Optimization and application of median filter corrections to relieve diverse spatial patterns in microtiter plate data.

The standard (STD) 5 × 5 hybrid median filter (HMF) was previously described as a nonparametric local backestimator of spatially arrayed microtiter plate (MTP) data. As such, the HMF is a useful tool for mitigating global and sporadic systematic error in MTP data arrays. Presented here is the first known HMF correction of a primary screen suffering from systematic error best described as gradient vectors. Application of the STD 5 × 5 HMF to the primary screen raw data reduced background signal deviation, thereby improving the assay dynamic range and hit confirmation rate. While this HMF can correct gradient vectors, it does not properly correct periodic patterns that may present in other screening campaigns. To address this issue, 1 × 7 median and a row/column 5 × 5 hybrid median filter kernels (1 × 7 MF and RC 5 × 5 HMF) were designed ad hoc, to better fit periodic error patterns. The correction data show periodic error in simulated MTP data arrays is reduced by these alternative filter designs and that multiple corrective filters can be combined in serial operations for progressive reduction of complex error patterns in a MTP data array.

Phenothiazine neuroleptics signal to the human insulin promoter as revealed by a novel high-throughput screen.

A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic beta-cell. A cell line from human islets in which the expression of insulin and other beta-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of beta-cell differentiated function.

Hybrid median filter background estimator for correcting distortions in microtiter plate data.

Microtiter plate (MTP) assays often exhibit distortions, such as caused by edge-dependent drying and robotic fluid handling variation. Distortions vary by assay system but can have both systematic patterns (predictable from plate to plate) and random (sporadic and unpredictable) components. Random errors can be especially difficult to resolve by assay optimization alone, and postassay algorithms reported to date have smoothing effects that often blunt hits. We implemented a 5 x 5 bidirectional hybrid median filter (HMF) as a local background estimator to scale each data point to the MTP global background median and compared it with a recently described Discrete Fourier Transform (DFT) technique for correcting errors on computationally and experimentally generated MTP datasets. Experimental data were generated from a 384-well format fluorescent bioassay using cells engineered to express eGFP and DsRED. MTP arrays were produced with and without control treatments used to simulate hits in random wells. The HMF demonstrated the greatest improvements in MTP coefficients of variation and dynamic range (defined by the ratio of average hit amplitude to standard deviation, SD) for all synthetic and experimental MTPs examined. After HMF application to a MTP of eGFP signal from mouse insulinoma (MIN6) cells obtained by a plate-reader, the assay coefficient of variation (CV) decreased from 8.0% in the raw dataset to 5.1% and the hit amplitudes were reduced by only 1% while the DFT method increased the CV by 36.0% and reduced the hit amplitude by 21%. Thus, our results show that the bidirectional HMF provides superior corrections of MTP data distortions while at the same time preserving hit amplitudes and improving dynamic range. The software to perform hybrid median filter MTP corrections is available at http://bccg.burnham.org/HTS/HMF_Download_Page.aspx, password is pbushway.