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Finding Homologs in Amino Acid Sequences Using Network BLAST Searches
Istvan Ladunga
BLAST, the Basic Local Alignment Search Tool, is used more frequently than any other biosequence database search program. We show how to run searches on the Web, and demonstrate how to increase performance by fine‐tuning arguments for a specific research project. We offer guidance for interpreting
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Using the Contextual Hub Analysis Tool (CHAT) in Cytoscape to Identify Contextually Relevant Network Hubs
Tanja Muetze, David J. Lynn
Highly connected nodes in biological networks are called network hubs. Hubs are topologically important to the structure of the network and have been shown to be preferentially associated with a range of phenotypes of interest. The relative importance of a hub node, however, can change depending on
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Using SQL Databases for Sequence Similarity Searching and Analysis
William R. Pearson, Aaron J. Mackey
Relational databases can integrate diverse types of information and manage large sets of similarity search results, greatly simplifying genome‐scale analyses. By focusing on taxonomic subsets of sequences, relational databases can reduce the size and redundancy of sequence libraries and improve the
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Data Analysis Pipeline for RNA‐seq Experiments: From Differential Expression to Cryptic Splicing
Hari Krishna Yalamanchili, Ying‐Wooi Wan, Zhandong Liu
RNA sequencing (RNA‐seq) is a high‐throughput technology that provides unique insights into the transcriptome. It has a wide variety of applications in quantifying genes/isoforms and in detecting non‐coding RNA, alternative splicing, and splice junctions. It is extremely important to comprehend the
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Using the PRIDE Database and ProteomeXchange for Submitting and Accessing Public Proteomics Datasets
Andrew F. Jarnuczak, Juan Antonio Vizcaíno
The ProteomeXchange (PX) Consortium is the unifying framework for world‐leading mass spectrometry (MS)–based proteomics repositories. Current members include the PRIDE database (U.K.), PeptideAtlas/PASSEL, and MassIVE (U.S.A.), and jPOST (Japan). The Consortium standardizes submission and
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Using ProteomeScout: A Resource of Post‐Translational Modifications, Their Experiments, and the Proteins That They Annotate
Arshag D. Mooradian, Jason M. Held, Kristen M. Naegle
Post‐translational modifications (PTMs) of protein amino acids are ubiquitous and important to protein function, localization, degradation, and more. In recent years, there has been an explosion in the discovery of PTMs as a result of improvements in PTM measurement techniques, including
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Cellular Detection of G‐Quadruplexes by Optical Imaging Methods
Souheila Amor, Sunny Y. Yang, Judy M. Y. Wong, David Monchaud
G‐quadruplexes (G4s) are higher‐order nucleic acid structures that fold from guanine (G)–rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of
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Lectin‐Array Blotting
Raquel Pazos, Juan Echevarria, Alvaro Hernandez, Niels‐Christian Reichardt
Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and
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In Vitro Reconstitution of the Endoplasmic Reticulum
Csilla‐Maria Ferencz, Gernot Guigas, Andreas Veres, Brigitte Neumann, Olaf Stemmann, Matthias Weiss
Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to
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Automated Tracking of Cell Migration with Rapid Data Analysis
Brian J. DuChez
Cell migration is essential for many biological processes including development, wound healing, and metastasis. However, studying cell migration often requires the time‐consuming and labor‐intensive task of manually tracking cells. To accelerate the task of obtaining coordinate positions of
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Determination of Membrane Protein Distribution on the Nuclear Envelope by Single‐Point Single‐Molecule FRAP
Krishna C. Mudumbi, Weidong Yang
Nuclear envelope transmembrane proteins (NETs) are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM) in eukaryotic cells. The abnormal distribution of NETs has been associated with many human diseases. However,
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Centrifugation‐Free Magnetic Isolation of Functional Mitochondria Using Paramagnetic Iron Oxide Nanoparticles
Bhabatosh Banik, Shanta Dhar
Subcellular fractionation techniques are essential for cell biology and drug development studies. The emergence of organelle‐targeted nanoparticle (NP) platforms necessitates the isolation of target organelles to study drug delivery and activity. Mitochondria‐targeted NPs have attracted the
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A Guide to Quantitative Biomarker Assay Development using Whispering Gallery Mode Biosensors
Heather M. Robison, Ryan C. Bailey
Whispering gallery mode (WGM) sensors are a class of powerful analytical techniques defined by the measurement of changes in the local refractive index at or near the sensor surface. When functionalized with target‐specific capture agents, analyte binding can be measured with very low limits of
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High‐Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor
Peter K. Foote, David T. Krist, Alexander V. Statsyuk
HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin
