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DNA Motif Databases and Their Uses
Gary D. Stormo
Transcription factors (TFs) recognize and bind to specific DNA sequences. The specificity of a TF is usually represented as a position weight matrix (PWM). Several databases of DNA motifs exist and are used in biological research to address important biological questions. This overview describes
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Protein Function Prediction: Problems and Pitfalls
William R. Pearson
The characterization of new genomes based on their protein sets has been revolutionized by new sequencing technologies, but biologists seeking to exploit new sequence information are often frustrated by the challenges associated with accurately assigning biological functions to newly identified
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Using RAxML to Infer Phylogenies
Alexandros Stamatakis
Inference of phylogenetic trees under the maximum likelihood (ML) criterion represents a routine task in biological data analysis. In this unit we describe how to plan analyses and use Randomized Accelerated Maximum Likelihood (RAxML) for phylogenetic inferences under ML, how to infer support values
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Mapping RNA‐seq Reads with STAR
Alexander Dobin, Thomas R. Gingeras
Mapping of large sets of high‐throughput sequencing reads to a reference genome is one of the foundational steps in RNA‐seq data analysis. The STAR software package performs this task with high levels of accuracy and speed. In addition to detecting annotated and novel splice junctions, STAR is
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Using PepExplorer to Filter and Organize De Novo Peptide Sequencing Results
Felipe da Veiga Leprevost, Valmir C. Barbosa, Paulo Costa Carvalho
PepExplorer aids in the biological interpretation of de novo sequencing results; this is accomplished by assembling a list of homolog proteins obtained by aligning results from widely adopted de novo sequencing tools against a target‐decoy sequence database. Our tool relies on pattern recognition to
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Unix Survival Guide
Lincoln D. Stein
Most bioinformatics software has been designed to run on Linux and other Unix‐like systems. Unix is different from most desktop operating systems because it makes extensive use of a text‐only command‐line interface. It can be a challenge to become familiar with the command line, but once a person
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Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts
Chad D. Williamson, Daniel S. Wong, Petros Bozidis, Aiping Zhang, Anamaris M. Colberg‐Poley
Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub‐domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria‐associated membrane (MAM)
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A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy
Keiko Mihara, Tomofumi Nakayama, Hisato Saitoh
Human myeloid HL‐60 cells are usually cultured in suspension in medium containing 5% to 10% fetal bovine serum (FBS) and thus are often difficult to adhere to a coverslip. In this unit, we describe how removal of FBS from the culture medium facilitates adhesion of HL‐60 cells to coverslips.
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Supported Lipid Bilayer Technology for the Study of Cellular Interfaces
Travis J. Crites, Michael Maddox, Kartika Padhan, James Muller, Calvin Eigsti, Rajat Varma
Glass‐supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell‐cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of
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Digestion, Purification, and Enrichment of Protein Samples for Mass Spectrometry
Victoria E. Hedrick, Mercedes N. LaLand, Ernesto S. Nakayasu, Lake N. Paul
Proteomic studies rely heavily on the use of liquid chromatography (LC)–mass spectrometry (MS and MS/MS) analyses to provide information about protein composition and function. Profiling the proteome can be the first step to understanding biological pathways, but the challenges scientists face with
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Use of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes (hiPSC‐CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways
Liang Guo, Sandy Eldridge, Mike Furniss, Jodie Mussio, Myrtle Davis
There is a need to develop mechanism‐based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization
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Genetic Code Expansion of Mammalian Cells with Unnatural Amino Acids
Kalyn A. Brown, Alexander Deiters
The expansion of the genetic code of mammalian cells enables the incorporation of unnatural amino acids into proteins. This is achieved by adding components to the protein biosynthetic machinery, specifically an engineered aminoacyl‐tRNA synthetase/tRNA pair. The unnatural amino acids are chemically
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Diabetic Retinopathy: Retina‐Specific Methods for Maintenance of Diabetic Rodents and Evaluation of Vascular Histopathology and Molecular Abnormalities
Alexander Veenstra, Haitao Liu, Chieh Allen Lee, Yunpeng Du, Jie Tang, Timothy S. Kern
Diabetic retinopathy is a major cause of visual impairment, which continues to increase in prevalence as more and more people develop diabetes. Despite the importance of vision, the retina is one of the smallest tissues in the body, and specialized techniques have been developed to study
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Exploration of Energy Metabolism in the Mouse Using Indirect Calorimetry: Measurement of Daily Energy Expenditure (DEE) and Basal Metabolic Rate (BMR)
Carola W. Meyer, Peter Reitmeir, Matthias H. Tschöp
