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The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses
Gil Stelzer, Naomi Rosen, Inbar Plaschkes, Shahar Zimmerman, Michal Twik, Simon Fishilevich, Tsippi Iny Stein, Ron Nudel, Iris Lieder, Yaron Mazor, Sergey Kaplan, Dvir Dahary, David Warshawsky, Yaron Guan‐Golan, Asher Kohn, Noa Rappaport, Marilyn Safran, Doron Lancet
GeneCards, the human gene compendium, enables researchers to effectively navigate and inter‐relate the wide universe of human genes, diseases, variants, proteins, cells, and biological pathways. Our recently launched Version 4 has a revamped infrastructure facilitating faster data updates,
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Comparative Protein Structure Modeling Using MODELLER
Benjamin Webb, Andrej Sali
Comparative protein structure modeling predicts the three‐dimensional structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target‐template alignment, model building,
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Inference of Episodic Changes in Natural Selection Acting on Protein Coding Sequences via CODEML
Joseph P. Bielawski, Jennifer L. Baker, Joseph Mingrone
This unit provides protocols for using the CODEML program from the PAML package to make inferences about episodic natural selection in protein‐coding sequences. The protocols cover inference tasks such as maximum likelihood estimation of selection intensity, testing the hypothesis of episodic
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Studying RNA Homology and Conservation with Infernal: From Single Sequences to RNA Families
Lars Barquist, Sarah W. Burge, Paul P. Gardner
Emerging high‐throughput technologies have led to a deluge of putative non‐coding RNA (ncRNA) sequences identified in a wide variety of organisms. Systematic characterization of these transcripts will be a tremendous challenge. Homology detection is critical to making maximal use of functional
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Using DrugBank for In Silico Drug Exploration and Discovery
David S. Wishart, Anthony Wu
DrugBank is a fully curated drug and drug target database that contains 8174 drug entries including 1944 FDA approved small‐molecule drugs, 198 FDA‐approved biotech (protein/peptide) drugs, 93 nutraceuticals, and over 6000 experimental drugs. Additionally, 4300 non‐redundant protein (i.e., drug
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Fluorescent Detection of Lipid Droplets and Associated Proteins
Laura L. Listenberger, Andrea M. Studer, Deborah A. Brown, Nathan E. Wolins
Excess lipid is stored in intracellular organelles known as lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets in fixed or live cells with BODIPY 493/503 are
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Immunoprecipitation
Juan S. Bonifacino, David C. Gershlick, Esteban C. Dell'Angelica
Selective immunoprecipitation of proteins is a useful tool for characterizing proteins and protein‐protein interactions. Clear step‐by‐step protocols are provided for preparing lysates of cells and yeast under a variety of conditions, for binding the antibody to a solid matrix, and for performing
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Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts
Janusz Franco‐Barraza, Dorothy A. Beacham, Michael D. Amatangelo, Edna Cukierman
Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell‐to‐matrix interactions are vital for vertebrate development. Disorders in
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Use of In Situ Proximity Ligation Assays for Systems Analysis of Signaling Pathways
Tzu‐Chi Chen, Chi‐Ying F. Huang
Understanding signaling pathway networks via protein‐protein interactions (PPIs) at the cellular level is a significant task that has not yet been completed. Here, a systems approach that computationally infers interlinked pathways from numerous PPIs is described. The endogenous PPIs can be
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Pseudo‐Ligandless Click Chemistry for Oligonucleotide Conjugation
Stephanie Mack, Munira F. Fouz, Sourav K. Dey, Subha R. Das
Particularly for its use in bioconjugations, the copper‐catalyzed (or copper‐promoted) azide‐alkyne cycloaddition (CuAAC) reaction or ‘click chemistry’, has become an essential component of the modern chemical biologist's toolbox. Click chemistry has been applied to DNA, and more recently, RNA
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Liquid Exfoliation of Layered Transition Metal Dichalcogenides for Biological Applications
Emily P. Nguyen, Torben Daeneke, Serge Zhuiykov, Kourosh Kalantar‐zadeh
