High-throughput Screening Applications Research Articles

In Silico Target-Specific siRNA Design Based on Domain Transfer in Heterogeneous Data

RNA interference via exogenous small interference RNAs (siRNA) is a powerful tool in gene function study and disease treatment. Designing efficient and specific siRNA on target gene remains the key issue in RNAi. Although various in silico models have been proposed for rational siRNA design, most of them focus on the efficiencies of selected siRNAs, while limited effort has been made to improve their specificities targeted on specific mRNAs, which is related to reducing off-target effects (OTEs) in RNAi. In our study, we propose for the first time that the enhancement of target specificity of siRNA design can be achieved computationally by domain transfer in heterogeneous data sources from different siRNA targets. A transfer learning based method i.e., heterogeneous regression (HEGS) is presented for target-specific siRNA efficacy modeling and feature selection. Based on the model, (1) the target regression model can be built by extracting information from related data in other targets/experiments, thus increasing the target specificity in siRNA design with the help of information from siRNAs binding to other homologous genes, and (2) the potential features correlated to the current siRNA design can be identified even when there is lack of experimental validated siRNA affinity data on this target. In summary, our findings present useful instructions for a better target-specific siRNA design, with potential applications in genome-wide high-throughput screening of effective siRNA, and will provide further insights on the mechanism of RNAi.

Inhibitors of the Yersinia protein tyrosine phosphatase through high throughput and virtual screening approaches

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design.

A method for high throughput determination of viable bacteria cell counts in 96-well plates

BackgroundThere are several methods for quantitating bacterial cells, each with advantages and disadvantages. The most common method is bacterial plating, which has the advantage of allowing live cell assessment through colony forming unit (CFU) counts but is not well suited for high throughput screening (HTS). On the other hand, spectrophotometry is adaptable to HTS applications but does not differentiate between dead and living bacteria and has low sensitivity.ResultsHere, we report a bacterial cell counting method termed Start Growth Time (SGT) that allows rapid and serial quantification of the absolute or relative number of live cells in a bacterial culture in a high throughput manner. We combined the methodology of quantitative polymerase chain reaction (qPCR) calculations with a previously described qualitative method of bacterial growth determination to develop an improved quantitative method. We show that SGT detects only live bacteria and is sensitive enough to differentiate between 40 and 400 cells/mL. SGT is based on the re-growth time required by a growing cell culture to reach a threshold, and the notion that this time is proportional to the number of cells in the initial inoculum. We show several applications of SGT, including assessment of antibiotic effects on cell viability and determination of an antibiotic tolerant subpopulation fraction within a cell population. SGT results do not differ significantly from results obtained by CFU counts.ConclusionSGT is a relatively quick, highly sensitive, reproducible and non-laborious method that can be used in HTS settings to longitudinally assess live cells in bacterial cell cultures.

Intracellular ATP Assay of Live Cells Using PTD-Conjugated Luciferase

Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recently, we developed a protein transduction domain-conjugated luciferase (PTD-Luc) for measuring cellular uptake efficacy. In this study, we evaluated the applicability of PTD-Luc to an intracellular ATP assay of live cells. The predominant fluorescence of Alexa 647-PTD-Luc was in the cytosol, whereas the fluorescence of Alexa 647-Luc was visualized surrounding the cell membrane, as confirmed by Western blot analysis. In vitro, PTD-Luc could detect less than 10–9 M ATP, and the correlation between the luciferase activity of PTD-Luc and the ATP content was strong (R = 0.999, p < 0.001). In vivo, luminescence signals of PTD-Luc detected intracellular ATP in as few as 50 HeLa cells, with a strong correlation between luminescence and cell number, suggesting high sensitivity and reliability. Furthermore, two blockers of the glycolytic pathway (2-deoxyglucose and iodoacetic acid) inhibited the signal in a dose-dependent manner, whereas potassium cyanide, an inhibitor of oxidative phosphorylation, had no effect on intracellular ATP in vivo, as seen with the PTD-Luc sensor. These data show that PTD-Luc can directly measure the intracellular ATP content in live cells, allowing real-time kinetic studies, suggesting that it is a promising tool for high-throughput drug screening and cytotoxicity assays.

