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Using the Arabidopsis Information Resource (TAIR) to Find Information About Arabidopsis Genes
Leonore Reiser, Shabari Subramaniam, Donghui Li, Eva Huala
The Arabidopsis Information Resource (TAIR; http://arabidopsis.org) is a comprehensive Web resource of Arabidopsis biology for plant scientists. TAIR curates and integrates information about genes, proteins, gene function, orthologs, gene expression, mutant phenotypes, biological materials such as
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Protein 3D Structure and Electron Microscopy Map Retrieval Using 3D‐SURFER2.0 and EM‐SURFER
Xusi Han, Qing Wei, Daisuke Kihara
With the rapid growth in the number of solved protein structures stored in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB), it is essential to develop tools to perform real‐time structure similarity searches against the entire structure database. Since conventional structure
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The HMMER Web Server for Protein Sequence Similarity Search
Ananth Prakash, Matt Jeffryes, Alex Bateman, Robert D. Finn
Protein sequence similarity search is one of the most commonly used bioinformatics methods for identifying evolutionarily related proteins. In general, sequences that are evolutionarily related share some degree of similarity, and sequence‐search algorithms use this principle to identify homologs.
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Prediction of Protein‐Protein Interactions
Max Kotlyar, Andrea E.M. Rossos, Igor Jurisica
The authors provide an overview of physical protein‐protein interaction prediction, covering the main strategies for predicting interactions, approaches for assessing predictions, and online resources for accessing predictions. This unit focuses on the main advancements in each of these areas over
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Using the Seven Bridges Cancer Genomics Cloud to Access and Analyze Petabytes of Cancer Data
Raunaq Malhotra, Isheeta Seth, Erik Lehnert, Jing Zhao, Gaurav Kaushik, Elizabeth H. Williams, Anurag Sethi, Brandi N. Davis‐Dusenbery
Next‐generation sequencing has produced petabytes of data, but accessing and analyzing these data remain challenging. Traditionally, researchers investigating public datasets like The Cancer Genome Atlas (TCGA) would download the data to a high‐performance cluster, which could take several weeks
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High Purity Isolation and Sensitive Quantification of Extracellular Vesicles Using Affinity to TIM4
Takeshi Yoshida, Takamasa Ishidome, Rikinari Hanayama
Almost all types of cells secrete extracellular vesicles (EVs), including exosomes and microvesicles. EVs carry various proteins, lipids, mRNAs, and microRNAs, and may participate in many aspects of physiological and pathophysiological processes. Various studies are currently being conducted to
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Live‐Cell Visualization of Calcium Flux in Vibratome‐Cut Thick Sections of Viable Tumor Tissue
James Koh, Joyce A. Hogue, Julie A. Sosa
This unit outlines a live‐cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium‐sensing receptor (CASR), a class C G protein–coupled receptor (GPCR). The primary assay readout, intracellular calcium release
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Combining Fluorescence and Bioluminescence Microscopy to Study the Series of Events from Cellular Signal Transduction to Gene Expression
Kazuhito Goda, Takeo Takahashi, Hirobumi Suzuki
The molecular interactions and translocation of signal transduction factors in individual cells can be imaged by fluorescence microscopy. Alternatively, downstream promoter activity in single cells can be imaged by bioluminescence microscopy. However, the same stimuli can lead to different gene
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In Vitro Dissection of Autophagy
Min Zhang, Dawei Liu, Liang Ge
Autophagy is an essential cellular process for bulk degradation of cytoplasmic components through the lysosome. Underlying this process is an intricate interaction between protein factors and the cell endomembrane system, leading to a gradual maturation of the autophagic membrane. This structure
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Microfluidics‐Assisted TIRF Imaging to Study Single Actin Filament Dynamics
Shashank Shekhar
Dynamic assembly of actin filaments is essential for many cellular processes. The rates of assembly and disassembly of actin filaments are intricately controlled by regulatory proteins that interact with the ends and the sides of filaments and with actin monomers. TIRF‐based single‐filament imaging
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Simple and Rapid Tissue Clearing Method for Three‐Dimensional Histology of the Pancreas
Hang Sheung Wong, Patrick Ka Kit Yeung, Hei Ming Lai, Karen Siu Ling Lam, Wu Wutian, Sookja Kim Chung
Previously, high‐resolution three‐dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central
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Fluorescence Lifetime Imaging of a Caspase‐3 Apoptosis Reporter
Johanna M. Buschhaus, Anne E. Gibbons, Kathryn E. Luker, Gary D. Luker
