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Application of Metabolomics in Lung Research

Application of Metabolomics in Lung Research

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作者： Nichole A. Reisdorph Charmion Cruickshank-Quinn Yasmeen Nkrumah-Elie Richard Reisdorph

Advancements in omics technologies have increased our potential to evaluate molecular changes in a rapid and comprehensive manner. This is especially true in mass spectrometry-based metabolomics where improvements, including ease of use, in high-performance liquid chromatography (HPLC), column chemistries, instruments, software, and molecular databases, have advanced the field considerably. Applications of this relatively new omics technology in clinical research include discovering disease biomarkers, finding new drug targets, and elucidating disease mechanisms. Here we describe a typical clinical metabolomics workflow, which includes the following steps: (1) extraction of metabolites from the lung, plasma, bronchoalveolar lavage, or cells; (2) sample analysis via liquid chromatography-mass spectrometry; and (3) data analysis using commercial and freely available software packages. Overall, the methods delineated here can help investigators use metabolomics to discovery novel biomarkers and to understand lung diseases. less

关键词： Mass Spectrometry Metabolomics Lavage HPLC Liquid Chromatography-mass Spectrometry Tissue Homogenization Protein Precipitation Liquid-liquid Extraction Hydrophobic Interaction Chromatography data Mining Statistical Calculation data visualization tools data analysis Homo sapiens Plasma Lung tissue BALF EBC

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Determination of DNA Methylation Levels Using Illumina HumanMethylation450 BeadChips

Determination of DNA Methylation Levels Using Illumina Human...

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作者： Melanie A. Carless

DNA methylation is a modifiable epigenetic phenomenon that has a strong influence over transcriptional regulation and as such has been consistently implicated in development and disease. Several platforms are targeted toward the identification of DNA methylation changes that might be pertinent to the disease process and include regional analysis (e.g., pyrosequencing) as well as genome-wide analysis (e.g., next-generation sequencing and microarray). The Illumina HumanMethylation450 BeadChip is one of the most comprehensive microarray platforms available, and due to the high costs associated with next-generation sequencing, it is becoming a widely used tool for the analysis of genome-wide DNA methylation levels. Providing quantitative DNA methylation levels at 482,421 CpG sites within CpG islands, shores, and shelves, as well as intergenic regions, the HumanMethylation450 BeadChip can allow accurate assessment of differential methylation across large studies. This chapter outlines the laboratory methodologies associated with performing the Illumina Infinium Methylation Assay, including bisulfite conversion, whole-genome amplification, BeadChip hybridization, XStain procedures, and imaging systems. Furthermore, this chapter provides an outline of data analysis tools, including the GenomeStudio pipeline, quality control measures, and additional statistical considerations. This comprehensive overview can aid not only in performing the Illumina Infinium Methylation Assay but also in the interpretation of data derived from this platform. less

关键词： Microarray Genome Amplification Image Analysis TEM Extrusion Methylation Analysis Genotyping data visualization tools Genome Alignment Bowtie Pyrosequencing PCR Liquid Handling data analysis DNA methylation Bisulfite conversion Infinium assay CpG site GenomeStudio

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Analysis of Transcriptomic data Generated from Drug-Treated Cancer Cell Line

Analysis of Transcriptomic Data Generated from Drug-Treated ...

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作者： Swarnima Kushwaha Sudeshna Mukherjee Rajdeep Chowdhury Shibasish Chowdhury

Resistance to chemotherapy is one major obstacle in current cancer treatment. Therefore, understanding the molecular basis of the acquisition of resistance is vital for the design and development of appropriate cancer therapy. Importantly, acquisition of resistance is not a single-step process, and the molecular signature of cells dynamically changes during this process. With the advent of next-generation omic technologies, today one can precisely map the molecular alterations not only in a population of tumor cells but also at the single-cell level as they attain chemo-resistance. In this chapter, we describe a detailed transcriptomic pipeline following next-generation sequencing for mapping alteration in expression during the process of attainment of resistance. We provide comprehensive information on the process to (1) track the differential expression of transcripts, (2) understand the gene ontology functions, (3) filter out candidate key genes, (4) identify the pathways regulated by them, and (5) generate a map of their probable interactions. We assume that our analytical method will be useful for research in this direction. less

关键词： RNA-seq RNA Extraction mRNA sequencing data analysis Gel Electrophoresis Deep Sequencing High-Throughput Sequencing TopHat Bowtie Programming Language Cufflinks Gene Expression Analysis Cuffdiff Cytoscape data visualization tools Cell And Tissue Culture Cancer Chemotherapy Drug resistance

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Metabolomics for Salinity Research

Metabolomics for Salinity Research

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作者： Ute Roessner Diane M. Beckles

