A statistical framework for high-content phenotypic profiling using cellular feature distributions.

High-content screening (HCS) uses microscopy images to generate phenotypic profiles of cell morphological data in high-dimensional feature space. While HCS provides detailed cytological information at single-cell resolution, these complex datasets are usually aggregated into summary statistics that do not leverage patterns of biological variability within cell populations. Here we present a broad-spectrum HCS analysis system that measures image-based cell features from 10 cellular compartments across multiple assay panels. We introduce quality control measures and statistical strategies to streamline and harmonize the data analysis workflow, including positional and plate effect detection, biological replicates analysis and feature reduction. We also demonstrate that the Wasserstein distance metric is superior over other measures to detect differences between cell feature distributions. With this workflow, we define per-dose phenotypic fingerprints for 65 mechanistically diverse compounds, provide phenotypic path visualizations for each compound and classify compounds into different activity groups.

FRET-Based Probe for High-Throughput DNA Intercalator Drug Discovery and In Vivo Imaging.

Molecules that bind DNA by intercalating its bases remain among the most potent cancer therapies and antimicrobials due to their interference with DNA-processing proteins. To accelerate the discovery of novel intercalating drugs, we designed a fluorescence resonance energy transfer (FRET)-based probe that reports on DNA intercalation, allowing rapid and sensitive screening of chemical libraries in a high-throughput format. We demonstrate that the method correctly identifies known DNA intercalators in approved drug libraries and discover previously unreported intercalating compounds. When introduced in cells, the oligonucleotide-based probe rapidly distributes in the nucleus, allowing direct imaging of the dynamics of drug entry and its interaction with DNA in its native environment. This enabled us to directly correlate the potency of intercalators in killing cultured cancer cells with the ability of the drug to penetrate the cell membrane. The combined capability of the single probe to identify intercalators in vitro and follow their function in vivo can play a valuable role in accelerating the discovery of novel DNA-intercalating drugs or repurposing approved ones.

Quantification of sterol-specific response in human macrophages using automated imaged-based analysis.

BACKGROUND: The transformation of normal macrophage cells into lipid-laden foam cells is an important step in the progression of atherosclerosis. One major contributor to foam cell formation in vivo is the intracellular accumulation of cholesterol. METHODS: Here, we report the effects of various combinations of low-density lipoprotein, sterols, lipids and other factors on human macrophages, using an automated image analysis program to quantitatively compare single cell properties, such as cell size and lipid content, in different conditions. RESULTS: We observed that the addition of cholesterol caused an increase in average cell lipid content across a range of conditions. All of the sterol-lipid mixtures examined were capable of inducing increases in average cell lipid content, with variations in the distribution of the response, in cytotoxicity and in how the sterol-lipid combination interacted with other activating factors. For example, cholesterol and lipopolysaccharide acted synergistically to increase cell lipid content while also increasing cell survival compared with the addition of lipopolysaccharide alone. Additionally, ergosterol and cholesteryl hemisuccinate caused similar increases in lipid content but also exhibited considerably greater cytotoxicity than cholesterol. CONCLUSIONS: The use of automated image analysis enables us to assess not only changes in average cell size and content, but also to rapidly and automatically compare population distributions based on simple fluorescence images. Our observations add to increasing understanding of the complex and multifactorial nature of foam-cell formation and provide a novel approach to assessing the heterogeneity of macrophage response to a variety of factors.

Non-invasive single-cell biomechanical analysis using live-imaging datasets.

The physiological state of a cell is governed by a multitude of processes and can be described by a combination of mechanical, spatial and temporal properties. Quantifying cell dynamics at multiple scales is essential for comprehensive studies of cellular function, and remains a challenge for traditional end-point assays. We introduce an efficient, non-invasive computational tool that takes time-lapse images as input to automatically detect, segment and analyze unlabeled live cells; the program then outputs kinematic cellular shape and migration parameters, while simultaneously measuring cellular stiffness and viscosity. We demonstrate the capabilities of the program by testing it on human mesenchymal stem cells (huMSCs) induced to differentiate towards the osteoblastic (huOB) lineage, and T-lymphocyte cells (T cells) of naïve and stimulated phenotypes. The program detected relative cellular stiffness differences in huMSCs and huOBs that were comparable to those obtained with studies that utilize atomic force microscopy; it further distinguished naïve from stimulated T cells, based on characteristics necessary to invoke an immune response. In summary, we introduce an integrated tool to decipher spatiotemporal and intracellular dynamics of cells, providing a new and alternative approach for cell characterization.

