A nonradioactive,high throughput assay for chitin synthase activity

Wheat germ agglutinin (WGA) binds with high affinity and specificity to several sites on chitin polymers. Based on these properties we have modified and adapted a previously patented (U.S. patent 5,888,757) nonradioactive, high throughput screening assay for antimicrobial agents, making it suitable as a quantitative enzymatic assay for the activity of individual chitin synthase isozymes in yeast. The procedure involves binding of synthesized chitin to a WGA-coated surface followed by detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity is then determined as an increment in absorbance at 600 nm. Absorbance values are converted to amounts of chitin using acid-solubilized chitin as a standard. The high sensitivity (lower limit of detection about 50 ng chitin), low dispersion (lower than 10%), and high throughput (96-well microtiter plate format) make this assay an excellent substitute for the conventional radioactive chitin synthase assay in cell-free extracts. We have applied this method to the differential assay of chitin synthase activities (Chs1, Chs2, and Chs3) in cell-free extracts of Saccharomyces cerevisiae. Analysis of Chs3 activity in chitosomal and plasma membrane fractions revealed that Chs3 in the plasma membrane fraction is about sixfold more active than in the chitosome.     

A yeast assay for high throughput screening of natural anti-viral agents

Over the last decade the yeast Saccharomyces cerevisiae has become a popular organism for studying heterologous gene expression and in vivo protein-protein interactions. Many variations of these basic systems have originated over the years. Besides these vast and varied applications of the yeast expression system, S. cerevisiae has also been used extensively in fundamental research as a model simple eukaryote. We have used the S. cerevisiae system to design a high throughput screen for anti-viral agents from natural sources. The design of the assay rests on the ability of the L-A helper virus and the M(1) satellite virus to detect small variations in -1 ribosomal frameshifting. A minor change in frameshifting efficiencies can be detected and clearly shown phenotypically in terms of zones of clearing on an agar plate. Using such a process, we have initiated a high throughput screening process for natural anti-viral agents.     

A colorimetric microplate assay method for high throughput analysis of lipase activity

The present work describes a colorimetric microplate assay for lipase activity based on the reaction between 5,5'-dithiobis(2-nitro benzoic acid) (DTNB) and the hydrolysis product of 2,3-dimercapto-1-propanol tributyrate (DMPTB). Reaction mixtures containing DTNB, DMPTB, and lipase were prepared in microplate wells, and the absorbance at 405nm was recorded after incubation at 37 degrees C for 30 min. A linear relationship was obtained in the range of 0.1-1 U of lipase activity by this method. The reaction conditions were also optimized for the range of 0.01-0.1 U or 1-10 U. When assaying crude tissue extracts, the reaction of DTNB with non-specific reducing agents created a major source of error. However, this error was corrected by the use of blank samples that did not contain DMPTB.     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to KV2.1 channels is inhibited by another KV2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel NaV1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-KV2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl KV channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of KV channels can be selectively targeted by small molecules to modify channel function.     

A KV2.1 gating modifier binding assay suitable for high throughput screening

Gating modifier peptides alter gating of voltage-gated potassium (KV) channels by binding to the voltage sensor paddle and changing the energetics of channel opening. Since the voltage sensor paddle is a modular motif with low sequence similarity across families, targeting of this region should yield highly specific channel modifiers. To test this idea, we developed a binding assay with the KV2.1 gating modifier, GxTX-1E. Monoiodotyrosine-GxTX-1E (125I-GxTX-1E) binds with high affinity (IC50 = 4 nM) to CHO cells stably expressing hKV2.1 channels, but not to CHO cells expressing Maxi-K channels. Binding of 125I-GxTX-1E to K_V2.1 channels is inhibited by another K_V2.1 gating modifier, stromatoxin (IC50 = 30 nM), but is not affected by iberiotoxin or charybdotoxin, pore blocking peptides of other types of potassium channels, or by ProTx-II, a selective gating modifier peptide of the voltage-gated sodium channel Na_V1.7. Specific 125I-GxTX-1E binding is not detectable when CHO-K_V2.1 cells are placed in high external potassium, suggesting that depolarization favors dissociation of the peptide. The binding assay was adapted to a 384-well format, allowing high throughput screening of large compound libraries. Interestingly, we discovered that compounds related to PAC, a di-substituted cyclohexyl K_V channel blocker, displayed inhibitory binding activity. These data establish the feasibility of screening large libraries of compounds in an assay that monitors the displacement of a gating modifier from the channel's voltage sensor. Future screens using this approach will ultimately test whether the voltage sensor of K_V channels can be selectively targeted by small molecules to modify channel function.     

