Protocols and programs for high-throughput growth and aging phenotyping in yeast.

in PloS one (2015), 10(3), 0119807

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines ... [more ▼]

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens. [less ▲]

Pro-inflammatory macrophages sustain pyruvate oxidation through pyruvate dehydrogenase for the synthesis of itaconate and to enable cytokine expression

in Journal of Biological Chemistry (2015)

Discovering pair-wise genetic interactions: an information theory-based approach.

in PloS one (2014), 9(3), 92310

Phenotypic variation, including that which underlies health and disease in humans, results in part from multiple interactions among both genetic variation and environmental factors. While diseases or ... [more ▼]

Phenotypic variation, including that which underlies health and disease in humans, results in part from multiple interactions among both genetic variation and environmental factors. While diseases or phenotypes caused by single gene variants can be identified by established association methods and family-based approaches, complex phenotypic traits resulting from multi-gene interactions remain very difficult to characterize. Here we describe a new method based on information theory, and demonstrate how it improves on previous approaches to identifying genetic interactions, including both synthetic and modifier kinds of interactions. We apply our measure, called interaction distance, to previously analyzed data sets of yeast sporulation efficiency, lipid related mouse data and several human disease models to characterize the method. We show how the interaction distance can reveal novel gene interaction candidates in experimental and simulated data sets, and outperforms other measures in several circumstances. The method also allows us to optimize case/control sample composition for clinical studies. [less ▲]

Automated nuclei clump splitting by combining local concavity orientation and graph partitioning

in International Conference on Biomedical and Health Informatics (2014)

Automated clump decomposition is essential for single cell based analysis of fluorescent microscopy images. This paper presents a new method for automatically splitting clumps of cell nuclei in ... [more ▼]

Automated clump decomposition is essential for single cell based analysis of fluorescent microscopy images. This paper presents a new method for automatically splitting clumps of cell nuclei in fluorescence microscopy images. Nuclei are first segmented using histogram concavity analysis. Clumps of nuclei are detected by fitting an ellipse to the segmented objects and examining objects where the fitted ellipse does not overlap accurately with the segmented object. These clumps are then further processed to find concave points on the object boundaries. The orientation of the detected concavities is subsequently calculated based on the local shape of the object border. Finally, a graph segmentation based approach is used to pair concavities that represent best candidates for splitting touching nuclei based on properties derived from the local concavity properties. This approach was validated by manual inspection and has shown promising results in the high throughput analysis of HeLa cell images. [less ▲]

Reliable encoding of stimulus intensities within random sequences of intracellular Ca2+ spikes.

in Science Signaling (2014), 7(331), 59

Ca(2+) is a ubiquitous intracellular messenger that regulates diverse cellular activities. Extracellular stimuli often evoke sequences of intracellular Ca(2+) spikes, and spike frequency may encode ... [more ▼]

Ca(2+) is a ubiquitous intracellular messenger that regulates diverse cellular activities. Extracellular stimuli often evoke sequences of intracellular Ca(2+) spikes, and spike frequency may encode stimulus intensity. However, the timing of spikes within a cell is random because each interspike interval has a large stochastic component. In human embryonic kidney (HEK) 293 cells and rat primary hepatocytes, we found that the average interspike interval also varied between individual cells. To evaluate how individual cells reliably encoded stimuli when Ca(2+) spikes exhibited such unpredictability, we combined Ca(2+) imaging of single cells with mathematical analyses of the Ca(2+) spikes evoked by receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP3). This analysis revealed that signal-to-noise ratios were improved by slow recovery from feedback inhibition of Ca(2+) spiking operating at the whole-cell level and that they were robust against perturbations of the signaling pathway. Despite variability in the frequency of Ca(2+) spikes between cells, steps in stimulus intensity caused the stochastic period of the interspike interval to change by the same factor in all cells. These fold changes reliably encoded changes in stimulus intensity, and they resulted in an exponential dependence of average interspike interval on stimulation strength. We conclude that Ca(2+) spikes enable reliable signaling in a cell population despite randomness and cell-to-cell variability, because global feedback reduces noise, and changes in stimulus intensity are represented by fold changes in the stochastic period of the interspike interval. [less ▲]

Aneuploidy underlies a multicellular phenotypic switch.

in Proceedings of the National Academy of Sciences of the United States of America (2013), 110(30), 12367-72

Although microorganisms are traditionally used to investigate unicellular processes, the yeast Saccharomyces cerevisiae has the ability to form colonies with highly complex, multicellular structures ... [more ▼]

