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PubMed2006

High-throughput fluorescent screening of transgenic animals: phenotyping and haplotyping.

BACKGROUND: Methods for genotyping transgenic animals currently consist of extracting genomic DNA from blood or tissue followed by PCR or Southern blot analysis. These methods when used to screen large numbers of animals can be time consuming and expensive. Therefore, we developed a novel method that allows high-throughput screening of phenotypic changes on leukocytes, resulting from the transgenic genotype. This technique allows investigators to quickly screen a large number of animals without the need to extract DNA from each one. Moreover, since blood is collected for the initial screening, putative homozygotes can be confirmed by conventional methods using the same blood samples. METHODS: We collected blood from wild-type alphagal positive and alphagal knockout mice and probed for the presence of Galalpha(1-->3)Gal (alphagal) epitopes. Also, alloantigen specific antibodies were used to determine the haplotype of our outbred mouse colony in order to develop an inbred line. RESULTS: alphagal epitopes were detected in wild-type but not alphagal knock-out samples. To validate these results, PCR was used to demonstrate the native alphagal gene in wild-type and the pGKneo construct in alphagal knock-out mice. Furthermore, haplotypes were determined and mice divided for backcrosses. CONCLUSIONS: This screening method is useful for both preliminary screening of transgenic mice and the development of an inbred mouse colony by rapid determination of MHC I haplotype. Here, we demonstrate the use of this technique and show how it can be a valuable tool, saving time and resources in both investigator effort and animal husbandry.

Animals↗
PubMed2002

High-throughput assay of ( R)-phenylacetylcarbinol synthesized by pyruvate decarboxylase.

A novel assay has been developed for the detection of ( R)-phenylacetylcarbinol, ( R)-PAC, a chiral intermediate in the industrial synthesis of ephedrine. It is the product of a biotransformation of benzaldehyde catalysed by the enzyme pyruvate decarboxylase. The assay, using 2,3,5-triphenyltetrazolium chloride, enables high-throughput photometric analysis of the activity of the enzyme thus avoiding time-consuming chromatographic procedures.

Acetone↗
PubMed2005

Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination.

The Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsis open reading frames into Escherichia coli expression vectors are presented along with an analysis of results from approximately 2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold-function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel Gateway protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the Gateway system.

Arabidopsis↗
PubMed2005

High-throughput screening-driven lead discovery: meeting the challenges of finding new therapeutics.

The pharmaceutical industry faces a number of challenges that have cumulatively led to a decline in productivity, despite increasing levels of investment in research and development. It is in this context that some researchers and business analysts are questioning the value of the high-throughput screening (HTS) paradigm for the discovery of new therapeutics. While this criticism may be premature, significant changes are occurring in HTS-driven lead discovery to address the failure of drug candidates in the clinic. This article highlights some of these changes and their potential impact.

Combinatorial Chemistry Techniques↗
PubMed2005

Development of a high-throughput method for preparing human urine for two-dimensional electrophoresis.

There is an increasing interest in analysing the human urinary proteome in the search for biomarkers. However, despite the ease of its collection, urine is a difficult fluid to analyse by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) because of its dilute protein content and high salt levels. Here, we describe a method for high-throughput processing of urine for 2-D PAGE. Urine is filtered and applied to solid phase extraction columns. After washing, the urinary proteins are eluted and freeze dried. The lyophilised powder can then be resuspended in an appropriate buffer for downstream proteomic analysis.

Buffers↗
PubMed2004

Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms.

Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC(50) below 100 microM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 microM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind (13)C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.

Apoptosis↗
PubMed2004

High-throughput tissue microarray analysis of c-myc activation in chronic liver diseases and hepatocellular carcinoma.

