ABSTRACT
High-throughput screening allows rapid identification of new candidate compounds for biological probe or drug development. Here, we describe a principled method to generate "assay performance profiles" for individual compounds that can serve as a basis for similarity searches and cluster analyses. Our method overcomes three challenges associated with generating robust assay performance profiles: (1) we transform data, allowing us to build profiles from assays having diverse dynamic ranges and variability; (2) we apply appropriate mathematical principles to handle missing data; and (3) we mitigate the fact that loss-of-signal assay measurements may not distinguish between multiple mechanisms that can lead to certain phenotypes (e.g., cell death). Our method connected compounds with similar mechanisms of action, enabling prediction of new targets and mechanisms both for known bioactives and for compounds emerging from new screens. Furthermore, we used Bayesian modeling of promiscuous compounds to distinguish between broadly bioactive and narrowly bioactive compound communities. Several examples illustrate the utility of our method to support mechanism-of-action studies in probe development and target identification projects.
Subject(s)
Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Small Molecule Libraries/chemistry , Algorithms , Animals , Bayes Theorem , Cell Line, Tumor , Cluster Analysis , Humans , Membrane Potential, Mitochondrial , Mice , Models, Molecular , Phenotype , Reproducibility of ResultsABSTRACT
Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.
Subject(s)
Fluorescent Dyes/chemistry , Image Processing, Computer-Assisted , Cell Line, Tumor , HumansABSTRACT
PURPOSE: To define alterations in the density of corneal basal epithelium in relation to diabetic retinopathic severity and the alteration of corneal innervation using confocal microscopy. DESIGN: Prospective case-control study. METHODS: Forty-two type II diabetic patients stratified into nondiabetic (NDR), nonproliferative diabetic (NPDR), and proliferative diabetic (PDR) retinopathy and 14 age-matched healthy control subjects were studied. Epithelial and stromal cell densities and subbasal nerves were investigated by confocal microscopy. RESULTS: Corneal basal epithelial cell density (P = .0001), nerve fiber density (NFD) (P < .0001), nerve branch density (NBD) (P = .0003), and tortuosity coefficient (Tc) (P < .0001) were significantly different among the four groups. Basal epithelial density was significantly correlated with NFD (r = 0.43, P = .0009), NBD (r = 0.36, P = .007), and TC (r = -0.58, P < .0001). CONCLUSIONS: Patients with diabetes show alterations in corneal innervations and basal epithelial cell density in different retinopathic stages. We demonstrate that reduced density in basal epithelial cell is correlated with changes in innervation.
Subject(s)
Cornea/innervation , Diabetic Retinopathy/complications , Epithelium, Corneal/pathology , Ophthalmic Nerve/pathology , Case-Control Studies , Cell Count , Corneal Stroma/pathology , Diabetes Mellitus, Type 2/complications , Female , Humans , Male , Microscopy, Confocal , Middle Aged , Prospective StudiesABSTRACT
PURPOSE: To evaluate the effects of tear film breakup on higher order aberrations of the anterior cornea in normal, dry, and post-LASIK eyes. METHODS: In a prospective study, 120 eyes (120 subjects) comprised three groups: post-LASIK eyes, dry eyes, and normal eyes. Tear film break-up time (BUT) was measured by fluorescein dye under slit-lamp microscopy. Corneal topographies were measured by videokeratography immediately after the following times: post-blink, 1/2 BUT, BUT, and after normal saline application. VOL-Pro software processed topographies to obtain and analyze higher order aberrations (3rd through 6th orders over 6 mm) of the anterior comeal surface. RESULTS: The measurement time scale was set by the BUT: significantly less for dry eyes (8.3 +/- 3.4 seconds) than post-LASIK eyes (11.3 +/- 5.1 seconds) or normal eyes (10.3 +/- 5.6 seconds). Normal and dry eye subjects showed a pattern of significant increase of aberrations from post-blink through BUT and decrease of aberrations after saline addition in coma, trefoil, and 3rd through 6th order aberrations. Post-LASIK eyes exhibited no significant difference between phases. CONCLUSIONS: Disruption of the tear film increases anterior corneal higher order aberrations in normal eyes and more rapidly in dry eyes. The increase in aberrations caused by conventional LASIK is greater than that of tear film-related effects.
