IgE epitope mapping of food allergens contributes to a better understanding of the pathogenesis of food allergy and may become an additional tool for food allergy diagnosis/prognosis. Microarray platforms which allow ...
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IgE epitope mapping of food allergens contributes to a better understanding of the pathogenesis of food allergy and may become an additional tool for food allergy diagnosis/prognosis. Microarray platforms which allow for simultaneous screening of a large number of peptides corresponding to the sequences of food allergens are ideally suited for large-scale IgE epitope mapping. Here we describe the method of performing a reliable and sensitive peptide microarray immunoassay, which was developed in our lab and results in the identification of candidate IgE epitope biomarkers useful in determining allergic disease severity and prognosis, as well as in the prediction of treatment outcomes. A gastric digestion model that can be applied to prescreen peptides and reduce costs in the peptide microarray is also described in this chapter. less
The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility ...
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The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility of enzymatic experiments and reusability of enzymatic data are challenging. We developed the XML-based markup language EnzymeML to enable storage and exchange of enzymatic data such as reaction conditions, the time course of the substrate and the product, kinetic parameters and the kinetic model, thus making enzymatic data findable, accessible, interoperable and reusable (FAIR). The feasibility and usefulness of the EnzymeML toolbox is demonstrated in six scenarios, for which data and metadata of different enzymatic reactions are collected and analyzed. EnzymeML serves as a seamless communication channel between experimental platforms, electronic lab notebooks, tools for modeling of enzyme kinetics, publication platforms and enzymatic reaction databases. EnzymeML is open and transparent, and invites the community to contribute. All documents and codes are freely available at https://*** . less
The initial stages of tandem mass spectral data interpretation for a pro-teomics project usually involve looking for exact matches between the query spectrum and theoretical fragmentation data derived from database se...
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The initial stages of tandem mass spectral data interpretation for a pro-teomics project usually involve looking for exact matches between the query spectrum and theoretical fragmentation data derived from database sequences (1–3). A number of programs are available that use the observed peptide mass as a filter for identifying portions of database sequences with the same calculated mass prior to evaluating the congruency between the database sequence candidates and the tandem mass spectrum (4–7). These programs generally fall short when analyzing peptides whose sequences differ in a manner that alters its mass compared with the database sequence (seeNote 1 ). For example, a single conservative substitution of isoleucine for valine is sufficient to thwart algorithms that rely on peptide mass prefilters. We commonly obtain high-quality tandem mass spectral data of peptides for which no exact database match can be made, and the question remains whether these nonmatching spectra are due to novel sequences, or are a result of less interesting possibilities such as interspecies variation, database sequence errors, or unexpected pro-teolytic cleavages. less
Fluorescent live cell time-lapse microscopy is steadily contributing to our better understanding of the relationship between cell signaling and fate. However, large volumes of time-series data generated in these exper...
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Fluorescent live cell time-lapse microscopy is steadily contributing to our better understanding of the relationship between cell signaling and fate. However, large volumes of time-series data generated in these experiments and the heterogenous nature of signaling responses due to cell-cell variability hinder the exploration of such datasets. The population averages insufficiently describe the dynamics, yet finding prototypic dynamic patterns that relate to different cell fates is difficult when mining thousands of time-series. Here we demonstrate a protocol where we identify such dynamic phenotypes in a population of PC-12 cells that respond to a range of sustained growth factor perturbations. We use Time-Course Inspector, a free R/Shiny web application to explore and cluster single-cell time-series. less
The level of gene expression is known to vary from cell to cell and even in the same cell over time. This variability provides cells with the ability to mitigate environmental stresses and genetic perturbations, and f...
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The level of gene expression is known to vary from cell to cell and even in the same cell over time. This variability provides cells with the ability to mitigate environmental stresses and genetic perturbations, and facilitates gene expression evolution. Recently, many valuable gene expression noise data measured at the single-cell level and gene expression variation measured for cell populations have become available. In this chapter, we show how to perform integrative analysis using these data. Specifically, we introduce how to apply a machine learning technique (support vector regression) to explore the relationship between gene expression variations and stochastic noise. less
DNA microarray technology is a useful tool to further our understanding of underlying biological pathways governing the behavior of cells and tissues. In fact, microarrays have evolved over the last 10 years or so to ...