Soil salinity devastates agriculture. It reduces crop yields and makes arable land unsuitable for later use. Many species have evolved highly efficient strategies to sense, transduce, and build up tolerance to high salinity and even sensitive species have endogenous mechanism for coping with this stress. These underlying physiological and metabolic mechanisms can be unraveled using metabolomics. Here we describe detailed protocols of how to extract polar metabolites for analysis using GC-MS and LC-MS. We also touch briefly on considerations that should be taken into account when designing the experiment and how the resulting data may be analyzed and visualized in a biological context. less

关键词： Metabolomics Gas Chromatography-mass Spectrometry Liquid Chromatography-mass Spectrometry Tissue Procurement Statistical Calculation HPLC Tandem Mass Spectrometry Principal Component Analysis data visualization tools Homogenization Quadrupole Time-of-flight Mass Spectrometry data analysis Metabolite extraction Polar metabolites

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Genomic Approaches to Analyze Alternative Splicing, A Key Regulator of Transcriptome and Proteome Diversity in

Genomic Approaches to Analyze Alternative Splicing, A Key Re...

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作者： Sonia Irigoyen Renesh H. Bedre Karen-Beth G. Scholthof Kranthi K. Mandadi

Alternative splicing (AS) promotes transcriptome and proteome diversity in plants, which influences growth and development, and host responses to stress. Advancements in next-generation sequencing, bioinformatics, and computational biology tools have allowed biologists to investigate AS landscapes on a genome-wide scale in several plant species. Furthermore, the development of Brachypodium distachyon (Brachypodium) as a model system for grasses has facilitated comparative studies of AS within the Poaceae. These analyses revealed a plethora of genes in several biological processes that are alternatively spliced and identified conserved AS patterns among monocot and dicot plants. In this chapter, using a Brachypodium-virus pathosystem as a research template, we provide an overview of genomic and bioinformatic tools that can be used to investigate constitutive and alternative splicing in plants. less

关键词： RNA-seq Library Preparation RNA Extraction Gel Electrophoresis Next Generation Sequencing Cufflinks Cuffdiff Genomics CummeRbund TopHat Bowtie Differential Expression Analysis Cuffcompare Splicing Model Analysis Gene Expression Analysis Gene Expression Profiling Reverse Transcription PCR PCR DNA Sequencing data visualization tools Microarray Paired-End RNA Sequencing data analysis StringTie Panicum mosaic virus Bioinformatics Alternative splicing Brachypodium

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Visualizing Patterns of Marine Eukaryotic Diversity from Metabarcoding data Using QIIME

Visualizing Patterns of Marine Eukaryotic Diversity from Met...

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作者： Matthieu Leray Nancy Knowlton

PCR amplification followed by deep sequencing of homologous gene regions is increasingly used to characterize the diversity and taxonomic composition of marine eukaryotic communities. This approach may generate millions of sequences for hundreds of samples simultaneously. Therefore, tools that researchers can use to visualize complex patterns of diversity for these massive datasets are essential. Efforts by microbiologists to understand the Earth and human microbiomes using high-throughput sequencing of the 16S rRNA gene has led to the development of several user-friendly, open-source software packages that can be similarly used to analyze eukaryotic datasets. Quantitative Insights Into Microbial Ecology (QIIME) offers some of the most helpful data visualization tools. Here, we describe functionalities to import OTU tables generated with any molecular marker (e.g., 18S, COI, ITS) and associated metadata into QIIME. We then present a range of analytical tools implemented within QIIME that can be used to obtain insights about patterns of alpha and beta diversity for marine eukaryotes. less

关键词： Taxonomic DNA Barcoding Principal Component Analysis PCR Deep Sequencing High-Throughput Sequencing data visualization tools data analysis QIIME Rarefaction Alpha diversity Beta diversity

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data Mining Methods in Omics-Based Biomarker Discovery

Data Mining Methods in Omics-Based Biomarker Discovery

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作者： Fan Zhang Jake Y. Chen

The advent of Omics technologies as genomics and proteomics has brought the hope of discovering novel biomarkers that can be used to diagnose, predict, and monitor the progress of disease. The importance of data mining to identify biological markers for the diagnostic classification and prognostic assessment in the context of microarray and proteomic data has been increasingly recognized. We present an overview of general data mining methods and their applications to biomarker discovery with particular focus on genomics and proteomics data. Two case studies are exemplarily presented, and relevant data mining terminology and techniques are explained. less

关键词： data Mining Genomics Proteomics Mass Spectrometry Microarray Electrocardiogram Neural Network Model Liquid Chromatography-tandem Mass Spectrometry Decision Tree SALDI MS data visualization tools Cytoscape MATLAB DNA Sequencing Genotyping Nucleic Acid Microarray Artificial Intelligence Machine Learning MALDI Mass Spectrometry Fractionation data analysis Biomarker discovery

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Functional Molecular Units for Guiding Biomarker Panel Design

Functional Molecular Units for Guiding Biomarker Panel Desig...