Amplification of 8q23-qter is common in human hepatocellular carcinoma (HCC). c-myc, an oncogene located on 8q24, may be important in hepatocarcinogenesis. The present study aimed to evaluate c-myc activation in hepatocarcinogenesis and its clinicopathological significance. High-throughput analysis of c-myc gene amplification and expression using dual-color fluorescence in situ hybridization and immunohistochemistry was performed on tissue microarrays consisting of 458 liver samples comprising HCCs, nontumorous livers and normal livers. HCCs demonstrated frequent c-myc amplification (30% when corrected for chromosome 8 aneusomy). In contrast, the noncancerous livers, which were mostly chronic hepatitis and cirrhosis, exhibited no c-myc amplification. Despite c-myc amplification, the HCCs exhibited less nuclear c-myc expression than the livers with chronic liver diseases and normal livers (P <0.001 and 0.004, respectively). The HCCs also had less cytoplasmic c-myc staining than the livers with chronic liver diseases (P = 0.002). Despite their absence of c-myc amplification, however, the livers with chronic disease had significantly increased expression of both nuclear and cytoplasmic c-myc protein compared with normal livers (P = 0.015 and 0.009, respectively). Clinicopathologically, the reduction in nuclear c-myc was more marked in HCCs with venous permeation and absence of tumor encapsulation (P = 0.013 and 0.021, respectively), whereas HCCs with cytoplasmic c-myc were positively associated with larger tumor size (P = 0.027). There was no significant association between c-myc amplification and protein expression levels in HCC. Our results suggest that overexpression of c-myc in chronic liver diseases may play an important role in the predisposition to hepatocarcinogenesis. Although c-myc was amplified in HCC, there appears to be a tight regulation by independent pathways of c-myc activation in hepatocarcinogenesis.

Adolescent↗
PubMed2002

Work-up strategies for high-throughput solution synthesis.

The key bottleneck in parallel synthesis has, in truth, always been the isolation and purification of the reaction products, rather than carrying out the reactions themselves. Solution-phase chemistry for parallel synthesis has recently been re-emphasized and has stimulated the development of a wide-range of practical tools for efficient high-throughput work-up, which are gaining increasing acceptance in medicinal chemistry groups.

Automation↗
PubMed2005

Functional genomics using high-throughput RNA interference.

RNA interference (RNAi) describes the post-transcriptional silencing of gene expression that occurs in response to the introduction of double-stranded RNA into cells. Application of RNAi in experimental systems has provided a great leap forward in the elucidation of gene function. To facilitate large-scale functional genomics studies using RNAi, several high throughput approaches have been developed based on microarray or microwell assays. Recent establishment of large libraries of RNAi reagents combined with a variety of detection assays further opens the door for genome-wide screens of gene function in mammalian cells.

Animals↗
PubMedDate not supplied

High throughput magnetic resonance imaging for evaluating targeted nanoparticle probes.

The ability to image specific molecular targets in vivo would have significant impact in allowing earlier disease detection and in tailoring molecular therapies. One of the rate-limiting steps in the development of novel compounds as reporter probes has been the lack of cell-based, biologically relevant, high throughput screening methods. Here we describe the development and validation of magnetic resonance imaging (MRI) as a technique to rapidly screen compounds that are potential MR reporter agents for their interaction with specific cellular targets. We show that MR imaging can (1) evaluate thousands of samples simultaneously and rapidly, (2) provide exceedingly accurate measurements, and (3) provide receptor binding/internalization data as validated by radioactive assays. The technique allows the screening of libraries of peptide-nanoparticle conjugates against target cells and the identification of conjugates that may be subsequently used as reporter agents in vivo. The technology should greatly accelerate the development of target-specific or cell-specific MR contrast agents.

Cell Line↗
PubMed2004

A novel class of inhibitors of peptide deformylase discovered through high-throughput screening and virtual ligand screening.

Peptide deformylase (PDF) has been identified as a promising antibacterial and herbicide target. A structurally novel class of inhibitors containing a 2-thioxo-thiazolidin-4-one heterocycle substituted by an arylidene group at the 5-position and a hexanoic acid side chain at the 3-position was discovered independently via high-throughput screening and virtual ligand screening. Data mining and analogue synthesis established a structure--activity relationship for the side chain region that is consistent with the docked structure.

Amidohydrolases↗
PubMed2006

Simple colorimetric inhibition assay of heme crystallization for high-throughput screening of antimalarial compounds.

Current assays for screening new antimalarials need initiators of beta-hematin formation that require laborious preparation, special devices, and substrates. In this study, based on reduction of heme absorption in beta-hematin formation, we developed a simple colorimetric assay using Tween 20 as an initiator and a microplate reader for high-throughput screening of inhibitors of beta-hematin formation.

Animals↗
PubMed2003

Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen ).

Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report.

Aggrecans↗
PubMed2005

Improving on nature's defenses: optimization & high throughput screening of antimicrobial peptides.