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DNA microarray technology is a useful tool to further our understanding of underlying biological pathways governing the behavior of cells and tissues. In fact, microarrays have evolved over the last 10 years or so to become one of the major technologies utilized in biomedical research. This chapter will provide a brief history of the development of DNA microarray technology, a brief survey of widely accepted microarray platforms, and then expand upon critical components of DNA microarray experimentation necessary to ensure optimal outcomes in a given microarray experiment. Topics covered will include the necessity of a good initial experimental design, quality control issues with respect to input RNA or DNA, appropriate use of biological replicates relevant to a given biological question, and analysis strategies employed to extract meaningful results from a microarray *** microarray technology is a useful tool to further our understanding of underlying biological pathways governing the behavior of cells and tissues. In fact, microarrays have evolved over the last 10 years or so to become one of the major technologies utilized in biomedical research. This chapter will provide a brief history of the development of DNA microarray technology, a brief survey of widely accepted microarray platforms, and then expand upon critical components of DNA microarray experimentation necessary to ensure optimal outcomes in a given microarray experiment. Topics covered will include the necessity of a good initial experimental design, quality control issues with respect to input RNA or DNA, appropriate use of biological replicates relevant to a given biological question, and analysis strategies employed to extract meaningful results from a microarray experiment.
Molecular descriptors encode a variety of molecular representations for computer-assisted drug discovery. Here, we focus on the Weighted Holistic Atom Localization and Entity Shape (WHALES) descriptors, which were ori...
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Molecular descriptors encode a variety of molecular representations for computer-assisted drug discovery. Here, we focus on the Weighted Holistic Atom Localization and Entity Shape (WHALES) descriptors, which were originally designed for scaffold hopping from natural products to synthetic molecules. WHALES descriptors capture molecular shape and partial charges simultaneously. We introduce the key aspects of the WHALES concept and provide a step-by-step guide on how to use these descriptors for virtual compound screening and scaffold hopping. The results presented can be reproduced by using the code freely available from URL: ***/ETHmodlab/scaffold_hopping_whales . less
Natural selection has molded the evolution of species across all taxa. Much more recently, on an evolutionary scale, human-oriented selection started to play an important role in shaping organisms, markedly so after t...
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Natural selection has molded the evolution of species across all taxa. Much more recently, on an evolutionary scale, human-oriented selection started to play an important role in shaping organisms, markedly so after the domestication of animals and plants. These selection processes have left traceable marks in the genome. Following from the recent advances in molecular genetics technologies, a number of methods have been developed to detect such signals, termed genomic signatures of selection. In this chapter we discuss a straightforward protocol based on the FST statistic to identify genomic regions that exhibit high variation in allelic frequency between groups, which is a characteristic of genomic regions that have gone through differential selection. How to define the borders of these regions and further explore its genetic content is then discussed. less
miRNAs are involved in various biological processes and different diseases through interacting with their target mRNAs. Therefore, deciphering miRNA targets is important for diagnostics and therapeutics. However, it i...
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miRNAs are involved in various biological processes and different diseases through interacting with their target mRNAs. Therefore, deciphering miRNA targets is important for diagnostics and therapeutics. However, it is still complex and expensive to do large scale screening for miRNA alterations and identify their downstream miRNA targets. Recently, integrating analysis of miRNA expression profiles and mRNA expression levels has been used to successfully identify the most prominent interactions. Here, we present a protocol that combines both expression data and computational prediction to refine the microarray result for identifying the miRNA targets. Many details are also provided to help researchers choose more appropriate tools and methods for a given type of application. Available web-based resources and experimental conditions for functional enrichment and validation of miRNA targets are also summarized. less
The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure–function rela...
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The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure–function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation. less
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