Current comprehensive mouse metabolic phenotyping involves studying energy balance in cohorts of mice via indirect calorimetry, which determines heat release from changes in respiratory air composition. Here, we describe the measurement of daily energy expenditure (DEE) and basal metabolic rate
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Phenotyping Circadian Rhythms in Mice
Kristin Eckel‐Mahan, Paolo Sassone‐Corsi
Circadian rhythms take place with a periodicity of 24 hr, temporally following the rotation of the earth around its axis. Examples of circadian rhythms are the sleep/wake cycle, feeding, and hormone secretion. Light powerfully entrains the mammalian clock and assists in keeping animals
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Mouse Necropsy
Piper M. Treuting, Jessica M. Snyder
Necropsy (also known as autopsy) is the post‐mortem dissection of bodies after euthanasia or death and is a scientific examination conducted to observe and dissect the organs, collect tissues, and determine the extent of grossly evident disease. Research necropsies are conducted to obtain specific
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An Overview of Typical Infections of Research Mice: Health Monitoring and Prevention of Infection
James R. Fahey, Haiyan Olekszak
There are many reasons to keep research mice healthy and free from infections. The two most important of these are to protect the health and welfare of research mice and to prevent infections from negatively impacting research. Just as the genetic integrity of a mouse strain will influence the
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Facile Access to 5′‐S‐(4,4′‐Dimethoxytrityl)‐2′,5′‐Dideoxyribonucleosides via Stable Disulfide Intermediates
Chandra Shekhar Reddy L., Vivek K. Sharma, Rajesh Kumar, Ankita Singh, Virinder S. Parmar, Yogesh S. Sanghvi, Ashok K. Prasad
Thionucleosides represent an important class of modified nucleos(t)ides that have found distinct applications in the chemical biology of synthetic oligonucleotides, but the use of these compounds is substantially lessened by the instability or high reactivity of the sulfhydryl group. This unit
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A New Nucleic Acid Prodrug Responsive to High Thiol Concentration: Synthesis of 2′‐O‐Methyldithiomethyl‐Modified Oligonucleotides by Post‐Synthetic Modification
Yosuke Ochi, Osamu Nakagawa, Junsuke Hayashi, Shun‐ichi Wada, Hidehito Urata
This unit describes the synthesis of 2′‐ O ‐methyldithiomethyluridine‐containing oligonucleotides, which can be deprotected to yield the parental oligoribonucleotides under high concentrations of glutathione similar in cytoplasm. The 2′‐ O ‐methyldithiomethyl group is sensitive to reductive
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Native Chemical Ligation of Hydrolysis‐Resistant 3′‐NH‐Cysteine‐Modified RNA
Anna‐Skrollan Geiermann, Ronald Micura
Hydrolysis‐resistant RNA‐peptide conjugates that contain a 3′‐NH linkage between the adenosine ribose and the C‐terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid‐phase oligonucleotide synthesis and
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Synthesis of Morpholino Monomers, Chlorophosphoramidate Monomers, and Solid‐Phase Synthesis of Short Morpholino Oligomers
Jhuma Bhadra, Sankha Pattanayak, Surajit Sinha
Phosphorodiamidate morpholino oligomers (PMOs) are a highly capable class of synthetic antisense oligonucleotides that are used to study gene functions in in vitro and in vivo models. This unit describes the synthesis of exocyclic‐amine‐protected 7′‐hydroxy and 7′‐chlorophosphoramidate‐activated
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Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist‐Induced Arrestin Recruitment to Modified and Unmodified G Protein‐Coupled Receptors
Prashant Donthamsetti, Jose Rafael Quejada, Jonathan A. Javitch, Vsevolod V. Gurevich, Nevin A. Lambert
G protein‐coupled receptors (GPCRs) represent ∼25% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which are involved in differential signaling pathways. Because functionally selective or biased ligands activate one of these two pathways, they may be
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Models of Inflammation: Carrageenan‐ or Complete Freund's Adjuvant (CFA)–Induced Edema and Hypersensitivity in the Rat
Kenneth E. McCarson
Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the processing of pain sensation at CNS sites, the anti‐inflammatory properties of agents such as nonsteroidal anti‐inflammatory drugs (NSAIDs), and the efficacy of putative analgesic
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Streptozotocin‐Induced Diabetic Models in Mice and Rats
Brian L. Furman
Streptozotocin (STZ) is an antibiotic that produces pancreatic islet β‐cell destruction and is widely used experimentally to produce a model of type 1 diabetes mellitus (T1DM). Detailed in this unit are protocols for producing STZ‐induced insulin deficiency and hyperglycemia in mice and rats. Also
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Overview of KRAS‐Driven Genetically Engineered Mouse Models of Non‐Small Cell Lung Cancer
Clare Sheridan, Julian Downward
KRAS, the most frequently mutated oncogene in non‐small cell lung cancer, has been utilized extensively to model human lung adenocarcinomas. The results from such studies have enhanced considerably an understanding of the relationship between KRAS and the development of lung cancer. Detailed in this
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Overview of Genetically Engineered Mouse Models of Breast Cancer Used in Translational Biology and Drug Development
Kirsty R. Greenow, Matthew J. Smalley
Breast cancer is a heterogeneous condition with no single standard of treatment and no definitive method for determining whether a tumor will respond to therapy. The development of murine models that faithfully mimic specific human breast cancer subtypes is critical for the development of
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