Quantitative high-content/high-throughput microscopy analysis of lipid droplets in subject-specific adipogenesis models.

Neutral lipids packed in lipid droplets (LDs) are essential as a source of fuel for organisms, and specialized storing cells, the adipocytes, provide a buffer for energy variations. Many modern-society-disorders are connected with excess accumulation or deficiency of LDs in adipose tissue. Intracellular LD number and size distribution reflect the tissue conditions, while the associated mechanisms and genes rs are still poorly understood. Large-scale genetic screens using human in vitro differentiated primary adipocytes require cell samples donated from many patients. The heterogeneity appearing between donors highlighted the need for high-throughput methods robust to individual variations. Previous image analysis algorithms failed to handle individual LDs, but focused on averages, hiding population heterogeneity. We present a new high-content analysis (HCA) technique for analysis of fat cell metabolism using data from a large-scale RNAi screen including images of more than 500 k in vitro differentiated adipocytes from three donors. The RNAi-based suppression of Perilipin 1 (PLIN1), a protein involved in the adipocyte lipid metabolism, served as a positive control, while cells treated with randomized RNA served as negative controls. We validate our segmentation by comparing our results to those of previously published methods: We also evaluate the discriminative power of different morphological features describing LD size distribution. Classification of cells as containing few large or many small LDs followed by calculating the percentage of cells in each class proved to discriminate the positive PLIN1-suppressed phenotype from the untreated negative control with an area under the receiver operating characteristic curve of 0.98. The results suggest that this HCA method offers improved segmentation and classification accuracy, and can, thus, be utilized to quantify changes in LD metabolism in response to treatment in many cell models relevant to a variety of diseases. © 2017 International Society for Advancement of Cytometry.

Forest-Scale Phenotyping: Productivity Characterisation Through Machine Learning.

Advances in remote sensing combined with the emergence of sophisticated methods for large-scale data analytics from the field of data science provide new methods to model complex interactions in biological systems. Using a data-driven philosophy, insights from experts are used to corroborate the results generated through analytical models instead of leading the model design. Following such an approach, this study outlines the development and implementation of a whole-of-forest phenotyping system that incorporates spatial estimates of productivity across a large plantation forest. In large-scale plantation forestry, improving the productivity and consistency of future forests is an important but challenging goal due to the multiple interactions between biotic and abiotic factors, the long breeding cycle, and the high variability of growing conditions. Forest phenotypic expression is highly affected by the interaction of environmental conditions and forest management but the understanding of this complex dynamics is incomplete. In this study, we collected an extensive set of 2.7 million observations composed of 62 variables describing climate, forest management, tree genetics, and fine-scale terrain information extracted from environmental surfaces, management records, and remotely sensed data. Using three machine learning methods, we compared models of forest productivity and evaluate the gain and Shapley values for interpreting the influence of categorical variables on the power of these methods to predict forest productivity at a landscape level. The most accurate model identified that the most important drivers of productivity were, in order of importance, genetics, environmental conditions, leaf area index, topology, and soil properties, thus describing the complex interactions of the forest. This approach demonstrates that new methods in remote sensing and data science enable powerful, landscape-level understanding of forest productivity. The phenotyping method developed here can be used to identify superior and inferior genotypes and estimate a productivity index for individual site. This approach can improve tree breeding and deployment of the right genetics to the right site in order to increase the overall productivity across planted forests.

Characterizing the DNA Damage Response by Cell Tracking Algorithms and Cell Features Classification Using High-Content Time-Lapse Analysis.

Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. Using automatic extraction of RIF imaging features and linear programming techniques, we were able to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolve with random motion following normal diffusion law. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We estimate repair domain sizes of 7.5 to 11 µm2 with a maximum number of ~15 domains per MCF10A cell. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair. Such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation.