to cohort studies (Chen, X., et al., Clin. Immunol. 2015).Arrays of High-Aspect-Ratio Microchannels for High-Throughput Isolation of Circulating Tumor CellsMicrosystem-based technologies are providing new opportunities in the area of in vitro diagnostics due to their ability to provide process automation enabling point-of-care operation. As an example, microsystems used for the isolation and analysis of circulating tumor cells (CTCs) from complex, heterogeneous samples in an automated fashion with improved recoveries and selectivity are providing new opportunities for this important biomarker. Unfortunately, many of the existing microfluidic systems lack the throughput capabilities and/or are too expensive to manufacture to warrant their widespread use in clinical testing scenarios.Hupert et al. describe a disposable, all-polymer, microfluidic system for the high-throughput (HT) isolation of CTCs directly from whole blood inputs. The device uses an array of high-aspect-ratio (HAR), parallel, sinusoidal microchannels (25 µm × 150 µm; W × D; AR = 6.0) with walls covalently decorated with anti-EpCAM antibodies to provide affinity-based isolation of CTCs. Channel width, which is similar to an average CTC diameter (12–25 µm), plays a critical role in maximizing the probability of cell–wall interactions and allows for achieving high CTC recovery. The extended channel depth allows for increased throughput at the optimized flow velocity (2 mm/s in a microchannel), maximizes cell recovery, and prevents clogging of the microfluidic channels during blood processing.Fluidic addressing of the microchannel array with a minimal device footprint is provided by large cross-sectional area feed and exit channels poised orthogonally to the network of the sinusoidal capillary channels (so-called Z geometry). Computational modeling is used to confirm uniform addressing of the channels in the isolation bed. Devices with various numbers of parallel microchannels ranging from 50 to 320 are successfully constructed. Cyclic olefin copolymer (COC) is chosen as the substrate material due to its superior properties during UV activation of the HAR microchannel surfaces prior to antibody attachment. Operation of the HT-CTC device is validated by isolation of CTCs directly from blood secured from patients with metastatic prostate cancer. High CTC sample purities [low number of contaminating white blood cells (WBCs)] allow for direct lysis and molecular profiling of isolated CTCs (Hupert, M. L., et al., Microsyst. Technol., 2014, 20 (10–11), 1815–1825).The Importance of Using Computational Resources for the Automation of Drug DiscoveryThe application of computational tools to drug discovery helps researchers to design and evaluate new drugs swiftly with reduced economic resources. To discover new potential drugs, computational chemistry incorporates automatization for obtaining biological data such as ADMET and drug mechanisms of action.This editorial looks at examples of these computational tools, including docking, molecular dynamics simulation, virtual screening, quantum chemistry, quantitative structural activity relationships, principal component analysis, and drug screening workflow systems. The authors then provide their perspectives on the importance of these techniques for drug discovery.Computational tools help researchers to design and discover new drugs for the treatment of several human diseases without side effects, thus allowing for the evaluation of millions of compounds with a reduced cost in both time and economic resources. The problem is that operating each program is difficult; researchers are required to use several programs and understand each of the properties being tested. In the future, it is possible that a single computer and software program will be capable of evaluating the complete properties (mechanisms of action and ADMET properties) of ligands. It is also possible that after submitting one target, this computer software will be capable of suggesting potential compounds, offering ways to synthesize them, and presenting biological models for testing (Rosales-Hernandez, M. C., and Correa-Basurto, J., Expert Opin Drug Discov. 2015, 10(3), 213–219).Automation in Next-Generation Sequencing ApplicationsFrom Single-Gene to Multiplex Analysis in Lung Cancer, Challenges and Accomplishments: A Review of a Single Institution’s ExperienceMolecular selection has led to the successful use of novel tyrosine kinase inhibitors (TKIs) in non–small cell lung cancers (NSCLCs). For instance, mutations in EGFR and translocations and fusions in ALK render tumor cells sensitive to some TKIs, leading to substantial clinical benefits. Molecular testing such as DNA sequencing or fragment analysis following PCR, and evaluation of copy number and gene positioning by fluorescence in situ hybridization, have been developed and used clinically to identify mutations and fusions. Meanwhile, TKIs to target actionable mutations and fusions in several other oncogenes are being evaluated. High-throughput sequencing can provide therapy-predictive information and identify novel, targetable genomic alterations. In this article, Zhao et al. report their experience of enabling single-gene testing and the evolution to panel-based next-generation sequencing (Zhao, W., et al., Lung Cancer Manag. 2014, 4 (3), 315–324).Quality Control of RNA-Seq ExperimentsDirect sequencing of complementary DNA (cDNA) using high-throughput sequencing technologies (RNA-seq) is widely used and allows for more comprehensive understanding of the transcriptome than microarrays. In theory, RNA-seq should be able to precisely identify and quantify all RNA species, small or large, at low or high abundance. However, RNA-seq is a complicated, multistep process involving reverse transcription, amplification, fragmentation, purification, adaptor ligation, and sequencing. Improper operations at any of these steps could create biased or even unusable data. In addition, RNA-seq intrinsic biases (e.g., GC bias and nucleotide composition bias) and transcriptome complexity can also make data imperfect. Therefore, comprehensive quality assessment is the first and most critical step for all downstream analyses and results interpretation.This report discusses the most widely used quality control metrics, including sequence quality, sequencing depth, reads of duplication rates (clonal reads), alignment quality, nucleotide composition bias, PCR bias, GC bias, ribosomal RNA (rRNA) and mitochondria contamination, coverage uniformity, and so on (Li, X., et al., Methods Mol. Biol. 2015, 1269, 137–146).Multiplatform Assessment of Transcriptome Profiling Using RNA-Seq in the ABRF Next-Generation Sequencing StudyHigh-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols, and performance capabilities has created a need for comprehensive reference data. Here, Li et al. describe the Association of Biomolecular Resource Facilities’ next-generation sequencing (ABRF-NGS) study on RNA-seq.Li et al. carried out replicate experiments among 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected, and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS, and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and interplatform (R > 0.83) concordance for expression measures among the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection among all platforms. For intact RNA, gene expression profiles from rRNA depletion and poly-A enrichment are similar.16In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation, and improvement of RNA-seq (Li, S., et al., Nat. Biotechnol. 