Chitosan Modified PSt-GMA Microspheres With/Without Spacer-Arms as Carriers: Their Influences on Kinetics, Stability, Optimal pH, Adsorption Behavior of Immobilized Trypsin

A series of microspheres, chitosan modified PSt-GMA microspheres with/without the spacer-arms, have been used as carriers to study related properties about immobilized trypsin, such as isothermal adsorption, stability, kinetics and optimal reaction pH. The experimental results showed that the immobilization process could fit the Freundlich model rather than the Langmuir model, indicating the real adsorption behavior was more complex than the ideal adsorption model. In addition, Km values of trypsin immobilized onto M5 or M8 were closer to that of free enzyme and lower than that onto other microspheres, suggesting the trypsin immobilized onto M5 or M8 possessed a higher affinity to substrate. Furthermore, the thermal and operational stabilities of immobilized trypsin, particularly onto M5 or M8, were enhanced compared to that of free trypsin. Compared to free trypsin, the optimal pH of immobilized trypsin didn't change; however,its pH profile was broader. These results have proved that chitosan modified microspheres could keep the conformation and activity of enzyme as well as enhance enzyme's properties. Therefore, it has potential application in high throughput screening of drugs (HTS) based on the scintillation proximity assay (SPA) method.

Self‐packed filter plates: A good alternative for pre‐packed filter plates for developing purification processes for therapeutic proteins

The use of high-throughput screening (HTS) has been successfully applied over the past years in downstream process development of therapeutic proteins. Different HTS applications have been introduced to speed up the development of purification processes for these proteins. In the light of these findings, studies were conducted to develop a controlled method of pipetting the right amount of resin into self-packed filter plates. In total, 13 plates were tested for their suitability in an HTS application. Based on these studies, the Seahorse plate was experimentally verified as the best filter plate. Suitable conditions for preparing a self-packed plate were established so that linear correlations between resin concentration (POROS 50HS) and UV adsorption (420 nm) were obtained. The accuracy of dispensing the slurry into a microtiter plate was controlled within 1% deviation under established conditions. Overall, self-packed filter plates are equipped to be used in the development of purification processes in HTS mode.

In vivo imaging and quantitative analysis of zebrafish embryos by digital holographic microscopy

Digital holographic microscopy (DHM) has been applied extensively to in vitro studies of different living cells. In this paper, we present a novel application of an off-axis DHM system to in vivo study of the development of zebrafish embryos. Even with low magnification microscope objectives, the morphological structures and individual cell types inside developing zebrafish embryos can be clearly observed from reconstructed amplitude images. We further study the dynamic process of blood flow in zebrafish embryos. A calibration routine and post-processing procedures are developed to quantify physiological parameters at different developmental stages. We measure quantitatively the blood flow as well as the heart rate to study the effects of elevated D-glucose (abnormal condition) on circulatory and cardiovascular systems of zebrafish embryos. To enhance our ability to use DHM as a quantitative tool for potential high throughput screening application, the calibration and post-processing algorithms are incorporated into an automated processing software. Our results show that DHM is an excellent non-invasive imaging technique for visualizing the cellular dynamics of organogenesis of zebrafish embryos in vivo.

Fluorescence-profile pre-definable quantum-dot barcodes in liquid-core microcapsules

Microparticles incorporated with quantum-dot (QD) barcodes for multiplexed bioassays attract a great attention due to their potential applications in drug discovery, gene profiling and clinic diagnostics. However, the existing QD barcodes lack a necessary optical stability to ambient fluids or a repeatability of fluorescent profiles. We developed a new QD barcode by loading an aqueous QD mixture as a liquid core into a monodispersed polymer microcapsule by a microfluidic method to avoid those problems. We found that the QDs in the liquid cores were able to maintain their original characteristics, especially the linear relation between photoluminescence intensity and concentration. In addition, we found that the fluorescent profiles of the QD-loaded liquid cores were the same as those of the QD mixtures before being loaded inside the microcapsules. With these two properties, the QD barcodes can be predefined directly by multiplexing the emission peaks and concentrations of the QDs in the liquid cores. Furthermore, the graphical information from fluorescent images of the microcapsules, such as the sizes and numbers of the QD-loaded liquid cores, offers another dimension for barcoding to increase the coding capacity. We also presented a microfluidic method to manufacture the QD-barcoded microcapsules of size ~30 μm. These pre-definable QD barcodes with stable fluorescent profiles can be used as a platform for various high-throughput screening applications in different bioassay buffers.

Development of functional biomaterials with micro- and nanoscale technologies for tissue engineering and drug delivery applications.

