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UniProt Tools
Sangya Pundir, Maria J. Martin, Claire O'Donovan,
The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data (UniProt Consortium, 2015). The UniProt Web site receives ∼400,000 unique visitors per month and is the primary means to access UniProt. Along with various datasets that you can search,
Finding Protein and Nucleotide Similarities with FASTA
William R. Pearson
The FASTA programs provide a comprehensive set of rapid similarity searching tools (fasta36, fastx36, tfastx36, fasty36, tfasty36), similar to those provided by the BLAST package, as well as programs for slower, optimal, local, and global similarity searches (ssearch36, ggsearch36), and for
Using PSEA‐Quant for Protein Set Enrichment Analysis of Quantitative Mass Spectrometry‐Based Proteomics
Mathieu Lavallée‐Adam, John R. Yates
PSEA‐Quant analyzes quantitative mass spectrometry–based proteomics datasets to identify enrichments of annotations contained in repositories such as the Gene Ontology and Molecular Signature databases. It allows users to identify the annotations that are significantly enriched for reproducibly
Introduction to Cheminformatics
David S. Wishart
Cheminformatics is a field of information technology that focuses on the collection, storage, analysis, and manipulation of chemical data. The chemical data of interest typically includes information on small molecule formulas, structures, properties, spectra, and activities (biological or
MetaboLights: An Open‐Access Database Repository for Metabolomics Data
Namrata S. Kale, Kenneth Haug, Pablo Conesa, Kalaivani Jayseelan, Pablo Moreno, Philippe Rocca‐Serra, Venkata Chandrasekhar Nainala, Rachel A. Spicer, Mark Williams, Xuefei Li, Reza M. Salek, Julian L. Griffin, Christoph Steinbeck
MetaboLights is the first general purpose, open‐access database repository for cross‐platform and cross‐species metabolomics research at the European Bioinformatics Institute (EMBL‐EBI). Based upon the open‐source ISA framework, MetaboLights provides Metabolomics Standard Initiative (MSI) compliant
Preparation of High‐Density Fibrillar Collagen Matrices That Mimic Desmoplastic Tumor Stroma
Vira V. Artym
The stroma of invasive tumors becomes enriched in dense fibrillar collagen as a result of the desmoplastic reaction. This desmoplastic collagen exerts profound effects on tumor and normal cells. In view of these findings, it is important to develop novel in vitro cell systems that mimic this
Analysis of the Stromal Cellular Components of the Solid Tumor Microenvironment Using Flow Cytometry
Michael Timaner, Ofrat Beyar‐Katz, Yuval Shaked
The tumor microenvironment consists of a variety of cell types. The contribution of each cell type to the tumor is an emerging subject in the field of cancer research. Here, we describe protocols for dissociating tumor tissues and Matrigel plugs into single cells for further analysis by flow
Three‐Dimensional Patterning of the ECM Microenvironment Using Magnetic Nanoparticle Self Assembly
Jiyun Kim, Kandice Tanner
This protocol describes a way to introduce topography to three‐dimensional (3D) biomaterials. The self‐assembling behavior of magnetic particles can be exploited to form nanoscale to microscale fibers, such that one can dissect the contribution of topography on cell behavior, which is independent of
RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells
Grigory S. Filonov, Samie R. Jaffrey
RNA spatial dynamics play a crucial role in cell physiology, and thus the ability to monitor RNA localization in live cells can provide insight into important biological problems. This unit focuses on imaging RNAs using an RNA mimic of GFP. This approach relies on an RNA aptamer called dimeric
Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes
Christina M. Woo, Carolyn R. Bertozzi
Protein glycosylation plays many critical roles in biological function and creates the most diversity of all post‐translational modifications (PTMs). Glycan structural diversity is directly correlated with difficulty in characterizing the intact glycoproteome by mass spectrometry (MS). In this
Hollow‐Fiber Methodology for Pharmacokinetic/Pharmacodynamic Studies of Antimalarial Compounds
Emily Caton, Elizabeth Nenortas, Rahul P. Bakshi, Theresa A. Shapiro
Knowledge of pharmacokinetic/pharmacodynamic (PK/PD) relationships can enhance the speed and economy of drug development by enabling informed and rational decisions at every step, from lead selection to clinical dosing. For anti‐infective agents in particular, dynamic in vitro hollow‐fiber cartridge
Analysis of Mouse Growth Plate Development
Laura Mangiavini, Christophe Merceron, Ernestina Schipani
To investigate skeletal development, pathophysiological mechanisms of cartilage and bone disease, and eventually assess innovative treatments, the mouse is a very important resource. During embryonic development, mesenchymal condensations are formed, and cells within these mesenchymal condensations
Cardiovascular Imaging in Mice
Colin K.L. Phoon, Daniel H. Turnbull
The mouse is the mammalian model of choice for investigating cardiovascular biology, given our ability to manipulate it by genetic, pharmacologic, mechanical, and environmental means. Imaging is an important approach to phenotyping both function and structure of cardiac and vascular components. This
Generating Mouse Models Using CRISPR‐Cas9‐Mediated Genome Editing
Wenning Qin, Peter M. Kutny, Richard S. Maser, Stephanie L. Dion, Jeffrey D. Lamont, Yingfan Zhang, Greggory A. Perry, Haoyi Wang
The CRISPR‐Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including mice. The CRISPR‐Cas9 reagents can be delivered directly into the mouse zygote to derive a mutant animal carrying targeted genetic modifications. The major components
Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE)
Pooja Jha, Xu Wang, Johan Auwerx
Mitochondria are cellular organelles that harvest energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the
Synthesis of S‐Adenosyl‐L‐Methionine Analogs with Extended Transferable Groups for Methyltransferase‐Directed Labeling of DNA and RNA
Viktoras Masevičius, Milda Nainytė, Saulius Klimašauskas
S ‐Adenosyl‐L‐methionine (AdoMet) is a ubiquitous methyl donor for a variety of biological methylation reactions catalyzed by methyltransferases (MTases). AdoMet analogs with extended propargylic chains replacing the sulfonium‐bound methyl group can serve as surrogate cofactors for many DNA and RNA
Solid‐Phase Synthesis of DNA and RNA 5′‐O‐Triphosphates Using cycloSal Chemistry
Ivo Sarac, Chris Meier
A chemical method for the synthesis of short strands of DNA and RNA 5′‐ O ‐triphosphates is described that makes use of the conventional coupling and oxidizing reagents that are readily available on standard DNA/RNA synthesizers. After automated solid‐phase synthesis of oligonucleotides and 5′‐ O
Solid‐Phase Synthesis of Oligo‐ADP‐Ribose
Hans A.V. Kistemaker, Nico J. Meeuwenoord, Herman S. Overkleeft, Gijsbert A. van der Marel, Dmitri V. Filippov
Solid‐phase methodology for synthesis of adenosine diphosphate ribose oligomers (ADPr‐oligomers) of defined length is described using an advanced 2‐ribosyl adenosine phosphoramidite building block that is prepared in solution via an expeditious and high‐yielding method. The methodology is based on
An Amphipathic trans‐Acting Phosphorothioate DNA Element Delivers Uncharged PNA and PMO Nucleic Acid Sequences in Mammalian Cells
Harsh V. Jain, Serge L. Beaucage
An innovative approach to the delivery of uncharged peptide nucleic acids (PNAs) and phosphorodiamidate morpholino (PMO) oligomers in mammalian cells is described and consists of extending the sequence of those oligomers with a short PNA‐polyA or PMO‐polyA tail. Recognition of the polyA‐tailed PNA
Synthesis of 5′‐Aldehyde Oligonucleotide
Rémy Lartia
Synthesis of oligonucleotide ending with an aldehyde functional group at their 5′‐end (5′‐AON) is possible for both DNA (5′‐AODN) and RNA (5′‐AORN) series irrespectively of the nature of the last nucleobase. The 5′‐alcohol of on‐support ODN is mildly oxidized under Moffat conditions. Transient
Models of Inflammation: Carrageenan Air Pouch
Djane B. Duarte, Michael R. Vasko, Jill C. Fehrenbacher
The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch
Methods of Inducing Inflammatory Bowel Disease in Mice
Byoungwook Bang, Lenard M. Lichtenberger
Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of the disease, identifying new targets for therapeutic intervention, and testing novel therapeutics. This unit provides detailed protocols for five widely used
Drug Combinations: Tests and Analysis with Isoboles
Ronald J. Tallarida
Described in this unit are experimental and computational methods to detect and classify drug interactions. In most cases, this relates to two drugs or compounds with overtly similar effects, e.g., two analgesics or two anti‐hypertensives. From the dose‐response data of the individual drugs, it is
Overview of Transgenic Glioblastoma and Oligoastrocytoma CNS Models and Their Utility in Drug Discovery
Fuyi Chen, Albert Becker, Joseph LoTurco
Many animal models have been developed to investigate the sources of central nervous system (CNS) tumor heterogeneity. Reviewed in this unit is a recently developed CNS tumor model using the piggyBac transposon system delivered by in utero electroporation, in which sources of tumor heterogeneity can
Overview of Genetically Engineered Mouse Models of Distinct Breast Cancer Subtypes
Jerry Usary, David Brian Darr, Adam D. Pfefferle, Charles M. Perou
Advances in the screening of new therapeutic options have significantly reduced the breast cancer death rate over the last decade. Despite these advances, breast cancer remains the second leading cause of cancer death among women. This is due in part to the complexity of the disease, which is
