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Integration of Plant Metabolomics Data with Metabolic Networks: Progresses and Challenges

Integration of Plant Metabolomics Data with Metabolic Networ...

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作者： Nadine Töpfer Samuel M. Seaver Asaph Aharoni

In the last decade, plant genome-scale modeling has developed rapidly and modeling efforts have advanced from representing metabolic behavior of plant heterotrophic cell suspensions to studying the complex interplay of cell types, tissues, and organs. A crucial driving force for such developments is the availability and integration of “omics” data (e.g., transcriptomics, proteomics, and metabolomics) which enable the reconstruction, extraction, and application of context-specific metabolic networks. In this chapter, we demonstrate a workflow to integrate gas chromatography coupled to mass spectrometry (GC-MS)-based metabolomics data of tomato fruit pericarp (flesh) tissue, at five developmental stages, with a genome-scale reconstruction of tomato metabolism. This method allows for the extraction of context-specific networks reflecting changing activities of metabolic pathways throughout fruit development and maturation. less

关键词： Metabolomics Gas Chromatography (GC) Mass Spectrometry Gas Chromatography-mass Spectrometry Python programming language Data integration Flux balance analysis Plant metabolic network Constraint-based optimization

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In Vivo Two-Photon Imaging of the Olfactory System in Insects

In Vivo Two-Photon Imaging of the Olfactory System in Insect...

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作者： Marco Paoli Albrecht Haase

This chapter describes how to apply two-photon neuroimaging to study the insect olfactory system in vivo. It provides a complete protocol for insect brain functional imaging, with some additional remarks on the acquisition of morphological information from the living brain. We discuss the most important choices to make when buying or building a two-photon laser-scanning microscope. We illustrate different possibilities of animal preparation and brain tissue labeling for in vivo imaging. Finally, we give an overview of the main methods of image data processing and analysis, followed by a short description of pioneering applications of this imaging modality. less

关键词： Two-photon Microscopy Calcium Imaging Two-photon In Vivo Imaging Image Analysis Neuroimaging In Vivo Imaging Optical Imaging In Vivo Labeling Wide-field Microscopy Dissection Confocal Microscopy Fluorescence Microscopy Image Processing Principal Component Analysis Statistical Calculation MATLAB Electrophysiology Recombinant Protein Expression In Situ Hybridization Chiral Separation Time-correlated Single Photon Counting 3D Imaging programming language Apis mellifera Drosophila Drosophila melanogaster Saccharomyces cerevisiae Ciona Insects Olfaction Antennal lobe Anti-bruchpilot

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Computational Prediction of

Computational Prediction of

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作者： Albert Ros-Lucas David Rioja-Soto Joaquim Gascón Julio Alonso-Padilla

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is considered a Neglected Tropical Disease. Limited investment is assigned to its study and control, even though it is one of the most prevalent parasitic infections worldwide. An innovative vaccination strategy involving an epitope-based vaccine that displays multiple immune determinants originating from different antigens could counteract the high biological complexity of the parasite and lead to a wide and protective immune response. In this chapter, we describe a computational reverse vaccinology pipeline applied to identify the most promising peptide sequences from T. cruzi proteins, prioritizing evolutionary conserved sequences, to finally select a list of T and B cell epitope candidates to be further tested in an experimental setting. less

关键词： Vaccination programming language Multiple Sequence Alignment Python SignalP Trypanosoma cruzi Homo sapiens Chagas disease Reverse vaccinology Epitopes

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Reconstruction of Archaeal Genomes from Short-Read Metagenomes

Reconstruction of Archaeal Genomes from Short-Read Metagenom...