Micro- and nanotechnologies have emerged as potentially effective fabrication tools for addressing the challenges faced in tissue engineering and drug delivery. The ability to control and manipulate polymeric biomaterials at the micron and nanometre scale with these fabrication techniques has allowed for the creation of controlled cellular environments, engineering of functional tissues and development of better drug delivery systems. In tissue engineering, micro- and nanotechnologies have enabled the recapitulation of the micro- and nanoscale detail of the cell's environment through controlling the surface chemistry and topography of materials, generating 3D cellular scaffolds and regulating cell-cell interactions. Furthermore, these technologies have led to advances in high-throughput screening (HTS), enabling rapid and efficient discovery of a library of materials and screening of drugs that induce cell-specific responses. In drug delivery, controlling the size and geometry of drug carriers with micro- and nanotechnologies have allowed for the modulation of parametres such as bioavailability, pharmacodynamics and cell-specific targeting. In this review, we introduce recent developments in micro- and nanoscale engineering of polymeric biomaterials, with an emphasis on lithographic techniques, and present an overview of their applications in tissue engineering, HTS and drug delivery.

High Throughput Single-cell and Multiple-cell Micro-encapsulation

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Exploring The Cancer Stem Cell Phenotype with High-Throughput Screening Applications

Several human cancer types consist of diverse cell populations that can differ in their tumor-driving potential. One breakthrough has been the identification of poorly differentiated tumor cells, herein termed cancer stem cells (CSCs). CSCs have been shown to initiate tumors in different model systems and have been implicated in cancer resistance to conventional therapies. The clinical relevance of CSCs has been increasingly recognized, and recent progress in their enrichment and characterization has paved the way for exploring CSC biology with high-throughput screening technologies. This article focuses on functional chemical and RNAi screens that have led to the identification of factors that control the CSC phenotype. Different experimental strategies, current challenges and perspectives in CSC drug discovery are discussed.

Development of a Dimethylarginine Dimethylaminohydrolase (DDAH) Assay for High-Throughput Chemical Screening

Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small-molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at a physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high-throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by inducible NOS) could play a role in idiopathic pulmonary fibrosis, sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high-throughput screen for DDAH modulators.

Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.

Parallel Synthesis of Peptide-Like Macrocycles Containing Imidazole-4,5-dicarboxylic Acid

We prepared a series of peptide-like 14-membered macrocycles containing an imidazole-4,5-dicarboxylic acid scaffold by using known coupling reagents and protecting group strategies. Yields of the purified macrocycles were poor on average, yet seemingly independent of amino acid substitution or stereochemistry. The macrocycles retain some level of conformational variability as observed by both molecular modeling and X-ray crystallography. These macrocycles represent a new class of structures for further development and for future application in high-throughput screening against a variety of biological targets.

Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests

Application of High Throughput Screening (HTS) to the regulatory safety assessment of chemicals is still in its infancy but shows great promise in terms of facilitating better understanding of toxicological modes-of-action, reducing the reliance on animal testing, and allowing more data-poor chemicals to be assessed at a reasonable cost. To promote the uptake and acceptance of HTS approaches, we describe in a stepwise manner how a well known cytotoxicity assay can be automated to increase throughput while maintaining reliability. Results generated with selected reference chemicals compared very favourably with data obtained from a previous international validation study concerning the prediction of acute systemic toxicity in rodents. The automated assay was then included in a formal ECVAM validation study to determine if the assay could be used for binary classification of chemicals with respect to their acute oral toxicity, using a threshold equivalent to a dose of 2000mg/kgb.w. in a rodent bioassay (LD50). This involved the blind-testing of 56 reference chemicals on the HTS platform to produce concentration–response and IC50 data. Finally, the assay was adapted to a format more suited to higher throughput testing without compromising the quality of the data obtained.

High Throughput Screening Technology and the Small Molecules Modulating Aging Related Signals

Aging and its related diseases are severe issues in modern society. Many efforts have been made to understand the mechanisms of aging and to find the ways to prevent age-related diseases. Identifying the compounds targeting aging-related signals is a challenging work because there are so many proteins and signals involved. Recently, alone with the progresses in high throughput screening (HTS) technology, increasing numbers of small molecules targeting aging-related pathologic processes have been identified. In this review, we introduce the basic workflow, classification and assay strategies of HTS technology, and sort out known small molecules identified via HTS technology by their roles in aging related diseases, such as neural degenerative diseases, diabetes and tumors. Given the fact that application of HTS on aging research is still at an early stage, we also summarize the cellular mechanisms about aging process, paralleled with the compounds which can modulate the functions of proteins important for aging signals. Finally, we briefly discuss some advanced HTS technologies for their potent applications on the discovery of anti-aging compounds. The main purpose of this review is to provide updated and useful information to those who are interested in pharmacology and HTS technology, but not familiar with aging biology, or vice versa.