Caspase‐3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase‐3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter
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In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target
Annamaria Ruscito, Erin M. McConnell, Anna Koudrina, Ranganathan Velu, Christopher Mattice, Vernon Hunt, Maureen McKeague, Maria C. DeRosa
Aptamers, synthetic oligonucleotide‐based molecular recognition probes, have found use in a wide array of biosensing technologies based on their tight and highly selective binding to a variety of molecular targets. However, the inherent challenges associated with the selection and characterization
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In Vivo Delivery of Nanoparticles into Plant Leaves
Honghong Wu, Israel Santana, Joshua Dansie, Juan P. Giraldo
Plant nanobiotechnology is an interdisciplinary field at the interface of nanotechnology and plant biology that aims to utilize nanomaterials as tools to study, augment or impart novel plant functions. The delivery of nanoparticles to plants in vivo is a key initial step to investigate plant
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The In Situ Enzymatic Screening (ISES) Approach to Reaction Discovery and Catalyst Identification
Robert A. Swyka, David B. Berkowitz
The importance of discovering new chemical transformations and/or optimizing catalytic combinations has led to a flurry of activity in reaction screening. The in situ enzymatic screening (ISES) approach described here utilizes biological tools (enzymes/cofactors) to advance chemistry. The protocol
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Evolving Aptamers with Unnatural Base Pairs
Michiko Kimoto, Ken‐ichiro Matsunaga, Ichiro Hirao
A novel technology, genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer
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Early‐Stage Formulation Considerations
Robert W. Lee, Mark Mitchnick
When a drug candidate—i.e., a new chemical entity (NCE) or new molecular entity (NME)—is discovered, there is a requirement to identify a vehicle for in vitro and/or in vivo evaluation to assess the activity and/or toxicity of the compound (here we refer to the biologically active compound as the
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X‐Ray Computed Tomography of Crop Plant Root Systems Grown in Soil
Stefan Mairhofer, Tony Pridmore, James Johnson, Darren M. Wells, Malcolm J. Bennett, Sacha J. Mooney, Craig J. Sturrock
Non‐destructive methods to quantify the root system architecture of a plant grown in soil are essential to aid our understanding of the factors that impact plant root development in natural environments. With environmental change threatening our ability to sustain agricultural productivity for an
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Medicago truncatula: Genetic and Genomic Resources
Marie Garmier, Laurent Gentzbittel, Jiangqi Wen, Kirankumar S. Mysore, Pascal Ratet
Medicago truncatula was chosen by the legume community, along with Lotus japonicus , as a model plant to study legume biology. Since then, numerous resources and tools have been developed for M. truncatula . These include, for example, its genome sequence, core ecotype collections,
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An Inexpensive, Easy‐to‐Use, and Highly Customizable Growth Chamber Optimized for Growing Large Plants
Thu M. Tran, David M. Braun
The ability to grow plants in highly controlled and reproducible environments is a critical factor for successful plant biology experiments. This protocol describes a simple and inexpensive method for constructing a fully automatic controlled growth chamber that can be easily adapted in plant
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Agrobacterium‐Mediated Transformation of Oilseed Rape (Brassica napus)
Ruth Bates, Melanie Craze, Emma J. Wallington
Oilseed rape ( Brassica napus ) is a commercially important member of the Brassicacea family. It is grown for its edible and industrial oils as well as for animal feed. Genetic transformation technology has been used to study gene function and produce oilseed rape with improved agronomic
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BiBAC Modification and Stable Transfer into Maize (Zea mays) Hi‐II Immature Embryos via Agrobacterium‐Mediated Transformation
Jon P. Cody, Nathaniel D. Graham, James A. Birchler
Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium‐ mediated transformation. However, the large size of BiBACs and low‐copy number in Escherichia coli (DH10B) and Agrobacterium tumefaciens make cloning
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Drug Affinity Responsive Target Stability (DARTS) to Resolve Protein–Small Molecule Interaction in Arabidopsis
Cecilia Rodriguez‐Furlan, Chunhua Zhang, Natasha Raikhel, Glenn R. Hicks
Target identification remains a challenging step in plant chemical genomics approaches. Drug affinity responsive target stability (DARTS) represents a straightforward technique to identify small molecules’ protein targets and assist in the characterization of interactions between small molecules and
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