Known to possess distinctive properties that differ greatly from their bulk form, layered two‐dimensional materials have been extensively studied and incorporated into many versatile applications ranging from optoelectronics to sensors. For biomedical research, two‐dimensional transition metal
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Combinatorial Library Screening Coupled to Mass Spectrometry to Identify Valuable Cyclic Peptides
Silvia A. Camperi, Silvana L. Giudicessi, María C. Martínez‐Ceron, Juan M. Gurevich‐Messina, Soledad L. Saavedra, Gerardo Acosta, Osvaldo Cascone, Rosa Erra‐Balsells, Fernando Albericio
Combinatorial library screening coupled to mass spectrometry (MS) analysis is a practical approach to identify useful peptides. Cyclic peptides can have high biological activity, selectivity, and affinity for target proteins, and high stability against proteolytic degradation. Here we describe two
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Optogenetic Control of Nuclear Protein Import in Living Cells Using Light‐Inducible Nuclear Localization Signals (LINuS)
Pierre Wehler, Dominik Niopek, Roland Eils, Barbara Di Ventura
Many biological processes are regulated by the timely import of specific proteins into the nucleus. The ability to spatiotemporally control the nuclear import of proteins of interest therefore allows study of their role in a given biological process as well as controlling this process in space and
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Intestinal Preparation Techniques for Histological Analysis in the Mouse
Jonathan M. Williams, Carrie A. Duckworth, Kate Vowell, Michael D. Burkitt, D. Mark Pritchard
The murine intestinal tract represents a difficult organ system to study due to its long convoluted tubular structure, narrow diameter, and delicate mucosa which undergoes rapid changes after sampling prior to fixation. These features do not make for easy histological analysis as rapid fixation in
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Development of a Representative Mouse Model with Nonalcoholic Steatohepatitis
Jef Verbeek, Ans Jacobs, Pieter Spincemaille, David Cassiman
Non‐alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in the Western world. It represents a disease spectrum ranging from isolated steatosis to non‐alcoholic steatohepatitis (NASH). In particular, NASH can evolve to fibrosis, cirrhosis, hepatocellular carcinoma, and liver
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Overview of Innovative Mouse Models for Imaging Neuroinflammation
Céline Caravagna, Alexandre Jaouën, Franck Debarbieux, Geneviève Rougon
Neuroinflammation demands a comprehensive appraisal in situ to gain in‐depth knowledge on the roles of particular cells and molecules and their potential roles in therapy. Because of the lack of appropriate tools, direct visualization of cells has been poorly investigated up to the present. In this
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Mouse Models of Asthma
Nincy Debeuf, Eline Haspeslagh, Mary van Helden, Hamida Hammad, Bart N. Lambrecht
Allergic asthma is a chronic inflammatory disease of the conducting airways characterized by the presence of allergen‐specific IgE, Th2 cytokine production, eosinophilic airway inflammation, bronchial hyperreactivity, mucus overproduction, and structural changes in the airways. Investigators have
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An Overview of Mouse Models of Nonalcoholic Steatohepatitis: From Past to Present
Ans Jacobs, Anne‐Sophie Warda, Jef Verbeek, David Cassiman, Pieter Spincemaille
Non‐alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world. It is associated with obesity and type 2 diabetes and represents a spectrum of histological abnormalities ranging from simple steatosis to non‐alcoholic steatohepatitis (NASH), which can further
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C‐C Bond Formation: Synthesis of C5 Substituted Pyrimidine and C8 Substituted Purine Nucleosides Using Water Soluble Pd‐imidate Complex
Vijay Gayakhe, Ajaykumar V. Ardhapure, Anant R. Kapdi, Yogesh S. Sanghvi, Jose Luis Serrano, Carola Schulzke
The synthesis of a highly efficient, water soluble [Pd(Sacc) 2 (TPA) 2 ] complex for C‐C bond formation is described. Additionally, application of the [Pd(Sacc) 2 (TPA) 2 ] complex for Suzuki‐Miyaura arylation of all four nucleosides (5‐iodo‐2′‐deoxyuridine [5‐IdU], 5‐iodo‐2′‐deoxycytidine [5‐IdC],
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Simple, High‐Yield Syntheses of DNA Duplexes Containing Interstrand DNA‐DNA Cross‐Links Between an N4‐Aminocytidine Residue and an Abasic Site
Jacqueline Gamboa Varela, Kent S. Gates