Antimicrobial peptides (AMPs) are ubiquitous in nature where they play important roles in host defense and microbial control. Despite their natural origin, antimicrobial spectrum and potency, the lead peptide candidates that so far have entered pharmaceutical development have all been further optimized by rational or semi-rational approaches. In recent years, several high throughput screening (HTS) systems have been developed to specifically address optimization of AMPs. These include a range of computational in silico systems and cell-based in vivo systems. The in silico-based screening systems comprise several computational methods such as Quantitative Structure/Activity Relationships (QSAR) as well as simulation methods mimicking peptide/membrane interactions. The in vivo-based systems can be divided in cis-acting and trans-acting screening systems. The cis-acting pre-screens, where the AMP exerts its antimicrobial effect on the producing cell, allow screening of millions or even billions of lead candidates for their basic antimicrobial or membrane-perturbating activity. The trans-acting screens, where the AMP is secreted or actively liberated from the producing cell and interacts with cells different from the producing cell, allow for screening under more complex and application-relevant conditions. This review describes the application of HTS systems employed for AMPs and lists advantages as well as limitations of these systems.

Animals↗
PubMed2006

A high-throughput mammalian protein expression, purification, aliquoting and storage pipeline to assemble a library of the human secretome.

In the post-human genome-sequencing era, the availability of recombinant proteins has become crucial for the identification of proteins with therapeutic potential. Based upon bioinformatic coding predictions of the genes for putative secreted proteins, we established a high-throughput protein pipeline (HTPP) for the production of a subset of the human secretome. The HTPP was based on a transient expression system in HEK293-EBNA cells at 100 to 500 mL culture scale, combined with an automated affinity purification procedure targeting >75% purity. This was followed by a semi-automated protein sample logistics to provide biologists with quality-controlled and 96 well formatted protein aliquots amenable to cell-based assays. Over a 4-year period, beginning in 2001, we performed over 7,500 transfections representing over 2,200 registered proteins, including both novel and reference proteins, at an average production of 280 proteins/month with a peak production of 320 proteins/month. All these proteins have been tested in more than 50 different cell-based assays. This article describes the major process steps and highlights the optimization required to maintain novel protein production while supporting both stock replenishment and scale-up productions.

Animals↗
PubMed2006

Development of a high-throughput assay to measure histidine decarboxylase activity.

Histamine is a well-known mediator of allergic, inflammatory, and neurological responses. More recent studies suggest a role for histamine and its receptors in a wide range of biological processes, including T-cell maturation and bone remodeling. Histamine serum levels are regulated mainly by the activity of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Despite the importance of this enzyme in many physiological processes, very few potent HDC inhibitors have been identified. HDC assays suitable for high-throughput screening have not been reported. The authors describe the development of a fluorescence polarization assay to measure HDC enzymatic activity. They used a fluorescein-histamine probe that binds with high affinity to an antihistamine antibody for detection. Importantly, they show that probe binding is fully competed by histamine, but no competition by the HDC substrate histidine was observed. The automated assay was performed in a total volume of 60 muL, had an assay window of 80 to 100 mP, and had a Z' factor of 0.6 to 0.7. This assay provides new tools to study HDC activity and pharmacological modulation of histamine levels.

Enzyme Inhibitors↗
PubMed2005

High-throughput analysis of vitamin C in human plasma with the use of HPLC with monolithic column and UV-detection.

Vitamin C plays a central role in the body. One of its important functions is its role as an antioxidant, and accurate measurements are important for interpretations of this role. However, its reactive nature and instability complicates the assessment, especially in biological samples. A high-throughput chromatographic method using monolithic column and UV-detection was developed for the assessment of plasma ascorbic acid and total ascorbic acid. The method showed excellent analytical sensitivity, specificity, precision, recovery and linearity during the validation study. The method was used for the assessment of ascorbic acid and total ascorbic acid during several clinical studies.

Adult↗
PubMed2006

Continuous-flow lithography for high-throughput microparticle synthesis.

Precisely shaped polymeric particles and structures are widely used for applications in photonic materials, MEMS, biomaterials and self-assembly. Current approaches for particle synthesis are either batch processes or flow-through microfluidic schemes that are based on two-phase systems, limiting the throughput, shape and functionality of the particles. We report a one-phase method that combines the advantages of microscope projection photolithography and microfluidics to continuously form morphologically complex or multifunctional particles down to the colloidal length scale. Exploiting the inhibition of free-radical polymerization near PDMS surfaces, we are able to repeatedly pattern and flow rows of particles in less than 0.1 s, affording a throughput of near 100 particles per second using the simplest of device designs. Polymerization was also carried out across laminar, co-flowing streams to generate Janus particles containing different chemistries, whose relative proportions could be easily tuned. This new high-throughput technique offers unprecedented control over particle size, shape and anisotropy.

Journal Article↗