Automated imaging MS: Toward high throughput imaging mass spectrometry

The term molecular histology has been used to convey the potential of imaging mass spectrometry to describe tissue by its constituent peptides and proteins, and to link this with established histological features. The low throughput of imaging mass spectrometry has been one of the factors inhibiting a full investigation of the clinical potential of molecular histology. Here we report the development of an automated set-up, consisting of a controlled environment sample storage chamber, a sample loading robot, and a MALDI-TOF/TOF mass spectrometer, all controlled by a single user interface. The automated set-up is demonstrated to have the positional stability and experimental reproducibility necessary for its clinical application.     

A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods

Background  

Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries.     

Tension, free space, and cell damage in a microfluidic wound healing assay

We use a novel, microfluidics-based technique to deconstruct the classical wound healing scratch assay, decoupling the contribution of free space and cell damage on the migratory dynamics of an epithelial sheet. This method utilizes multiple laminar flows to selectively cleave cells enzymatically, and allows us to present a 'damage free' denudation. We therefore isolate the influence of free space on the onset of sheet migration. First, we observe denudation directly to measure the retraction in the cell sheet that occurs after cell-cell contact is broken, providing direct and quantitative evidence of strong tension within the sheet. We further probe the mechanical integrity of the sheet without denudation, instead using laminar flows to selectively inactivate actomyosin contractility. In both cases, retraction is observed over many cell diameters. We then extend this method and complement the enzymatic denudation with analogies to wounding, including gradients in signals associated with cell damage, such as reactive oxygen species, suspected to play a role in the induction of movement after wounding. These chemical factors are evaluated in combination with the enzymatic cleavage of cells, and are assessed for their influence on the collective migration of a non-abrasively denuded epithelial sheet. We conclude that free space alone is sufficient to induce movement, but this movement is predominantly limited to the leading edge, leaving cells further from the edge less able to move towards the wound. Surprisingly, when coupled with a gradient in ROS to simulate the chemical effects of abrasion however, motility was not restored, but further inhibited.     

Adaptation of aequorin functional assay to high throughput screening

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.     

A colourimetric microplate assay for simple,high throughput assessment of synthetic and biological nitrification inhibitors

Aim

A simple, rapid, colourimetric method for screening biological nitrification inhibitors in plants is presented.

Methods

Our approach combines the use of the Griess assay to track the rate of nitrite (NO₂ ^?) production by pure cultures of ammonia oxidising bacteria in the presence and absence of nitrification inhibitors with a simple method for collecting root exudates from plants. NO₂ ^? formation was tracked colourimetically on a microplate reader over 9 h of incubation. The advantage of this method is that it provides a simple, high throughput means of measuring biological nitrification inhibition in root exudates, using wild-type bacterial cultures.

Results

NO₂ ^? formation rates and inhibition levels measured using the high through-put method were highly correlated with those measured by tracking NO₂ ^? formation using a segmented flow analyser. The method was able to quantify inhibition of Nitrosomonas europaea by the synthetic nitrification inhibitors allythiourea (AT), dicyandiamide (DCD) and 3,4,-dimethylpyrazole phosphate (DMPP) with IC50 values similar to those reported in the literature. The method detected biological nitrification inhibition (BNI) in root exudates from Brachiaria humidicola and the lack of BNI in root exudates from wheat cv. Janz with minimal alteration of the exudates prior to testing. The results also showed that the more common soil ammonia oxidising bacterium (AOB), Nitrosospira multiformis, was much less sensitive to AT and DCD than N. europaea but had similar sensitivity to DMPP.