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作者： Andreas Heinzel Irmgard Mühlberger Raul Fechete Bernd Mayer Paul Perco

The field of biomarker research has experienced a major boost in recent years, and the number of publications on biomarker studies evaluating given, but also proposing novel biomarker candidates is increasing rapidly for numerous clinically relevant disease areas. However, individual markers often lack sensitivity and specificity in the clinical context, resting essentially on the intra-individual phenotype variability hampering sensitivity, or on assessing more general processes downstream of the causative molecular events characterizing a disease term, in consequence impairing disease specificity. The trend to circumvent these shortcomings goes towards utilizing multimarker panels, thus combining the strength of individual markers to further enhance performance regarding both sensitivity and specificity. A way of identifying the optimal composition of individual markers in a panel approach is to pick each marker as representative for a specific pathophysiological (mechanistic) process relevant for the disease under investigation, hence resulting in a multimarker panel for covering the set of pathophysiological processes underlying the frequently multifactorial composition of a clinical phenotype. Here we outline a procedure of identifying such sets of disease-specific pathophysiological processes (units) delineated on the basis of disease-associated molecular feature lists derived from literature mining as well as aggregated, publicly available Omics profiling experiments. With such molecular units in hand, providing an improved reflection of a specific clinical phenotype, biomarker candidates can then be assigned to or novel candidates are to be selected from these units, subsequently resulting in a multimarker panel promising improved accuracy in disease diagnosis as well as prognosis. less

关键词： Transcriptomics Gene Expression Profiling Pathway Or Network visualization Cytoscape Statistical Calculation Proteomics Metabolomics Genetic Association Studies Text Mining Genome Assembly Affinity Purification data visualization tools Microarray GenBank Ensembl Splicing Assay Homo sapiens Protein–protein interaction networks Literature mining Gene sets Omics profiles Network modules Biomarker candidates Biomarker panels

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Metagenomics data visualization Using R

Metagenomics Data Visualization Using R

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作者： Alex Coleman Anupam Bose Suparna Mitra

Communicating key finds is a crucial part of the research process. data visualization is the field of graphically representing data to help communicate key findings. Building on previous chapters around data manipulating using the R programming language this, chapter will explore how to use R to plot data and generate high-quality graphics. It will cover plotting using the base R plotting functionality and introduce the famous ggplot2 package [2] that is widely used for data visualization in R. After this general introduction to data visualization tools, the chapter will explore more specific data visualization techniques for metagenomics data and their use cases using these basic packages. less

关键词： Programming Language Metagenomics data visualization tools data visualization ggplot2 Plotting Communication Research outputs Research visualization

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CRISPR Screen to Identify Factors that Render Tumor Cells Sensitive or Resistant to Killing by NK Cells

CRISPR Screen to Identify Factors that Render Tumor Cells Se...

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作者： Xiaoxuan Zhuang Eric O. Long

Natural killer (NK) cells are an important component of the cancer immune surveillance system. They are regulated by germline-encoded receptors that activate and inhibit their effector function, such as secretion of cytokines and direct lysis of tumor cells and virus-infected cells. Without the need to be primed by prior exposure to tumor antigen, NK cells can detect ligands expressed on tumor cells and selectively kill these cells. NK cells are under strict control by inhibitory receptors that bind to HLA class I on target cells and block early activation signals, thus preventing lysis of target cells. The sensitivity to lysis by NK cells is therefore determined to a large extent by the expression of HLA class I molecules on tumor cells. In addition to receptor–ligand interactions that occur at NK–target cell synapses, many other factors determine the sensitivity of tumor cells to lysis by NK. Intrinsic properties of tumor cells, such as their metabolism and signaling networks establish a threshold above which they will succumb to the death pathways triggered by NK cell attack. Here we provide a protocol for a genome-wide CRISPR screen in tumor cells to identify factors that regulate their sensitivity to primary human NK cells. Tumor cells first transduced for expression of Cas9 are then transduced with a guide RNA (gRNA) library and co-cultured with NK cells. Deep sequencing of the library generated from the genome of tumor cells that survived the selection by NK cells and analysis of the distribution of guide RNAs is performed to identify genes that promote either sensitivity or resistance to NK-mediated killing. The contribution of individual genes to tumor sensitivity can be validated by knockouts using individual gRNAs. The techniques and workflow described here could be applied to primary tumors from cancer patients and reveal tumor-specific points of vulnerability that could be exploited for cancer immunotherapy, such as checkpoint blockade or expression of

关键词： CRISPR Cell Lysis Cell Separation And Isolation Deep Sequencing Cell And Tissue Culture Cell Enrichment Electroporation Freeze-thaw Method Cell Proliferation Assay Lentivirus Production Transfection Western Blot Immunofluorescence Microscopy Flow Cytometry Immunofluorescence Co-culture DNA Extraction Protein Precipitation PCR data visualization tools DNA Sequencing Gene Knockout Cytotoxicity Assay Leukapheresis Library Preparation Homo sapiens Escherichia coli Lentivirus K-562 BOSC-23 Cancer Lymphocyte Natural killer cell Resistant tumor cell Anti-CD3 Anti-caspase 9

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