The protocol describes the preparation and purification of interstrand DNA‐DNA cross‐links derived from the reaction of an N 4 ‐aminocytidine residue with an abasic site in duplex DNA. The procedures employ inexpensive, commercially available chemicals and enzymes to carry out post‐synthetic
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Diffraction Techniques in Structural Biology
Martin Egli
A detailed understanding of chemical and biological function and the mechanisms underlying the molecular activities ultimately requires atomic‐resolution structural data. Diffraction‐based techniques such as single‐crystal X‐ray crystallography, electron microscopy, and neutron diffraction are well
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Pre‐Steady‐State Kinetic Analysis of Single‐Nucleotide Incorporation by DNA Polymerases
Yan Su, F. Peter Guengerich
Pre‐steady‐state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step‐by‐step procedure for pre‐steady‐state kinetic analysis of single‐nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA
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Selection of Natural and Base‐Modified DNA Aptamers for a Camptothecin Derivative
Hiroto Fujita, Masayasu Kuwahara
Nucleic acid aptamers for small molecules are currently being developed and have a potential role in diverse applications including biosensing, diagnostics, and therapeutics involving low‐molecular‐weight biomarkers and drugs. To enhance and broaden their functions through chemical modification,
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Measurement of Enzyme Activities and Optimization of Continuous and Discontinuous Assays
Camille Bénard, Yves Gibon
The measurement of enzyme activities represents an important step towards the understanding of biological networks. Continuous or discontinuous assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, two protocols for
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Generation of Stable Transgenic Rice (Oryza sativa L.) by Agrobacterium‐Mediated Transformation
Yi Zhang, Jun Li, Caixia Gao
Rice ( Oryza sativa L .) is the staple food for more than half of the world's population, and has also become an important model monocot. As a result, numerous genetic transformation protocols have been developed to improve and better understand this particular agronomic plant. Here we introduce a
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Virus‐Induced Gene Silencing and Transient Gene Expression in Soybean (Glycine max) Using Bean Pod Mottle Virus Infectious Clones
Steven A. Whitham, Lori M. Lincoln, R. V. Chowda‐Reddy, Jaime D. Dittman, Jamie A. O'Rourke, Michelle A. Graham
Virus‐induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200 to 300 base pair size range. The recombinant viruses are used
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An Improved Transient Expression System Using Arabidopsis Protoplasts
Yangrong Cao, Hong Li, An Q. Pham, Gary Stacey
Transient gene expression in protoplasts provides a powerful tool to study protein expression, protein localization, protein‐protein association, and gene expression regulation, etc. There are several methods including electroporation, which have been reported to introduce DNA into protoplasts.
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Profiling of Transcription Factor Binding Events by Chromatin Immunoprecipitation Sequencing (ChIP‐seq)
Liang Song, Yusuke Koga, Joseph R. Ecker
In multi‐cellular organisms, gene expression is orchestrated by thousands of transcription factors (TF). Chromatin immunoprecipitation followed by sequencing (ChIP‐seq) is a robust tool to investigate gene expression because this technique profiles in vivo protein‐DNA interaction at a genome‐wide
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Soybean (Glycine max) Mutant and Germplasm Resources: Current Status and Future Prospects
Benjamin W. Campbell, Robert M. Stupar
Genetic bottlenecks during domestication and modern breeding limited the genetic diversity of soybean ( Glycine max (L.) Merr.). Therefore, expanding and diversifying soybean genetic resources is a major priority for the research community. These resources, consisting of natural and induced genetic
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An Efficient Targeted Mutagenesis System Using CRISPR/Cas in Monocotyledons
Zhen Liang, Yuan Zong, Caixia Gao
Precise genome modification using artificial nucleases is a powerful tool for in‐depth understanding of gene functions and for creating new varieties. The CRISPR/Cas system, derived from an adaptive immunity system in bacteria and archaea, can introduce DNA double‐strand breaks (DSBs) into