Although microorganisms are traditionally used to investigate unicellular processes, the yeast Saccharomyces cerevisiae has the ability to form colonies with highly complex, multicellular structures. Colonies with the "fluffy" morphology have properties reminiscent of bacterial biofilms and are easily distinguished from the "smooth" colonies typically formed by laboratory strains. We have identified strains that are able to reversibly toggle between the fluffy and smooth colony-forming states. Using a combination of flow cytometry and high-throughput restriction-site associated DNA tag sequencing, we show that this switch is correlated with a change in chromosomal copy number. Furthermore, the gain of a single chromosome is sufficient to switch a strain from the fluffy to the smooth state, and its subsequent loss to revert the strain back to the fluffy state. Because copy number imbalance of six of the 16 S. cerevisiae chromosomes and even a single gene can modulate the switch, our results support the hypothesis that the state switch is produced by dosage-sensitive genes, rather than a general response to altered DNA content. These findings add a complex, multicellular phenotype to the list of molecular and cellular traits known to be altered by aneuploidy and suggest that chromosome missegregation can provide a quick, heritable, and reversible mechanism by which organisms can toggle between phenotypes. [less ▲]

New methods for finding associations in large data sets: Generalizing the maximal information coefficient (MIC)

in Proceedings of the Ninth International Workshop on Computational Systems Biology (2012)

We propose here a natural, but substantive, extension of the MIC. Defined for two variables, MIC has a distinct advance for detecting potentially complex dependencies. Our extension provides a similar ... [more ▼]

We propose here a natural, but substantive, extension of the MIC. Defined for two variables, MIC has a distinct advance for detecting potentially complex dependencies. Our extension provides a similar means for dependencies among three variables. This itself is an important step for practical applications. We show that by merging two concepts, the interaction information, which is a generalization of the mutual information to three variables, and the normalized information distance, which measures informational sharing between two variables, we can extend the fundamental idea of MIC. Our results also exhibit some attractive properties that should be useful for practical applications in data analysis. Finally, the conceptual and mathematical framework presented here can be used to generalize the idea of MIC to the multi-variable case. [less ▲]

Carbohydrate-active enzymes exemplify entropic principles in metabolism

in Molecular Systems Biology (2011), 7(542),

Glycans comprise ubiquitous and essential biopolymers, which usually occur as highly diverse mixtures. The myriad different structures are generated by a limited number of carbohydrate-active enzymes ... [more ▼]

Glycans comprise ubiquitous and essential biopolymers, which usually occur as highly diverse mixtures. The myriad different structures are generated by a limited number of carbohydrate-active enzymes (CAZymes), which are unusual in that they catalyze multiple reactions by being relatively unspecific with respect to substrate size. Existing experimental and theoretical descriptions of CAZyme-mediated reaction systems neither comprehensively explain observed action patterns nor suggest biological functions of polydisperse pools in metabolism. Here, we overcome these limitations with a novel theoretical description of this important class of biological systems in which the mixing entropy of polydisperse pools emerges as an important system variable. In vitro assays of three CAZymes essential for central carbon metabolism confirm the power of our approach to predict equilibrium distributions and non-equilibrium dynamics. A computational study of the turnover of the soluble heteroglycan pool exemplifies how entropy-driven reactions establish a metabolic buffer in vivo that attenuates fluctuations in carbohydrate availability. We argue that this interplay between energy- and entropy-driven processes represents an important regulatory design principle of metabolic systems. [less ▲]

Statistical Analysis of Calcium Oscillations

in European Physical Journal. Special Topics (2010), 187

Modeling the complex dynamics of enzyme-pathway coevolution.

in Chaos (2010), 20(4), 045115

Metabolic pathways must have coevolved with the corresponding enzyme gene sequences. However, the evolutionary dynamics ensuing from the interplay between metabolic networks and genomes is still poorly ... [more ▼]

Metabolic pathways must have coevolved with the corresponding enzyme gene sequences. However, the evolutionary dynamics ensuing from the interplay between metabolic networks and genomes is still poorly understood. Here, we present a computational model that generates putative evolutionary walks on the metabolic network using a parallel evolution of metabolic reactions and their catalyzing enzymes. Starting from an initial set of compounds and enzymes, we expand the metabolic network iteratively by adding new enzymes with a probability that depends on their sequence-based similarity to already present enzymes. Thus, we obtain simulated time courses of chemical evolution in which we can monitor the appearance of new metabolites, enzyme sequences, or even entire organisms. We observe that new enzymes do not appear gradually but rather in clusters which correspond to enzyme classes. A comparison with Brownian motion dynamics indicates that our system displays biased random walks similar to diffusion on the metabolic network with long-range correlations. This suggests that a quantitative molecular principle may underlie the appearance of punctuated equilibrium dynamics, whereby enzymes occur in bursts rather than by phyletic gradualism. Moreover, the simulated time courses lead to a putative time-order of enzyme and organism appearance. Among the patterns we detect in these evolutionary trends is a significant correlation between the time of appearance and their enzyme repertoire size. Hence, our approach to metabolic evolution may help understand the rise in complexity at the biochemical and genomic levels. [less ▲]