Conclusions

This method provides a potentially useful way of screening large numbers of root exudate samples allowing for phenotyping of the BNI trait in crop and pasture populations which will be required for the trait to be introduced into commercial varieties.

     

An introduction to the wound healing assay using live-cell microscopy

Abstract

The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results.     

An image-based assay for high throughput screening of Giardia lamblia

Giardia lamblia is a protozoan parasite that causes widespread gastrointestinal illness. Drugs to treat giardiasis are limited, but efforts to discover new anti-giardial compounds are constrained by the lack of a facile system for cell culture and inhibitor testing. We achieved robust and reproducible growth of G. lamblia in 384-well tissue culture plates in a modified TYI-S-33 medium. A high throughput assay for the screening of potential anti-giardial compounds was developed utilizing the WB strain of G. lamblia and automated optical detection of parasites after growth with tested inhibitors. We screened a library of 1600 known bioactive molecules and identified 12 compounds that inhibited growth of G. lamblia at low- or sub-micromolar concentrations. Our high throughput assay should facilitate evaluation of available chemical libraries for novel drugs to treat giardiasis.     

GFP-reporter for a high throughput assay to monitor estrogenic compounds

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor α (ERα) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing β-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.     

Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and F?rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new all-solid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of alpha-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant alpha-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation.     

Development of a high throughput Pseudomonas aeruginosa epithelial cell adhesion assay

Pseudomonas aeruginosa colonizes the lungs of cystic fibrosis and mechanically ventilated patients by binding to specific carbohydrate residues on the surface of lung epithelial cells. Studies have shown that blocking this interaction may have therapeutic effects in vivo. To test compounds that may have an effect on the binding of P. aeruginosa to epithelial cells, we have developed a pseudomonal adhesion assay that is compatible with high throughput technology. This assay utilizes a 96-well culture plate assay and P. aeruginosa strains that have been modified to bioluminesce. This method has proven to be a rapid, sensitive and reproducible system for screening agents that inhibit bacterial adhesion.     

Design and analysis of experiments with high throughput biological assay data

The design and analysis of experiments using gene expression microarrays is a topic of considerable current research, and work is beginning to appear on the analysis of proteomics and metabolomics data by mass spectrometry and NMR spectroscopy. The literature in this area is evolving rapidly, and commercial software for analysis of array or proteomics data is rarely up to date, and is essentially nonexistent for metabolomics data. In this paper, I review some of the issues that should concern any biologists planning to use such high-throughput biological assay data in an experimental investigation. Technical details are kept to a minimum, and may be found in the referenced literature, as well as in the many excellent papers which space limitations prevent my describing. There are usually a number of viable options for design and analysis of such experiments, but unfortunately, there are even more non-viable ones that have been used even in the published literature. This is an area in which up-to-date knowledge of the literature is indispensable for efficient and effective design and analysis of these experiments. In general, we concentrate on relatively simple analyses, often focusing on identifying differentially expressed genes and the comparable issues in mass spectrometry and NMR spectroscopy (consistent differences in peak heights or areas for example). Complex multivariate and pattern recognition methods also need much attention, but the issues we describe in this paper must be dealt with first. The literature on analysis of proteomics and metabolomics data is as yet sparse, so the main focus of this paper will be on methods devised for analysis of gene expression data that generalize to proteomics and metabolomics, with some specific comments near the end on analysis of metabolomics data by mass spectrometry and NMR spectroscopy.     

A novel high throughput chemiluminescent assay for the measurement of cellular cyclic adenosine monophosphate levels

The second messenger 3', 5'-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.     

A high throughput assay for inhibitors of HIV-1 protease. Screening of microbial metabolites

A novel method for discovery of HIV-1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV-1 Gag polyprotein comprising the p17-p24 cleavage site, fused to E. coli beta-galactosidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV-1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96-well microtiter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV-1 protease inhibitory activities have been detected. One of these has been studied in detail.