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Painting of Arabidopsis Chromosomes with Chromosome‐Specific BAC Clones
Terezie Mandáková, Martin A. Lysak
Chromosome painting (CP) refers to fluorescence in situ hybridization (FISH) of chromosome‐specific DNA probes to identify large chromosome regions, chromosome arms, and whole chromosomes. For CP and CCP (comparative chromosome painting) in plants, most often, contigs of chromosome‐specific
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RNA Extraction and Preparation in Rice (Oryza sativa)
Cui‐Cui Yin, Biao Ma, Wei Wang, Qing Xiong, He Zhao, Shou‐Yi Chen, Jin‐Song Zhang
There has been a surge of interest in the molecular biology of RNA. We describe a simple, fast, and convenient protocol for isolating total RNA from rice (Oryza sativa) seedlings and young tissues/organs. RNA obtained using this method can be used for reverse transcription‐polymerase chain reaction
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Extraction and Analysis of Terpenes/Terpenoids
Zuodong Jiang, Chase Kempinski, Joe Chappell
Terpenes/terpenoids constitute one of the largest classes of natural products, due to the incredible chemical diversity that can arise from the biochemical transformations of relatively simple prenyl diphosphate starter units. All terpenes/terpenoids comprise a hydrocarbon backbone generated from
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Creation and Genomic Analysis of Irradiation Hybrids in Populus
Matthew Zinkgraf, Kathy Haiby, Meric C. Lieberman, Luca Comai, Isabelle M. Henry, Andrew Groover
Establishing efficient functional genomic systems for creating and characterizing genetic variation in forest trees is challenging. Here we describe protocols for creating novel gene‐dosage variation in Populus through gamma‐irradiation of pollen, followed by genomic analysis to identify chromosomal
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Rice (Oryza sativa) Protoplast Isolation and Its Application for Transient Expression Analysis
Feng He, Songbiao Chen, Yuese Ning, Guo‐Liang Wang
Rice ( Oryza sativa ) is not only the staple food for half of the world's population but also a model monocot plant for molecular biology studies. Although rice genes have been extensively investigated in the last two decades, the functions of many genes in the rice genome are still not known. One
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Analysis and Quantification of Glucosinolates
Christoph Crocoll, Barbara Ann Halkier, Meike Burow
Recent advances in liquid chromatography and mass spectrometry have made it possible to increase the throughput of phytochemical analyses at high sensitivity. These improvements have made it more feasible to monitor metabolic processes at the metabolite level. Glucosinolates, the primary defense
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Quantitative Auxin Metabolite Profiling Using Stable Isotope Dilution UHPLC‐MS/MS
Ondřej Novák, Aleš Pěnčík, Ota Blahoušek, Karin Ljung
Auxin, a plant hormone, is able to stimulate growth and to control developmental processes. To get a deeper level of understanding of how auxin activity is regulated in planta, we have developed a sensitive and selective mass spectrometry‐based protocol for multiplex quantification of indole
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Mammalian Cell Tissue Culture Techniques
Katy Phelan, Kristin M. May
Cultured tissues and cells are used extensively in physiological and pharmacological studies. In vitro cultures provide a means of examining cells and tissues without the complex interactions that would be present if the whole organism were studied. A number of special skills are required in order
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Genetically Engineered Mouse Models of Pancreatic Cancer: The KPC Model (LSL‐KrasG12D/+;LSL‐Trp53R172H/+;Pdx‐1‐Cre), Its Variants, and Their Application in Immuno‐oncology Drug Discovery
Jae W. Lee, Chad A. Komar, Fee Bengsch, Kathleen Graham, Gregory L. Beatty
Pancreatic ductal adenocarcinoma (PDAC) ranks fourth among cancer‐related deaths in the United States. For patients with unresectable disease, treatment options are limited and lack curative potential. Preclinical mouse models of PDAC that recapitulate the biology of human pancreatic cancer offer an
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Assays for Determination of Protein Concentration
Bradley J.S.C. Olson
Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3‐(4‐carboxybenzoyl)quinoline‐2‐carboxaldehyde (CBQCA)
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