2014, 32 (9), 915–925).Emerging Genomics and Systems Biology Research Leads to Systems Genomics StudiesSynergistically integrating multilayer genomic data at the systems level not only can lead to deeper insights into the molecular mechanisms related to disease initiation and progression, but also can guide pathway-based biomarker and drug target identification. With the advent of high-throughput next-generation sequencing technologies, sequencing both DNA and RNA has generated multilayer genomic data that can provide DNA polymorphism, noncoding RNA, messenger RNA, gene expression, isoform, and alternative splicing information.Systems biology, on the other hand, studies complex biological systems, particularly the systematic study of complex molecular interactions within specific cells or organisms. Genomics and molecular systems biology can be merged into the study of genomics profiles and implicated biological functions at the cellular or organism level. The prospectively emerging field can be referred to as systems genomics or genomic systems biology (Yang, M., et al., BMC Genomics. 2014, 15 (Suppl. 11), I1). Laboratory Automation and High-Throughput ChemistryDevelopment of a Semiautomated High-Throughput Transient Transfection SystemTransient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high-throughput protein production platform that could be used to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies, including proof of concept, antibody development, and biological structure and function.Bos et al. describe such a platform: a semiautomated process for polyethylenimine (PEI)-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid-handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods are developed for the liquid-handling system to automate the different processes associated with transient transfections, such as initial cell seeding, DNA–PEI complex activation, and DNA–PEI complex addition to the cells. Increasing the DNA–PEI complex incubation time results in lower protein expression. To minimize protein production variability, the methods are further optimized to achieve consistent cell seeding, control the DNA–PEI incubation time, and prevent cross-contamination among different tubespins. This semiautomated transfection process is applied to express 520 variants of a human IgG1 (hu IgG1) antibody (Bos, A. B., et al., J. Biotechnol. 2014, 180, 10–16).Automated Analysis of Dynamic Behavior of Single Cells in Picoliter DropletsKhorshidi et al. present a droplet-based microfluidic platform to automatically track and characterize the behavior of single cells throughout time. This high-throughput assay allows encapsulation of single cells in microdroplets and traps intact droplets in arrays of miniature wells on a polydimethylsiloxane (PDMS)–glass chip. Automated time-lapse fluorescence imaging and image analysis of the incubated droplets on the chip allow the determination of the viability of individual cells throughout time. To automatically track the droplets containing cells, the authors present a simple method based on circular Hough transform to identify droplets in images and quantify the number of live and dead cells in each droplet. Here, the authors study the viability of several hundred single isolated HEK293T cells throughout time and demonstrate a high survival rate of the encapsulated cells for up to 11 hours. The presented platform has a wide range of potential applications for single-cell analysis (e.g., monitoring heterogeneity of drug action throughout time and rapidly assessing the transient behavior of single cells under various conditions and treatments in vitro) (Khorshidi, M. A., et al., Lab Chip. 2014, 14 (5), 931–937).A Fully Automated, Dual-Online, Multifunctional, Ultra-High-Pressure Liquid Chromatography System for High-Throughput Proteomics AnalysisA fully automated, dual-online, multifunctional, ultra-high-pressure liquid chromatography (DO-MULTI-UPLC) system has been developed for high-throughput proteome analyses of complex peptide mixtures. The system uses two online solid phase extraction (SPE) columns (150 µm inner diameter × 3 cm), two capillary reverse phase (RP) columns (75 µm × 100 cm), and a strong cation exchange (SCX) column (150 µm × 15 cm) on a single system using one binary pump and one isocratic pump.With the automated operation of six switching valves, the selection of LC experiments between single-dimensional reverse phase liquid chromatography (RPLC) and online two-dimensional SCX/RPLC is achieved automatically, without manual intervention, while two RPLC columns are used independently and alternatively. By essentially removing the dead time for column equilibration between experiments, in either one-dimensional (1D) mode or 2D experimental mode, the current system demonstrates how it can increase the experimental throughput about twofold while keeping the intercolumn reproducibility of peptide elution time to less than 1% of the gradient time. The advantageous features of the proposed system are demonstrated by its application to proteome samples of varying complexities (Lee, H., et al., J. Chromatogr. A. 2014, 1329, 83–89).Large-Scale Cell Production of Stem Cells for Clinical Application Using the Automated Cell-Processing MachineCell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process.To solve this problem, Kami et al. focus on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported to date. The AutoCulture system used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. The authors further examine the feasibility of this system in a clinical setting.The authors observe similar characteristics by both the culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture is a feasible method for the cultivation of human cells for regenerative medicine.An automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products (Kami, D., et al., BMC Biotechnol. 2013, 13, 102).Microplates in Liquid Chromatography: A New Solution in Clinical Research? A ReviewMicroplates are routinely used in radio- or immuno-assays. Recently, microplates have found use not only in the analytical phase but also in the pre-analytical phase in bioanalyses (e.g., sample storage and preparation). This new connection of this technology to LC could be an economical, fast, and simple solution for many routine laboratories that handle large sequences of" @default.
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W4242887310 title "Automation Highlights from the Literature" @default.
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