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作者： Till L. Bornemann Panagiotis S. Adam Alexander J. Probst

As the majority of biological diversity remains unexplored and uncultured, investigating it requires culture-independent approaches. Archaea in particular suffer from a multitude of issues that make their culturing problematic, from them being frequently members of the rare biosphere, to low growth rates, to them thriving under very specific and often extreme environmental and community conditions that are difficult to replicate. OMICs techniques are state of the art approaches that allow direct high-throughput investigations of environmental samples at all levels from nucleic acids to proteins, lipids, and secondary metabolites. Metagenomics, as the foundation for other OMICs techniques, facilitates the identification and functional characterization of the microbial community members and can be combined with other methods to provide insights into the microbial activities, both on the RNA and protein levels. In this chapter, we provide a step-by-step workflow for the recovery of archaeal genomes from metagenomes, starting from raw short-read sequences. This workflow can be applied to recover bacterial genomes as well. less

关键词： Metagenomics Statistical Calculation Bowtie Coding Region Prediction Sequence Editing CRISPR DNA Extraction Microscopy DNA Fragmentation Assay programming language Python SNP Calling Genome Annotation Base-calling Prokaryotes Genome curation Short-read sequencing Rare biosphere

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Detection, Discrimination & Localization: The Psychophysics of Touch

Detection, Discrimination & Localization: The Psychophysics ...

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作者： Nicholas Paul Holmes Luigi Tamè

Detecting and discriminating touches on your fingertip and other highly sensitive body parts has been a paradigm in somatosensory science since the birth of psychophysics in the nineteenth century. By isolating a body part and applying discrete stimuli over many repetitions, the limits of somatosensation and bodily perception can be discovered. This chapter will focus on two methods of studying discriminative touch in the temporal and spatial domains: vibrotactile perception and spatial acuity. Different psychophysical approaches and experimental designs will be described and evaluated in terms of their validity, efficiency, and reliability. Practical and participant-specific difficulties will be noted. Vibrotactile and spatial acuity methods offer relatively cheap and reliable measures of somatosensation, often suitable for undergraduate student projects. Yet care and experimentation is required to ensure that the experimental design is adequate, and the data collection is sufficient to answer your theoretical question. less

关键词： MRI programming language Interferometry Homo sapiens Detection Behavior Discrimination Cutaneous Vibrotactile Fingers Grating orientation task Two-point discrimination

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Large-Scale Molecular Dynamics Simulations of Cellular Compartments

Large-Scale Molecular Dynamics Simulations of Cellular Compa...

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作者： Eric Wilson John Vant Jacob Layton Ryan Boyd Hyungro Lee Matteo Turilli Benjamín Hernández Sean Wilkinson Shantenu Jha Chitrak Gupta Daipayan Sarkar Abhishek Singharoy

Molecular dynamics or MD simulation is gradually maturing into a tool for constructing in vivo models of living cells in atomistic details. The feasibility of such models is bolstered by integrating the simulations with data from microscopic, tomographic and spectroscopic experiments on exascale supercomputers, facilitated by the use of deep learning technologies. Over time, MD simulation has evolved from tens of thousands of atoms to over 100 million atoms comprising an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium. In this chapter, we present a step-by-step outline for preparing, executing and analyzing such large-scale MD simulations of biological systems that are essential to life processes. All scripts are provided via GitHub. less

关键词： Molecular Dynamics Simulation Deep learning X-ray Crystallography Cryo-EM Mass Spectrometry Tomography Protein Structure Analysis Python programming language High-performance computing NAMD VMD Multiscale simulation Photosynthetic chromatophore Ensemble toolkit

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Integrated Microarray-based Tools for Detection of Genomic DNA Damage and Repair Mechanisms

Integrated Microarray-based Tools for Detection of Genomic D...

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作者： Patrick Eijk Yumin Teng Mark R. Bennet Katie E. Evans James R. Powell Richard M. Webster Simon H. Reed