A spatiotemporal characterization method for the dynamic cytoskeleton.

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (Df ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to Df , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in Df and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. © 2016 Wiley Periodicals, Inc.

Phylogenetic prediction of the maximum per capita rate of population growth.

The maximum per capita rate of population growth, r, is a central measure of population biology. However, researchers can only directly calculate r when adequate time series, life tables and similar datasets are available. We instead view r as an evolvable, synthetic life-history trait and use comparative phylogenetic approaches to predict r for poorly known species. Combining molecular phylogenies, life-history trait data and stochastic macroevolutionary models, we predicted r for mammals of the Caniformia and Cervidae. Cross-validation analyses demonstrated that, even with sparse life-history data, comparative methods estimated r well and outperformed models based on body mass. Values of r predicted via comparative methods were in strong rank agreement with observed values and reduced mean prediction errors by approximately 68 per cent compared with two null models. We demonstrate the utility of our method by estimating r for 102 extant species in these mammal groups with unknown life-history traits.

Mathematical modeling of axonal formation. Part I: Geometry.

A stochastic model is proposed for the position of the tip of an axon. Parameters in the model are determined from laboratory data. The first step is the reduction of inherent error in the laboratory data, followed by estimating parameters and fitting a mathematical model to this data. Several axonogenesis aspects have been investigated, particularly how positive axon elongation and growth cone kinematics are coupled processes but require very different theoretical descriptions. Preliminary results have been obtained through a series of experiments aimed at isolating the response of axons to controlled gradient exposures to guidance cues and the effects of ethanol and similar substances. We show results based on the following tasks; (A) development of a novel filtering strategy to obtain data sets truly representative of the axon trail formation; (B) creation of a coarse graining method which establishes (C) an optimal parameter estimation technique, and (D) derivation of a mathematical model which is stochastic in nature, parameterized by arc length. The framework and the resulting model allow for the comparison of experimental and theoretical mean square displacement (MSD) of the developing axon. Current results are focused on uncovering the geometric characteristics of the axons and MSD through analytical solutions and numerical simulations parameterized by arc length, thus ignoring the temporal growth processes. Future developments will capture the dynamic growth cone and how it behaves as a function of time. Qualitative and quantitative predictions of the model at specific length scales capture the experimental behavior well.

Computer simulations of heterologous immunity: highlights of an interdisciplinary cooperation.

The relationship between biological research and mathematical modeling is complex, critical, and vital. In this review, we summarize the results of the collaboration between two laboratories, exploring the interaction between mathematical modeling and wet-lab immunology. During this collaboration several aspects of the immune defence against viral infections were investigated, focusing primarily on the subject of heterologous immunity. In this manuscript, we emphasize the topics where computational simulations were applied in conjunction with experiments, such as immune attrition, the growing and shrinking of cross-reactive T cell repertoires following repeated infections, the short and long-term effects of cross-reactive immunological memory, and the factors influencing the appearance of new clonal specificities. For each topic, we describe how the mathematical model used was adapted to answer specific biological questions, and we discuss the hypotheses that were generated by simulations. Finally, we propose rules for testing hypotheses that emerge from model experimentation in the wet lab, and vice-versa.

Systematic simulation of cross-reactivity predicts ambiguity in Tk memory: it may save lives of the infected, but limits specificities vital for further responses.

The present study uses the agent-based model IMMSIM to simulate immune responses to a viral infection, with a focus on the impact of preformed memory (homologous and heterologous) on the quality and the efficacy of the response. The in machina results confirm the observed thwarting of new, naïve responses exerted by cross-reacting memory, but they also reveal that the competitive inhibition is made possible by the different time frame used by the primary and the secondary response, a well-known fact, epitomized by the interval of about 75 time steps between their peaks. This novel finding justifies the depression of naïve responses and the long-term consequences it could bring about and the role of memory as a player in a survival of the fittest game.