Abstract C52: Semi-high-throughput whole-animal bioimaging assay identifies novel players in prostate cancer progression

Abstract Although genetic mouse models for cancer and human tumor cell xenotransplantation models in rodents remain essential, such systems are costly, slow, and not amenable to high-throughput assays for cancer drug target discovery. There is a clear need to develop fast, semi-automated in vivo systems for medium to high-throughput screening applications in preclinical target discovery and lead compound identification. In this study, we developed an automated, semi-high throughput, noninvasive and quantitative platform for imaging one critical aspect of cancer progression, namely tumor cell dissemination, in a zebrafish xenotransplantation model. This is the first semi-automated whole organism based bio-imaging assay of tumor progression in a vertebrate. We have used this platform to screen for prostate cancer relevant drug and gene targets, and have identified novel regulators of prostate cancer dissemination. Using 3D in vitro invasion assays, we show a critical role for these molecules in prostate cancer migration strategies. Further validation of these targets is underway in mouse models and human cancer specimens. Citation Format: Veerander Paul Singh Ghotra, Shuning H.E., Hans De Bont, H.P. Spaink, Bob Van de Water, B. Ewa Snaar Jagalska, Erik H.J. Danen. Semi-high-throughput whole-animal bioimaging assay identifies novel players in prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C52.

TR-FRET-Based Duplex Immunoassay Reveals an Inverse Correlation of Soluble and Aggregated Mutant huntingtin in Huntington's Disease

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the amplification of a polyglutamine stretch at the N terminus of the huntingtin protein. N-terminal fragments of the mutant huntingtin (mHtt) aggregate and form intracellular inclusions in brain and peripheral tissues. Aggregates are an important hallmark of the disease, translating into a high need to quantify them invitro and invivo. We developed a one-step TR-FRET-based immunoassay to quantify soluble and aggregated mHtt incell and tissue homogenates. Strikingly, quantification revealed a decrease of soluble mHtt correlating with an increase of aggregated protein in primary neuronal cell cultures, transgenic R6/2, and HdhQ150 knock-in HD mice. These results emphasize the assay's efficiency for highly sensitive and quantitative detection of soluble and aggregated mHtt and its application in high-throughput screening and characterization of HD models.

A Practical Method for Patterning Lumens through ECM Hydrogels via Viscous Finger Patterning

Extracellular matrix (ECM) hydrogels with patterned lumens have been used as a framework to generate more physiologically relevant models of tissues, such as vessels and mammary ducts, for biological investigations. However, these models have not found widespread use in research labs or in high-throughput screening applications in large part because the basic methods for generating the lumen structures are generally cumbersome and slow. Here we present viscous finger patterning, a technique to generate lumens through ECM hydrogels in microchannels that can be accomplished using manual or automated pipetting. Passive pumping is used to flow culture media through an unpolymerized hydrogel, creating a lumen through the hydrogel that is subsequently polymerized. Viscous finger patterning takes advantage of viscous fingering, the fluid dynamics phenomenon where a less viscous fluid will flow through and displace a more viscous fluid. We have characterized the technique and used it to create a variety of channel geometries and ECM hydrogel compositions, as well as for the generation of lumens surrounded by multiple hydrogel layers. Because viscous finger patterning can be performed with automated liquid handling systems, high-throughput generation of ECM hydrogels with patterned lumen is enabled. The ability to rapidly and cost-effectively create large numbers of lumens in natural polymers overcomes a critical barrier to the use of more physiologically relevant tissue models in a variety of biological studies and drug screening applications.

A 24-microwell plate with improved mixing and scalable performance for high throughput cell cultures

Multi-well plates are widely used in high throughput drug screening, cell clone development, media design and cell culture optimization in the biotechnology industry. The reproducibility and data quality of cell cultures in multi-well plates are greatly affected by mixing, aeration, and evaporation. A novel 24-microwell plate (MWP) with static mixers for improved mixing and aeration, and gas permeable lids for reduced evaporation was developed for cell cultures. Mixing, oxygen transfer, evaporation, and cell proliferation as affected by the static mixer, shape of the well and agitation rate were studied. The static mixer improved mixing pattern and reduced cell aggregation under orbital shaking conditions. Consequently, the static mixer also improved cell proliferation with a significantly higher specific growth rate in round wells. In general, consistent growth kinetics was observed for cells cultured on the plate. Overall, the MWP improved the data quality with smaller standard deviations and better reproducibility. Furthermore, CHO cells cultured in the MWP gave similar kinetics in glucose consumption, lactate production, cell growth and viability, and antibody production in a serum-free medium to those cultured in spinner flasks, demonstrating its scalable performance and potential application in high throughput screening for cell culture process development.