The genetic information contained within the DNA molecule is highly susceptible to chemical and physical insult, caused by both endogenous and exogenous sources that can generate in the order of thousands of lesions a day in each of our cells (Lindahl, Nature 362(6422):709–715, 1993). DNA damages interfere with DNA metabolic processes such as transcription and replication and can be potent inhibitors of cell division and gene expression. To combat these regular threats to genome stability, a host of DNA repair mechanisms have evolved. When DNA lesions are left unrepaired due to defects in the repair pathway, mutations can arise that may alter the genetic information of the cell. DNA repair is thus fundamental to genome stability and defects in all the major repair pathways can lead to cancer predisposition. Therefore, the ability to accurately measure DNA damage at a genomic scale and determine the level, position, and rates of removal by DNA repair can contribute greatly to our understanding of how DNA repair in chromatin is organized throughout the genome. For this reason, we developed the 3D-DIP-Chip protocol described in this chapter. Conducting such measurements has potential applications in a variety of other fields, such as genotoxicity testing and cancer treatment using DNA damage inducing chemotherapy. Being able to detect and measure genomic DNA damage and repair patterns in individuals following treatment with chemotherapy could enable personalized medicine by predicting response to therapy. less

关键词： Immunoprecipitation Real-time PCR Cell And Tissue Culture Harvesting Cells DNA Extraction Sonication Microarray Hybridization DNA Purification Nucleic Acid Microarray Cross-linking Cell Lysis Snap Freeze Agarose Gel Electrophoresis Quantitative Reverse Transcription PCR ChIP-on-chip Ligation-Mediated PCR Genome Amplification Library Preparation Genomics ChIP Assay Microarray Data analysis programming language Saccharomyces cerevisiae Homo sapiens Chemotherapy Nucleotide excision repair DNA repair DNA damage Genotoxicity Genome stability 3D-DIP-Chip Anti-rat Anti-mouse Anti-CPD

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Interpreting Microarray Results With Gene Ontology and MeSH

Interpreting Microarray Results With Gene Ontology and MeSH

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作者： John D. Osborne Lihua (Julie) Zhu Simon M. Lin Warren A. Kibbe

Methods are described to take a list of genes generated from a microarray experiment and interpret these results using various tools and ontologies. A workflow is described that details how to convert gene identifiers with SOURCE and MatchMiner and then use these converted gene lists to search the gene ontology (GO) and the medical subject headings (MeSH) ontology. Examples of searching GO with DAVID, EASE, and GOMiner are provided along with an interpretation of results. The mining of MeSH using high-density array pattern interpreter with a set of gene identifiers is also described. less

关键词： Microarray Statistical Calculation GenBank programming language protocol MeSH DAVID GO HAPI SOURCE MatchMiner Interpret

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Mapping the

Mapping the

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作者： Daniel J. Nightingale Stephen G. Oliver Kathryn S. Lilley

The subcellular localization of proteins is a posttranslational modification of paramount importance. The ability to study subcellular and organelle proteomes improves our understanding of cellular homeostasis and cellular dynamics. In this chapter, we describe a protocol for the unbiased and high-throughput study of protein subcellular localization in the yeast Saccharomyces cerevisiae: hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT), which involves biochemical fractionation of Saccharomyces cerevisiae and high resolution mass spectrometry-based protein quantitation using TMT 10-plex isobaric tags. This protocol enables the determination of the subcellular localizations of thousands of proteins in parallel in a single experiment and thereby deep sampling and high-resolution mapping of the spatial proteome. less

关键词： Subcellular Fractionation Cell And Tissue Culture Solid-phase Extraction (SPE) Mass Spectrometry Localization Of Organelle Proteins By Isotope Tagging Fractionation Isobaric Mass Labeling Sonication BCA assay Western Blot Chromatography hyperLOPIT Spatial proteomics programming language Saccharomyces cerevisiae Protein localization Organelle Anti-Cox2p

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qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development

qFlow Cytometry-Based Receptoromic Screening: A High-Through...

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作者： Si Chen Jared Weddell Pavan Gupta Grace Conard James Parkin Princess I. Imoukhuede

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression. less

关键词： Immunolabeling Flow Cytometry Cell And Tissue Culture Immunohistochemistry Cell Labeling MATLAB Data analysis programming language Homo sapiens Angiogenesis Systems biology Background subtraction Heterogeneity qFlow cytometry Vascular Endothelial Growth Factor (VEGF) Platelet-Derived Growth Factor (PDGF) Mixture modeling

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