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Optimized PEI‐based Transfection Method for Transient Transfection and Lentiviral Production
Shaozhe Yang, Xiaoling Zhou, Rongxiang Li, Xiuhong Fu, Pingnan Sun
Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI‐based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI‐based transfection is affected by numerous factors,
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Characterizing the Nano‐Bio Interface Using Microscopic Techniques: Imaging the Cell System is Just as Important as Imaging the Nanoparticle System
Christie M. Sayes, Henry Lujan
The rapid growth of nanotechnology and its industries has elevated the need to understand the risks associated with handling, using, and disposing of nanomaterials. These risks can be assessed through exposure measurement and hazard identification. One of the common challenges associated with
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Overview of Methods and Strategies for Conducting Virtual Small Molecule Screening
Xavier Fradera, Kerim Babaoglu
Virtual screening (VS) in the context of drug discovery is the use of computational methods to discover novel ligands with a desired biological activity from within a larger collection of molecules. These techniques have been in use for many years, there is a wide range of methodologies available,
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Pathology Evaluation of Developmental Phenotypes in Neonatal and Juvenile Mice
Brad Bolon, Susan Newbigging, Kelli L. Boyd
Necropsy (or autopsy) is the post mortem dissection of an animal to examine and collect organs and tissues in order to understand the effects and causes of disease. The systematic harvesting of samples at necropsy is an essential step in defining the reason for an unexpected death and in
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Profiling of Single‐Cell Transcriptomes
Wanze Chen, Vincent Gardeux, Antonio Meireles‐Filho, Bart Deplancke
Complex biological systems are composed of multiple cell types whose transcriptional activity can vary due to differences in cell state, environmental stimulation, or intrinsic programs. Conventional bulk analysis methods capture the average transcriptional programs of the cell population, thus
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Using Vascular Landmarks to Orient 3D Optical Coherence Tomography Images of the Mouse Eye
Mark P. Krebs
Comparing 3D structural information obtained by optical coherence tomography (OCT) requires accurate alignment of images acquired from individual subjects. Despite the widespread use of OCT to image the anterior and posterior mouse eye, few approaches to align the resulting image data have been
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Synthesis of 4′‐Selenoribonucleosides, the Building Blocks of 4′‐SelenoRNA, Using a Hypervalent Iodine
Noriko Saito‐Tarashima, Masashi Ota, Noriaki Minakawa
Herein is described a detailed protocol for the synthesis of 4′‐selenoribonucleoside derivatives that involves the use of a hypervalent iodine species. These derivatives are versatile units for the preparation of 4′‐selenoRNA. Large‐scale synthesis of a 4‐selenosugar starting from D ‐ribose is
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Vibration Ball Milling for the Synthesis of 5′‐Thioadenosine 5′‐Pyrophosphate (P′→5′) Adenosine (dASppA)
Olga Eguaogie, Joseph S. Vyle
Using vibration ball milling, 5′‐chloro‐5′‐deoxyadenosine (CldA) reacts cleanly with 4‐methoxybenzyl mercaptan (MobSH), under basic conditions, to the corresponding thioether (MobSdA), which is isolated following precipitation and trituration. Under acidic conditions, in a one‐pot, two‐step process,
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Synthesis of the Phosphoramidite Units for Benzene‐Glycol Nucleic Acid
Yusuke Maeda, Nazuki Niwa, Yoshihito Ueno
Benzene‐glycol nucleic acid‐DNA chimeras form thermally and thermodynamically stable duplexes with complementary RNAs, and have base‐discriminating abilities. This unit describes the synthesis of four nucleoside analogs, an adenine, cytosine, thymine, and guanine analogs with base‐benzene‐glycol
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Nucleoside‐O‐Methyl‐(H)‐Phosphinates: Novel Monomers for the Synthesis of Methylphosphonate Oligonucleotides Using H‐Phosphonate Chemistry
Ondřej Kostov, Ondřej Páv, Ivan Rosenberg
This unit comprises the straightforward synthesis of protected 2′‐deoxyribonucleoside‐ O ‐methyl‐(H)‐phosphinates in both 3′‐ and 5′‐series. These compounds represent a new class of monomers compatible with the solid‐phase synthesis of oligonucleotides using H‐phosphonate chemistry and are suitable
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Solid‐Phase Synthesis of RNA Analogs Containing Phosphorodithioate Linkages
Xianbin Yang
The oligoribonucleotide phosphorodithioate (PS2‐RNA) modification uses two sulfur atoms to replace two non‐bridging oxygen atoms at an internucleotide phosphorodiester backbone linkage. Like a natural phosphodiester RNA backbone linkage, a PS2‐modified backbone linkage is achiral at phosphorus.
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Probing RNA Folding Pathways by RNA Fingerprinting
Sarah A. Woodson
This unit provides protocols for using native polyacrylamide gel electrophoresis to distinguish folding and unfolding conformers of RNA. It is useful for studying conformers that can exchange in a period of minutes or seconds, and that are thus difficult to study by solution‐based methods.
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Quantitative Analysis of Nucleic Acid Stability with Ligands Under High Pressure to Design Novel Drugs Targeting G‐Quadruplexes
Shuntaro Takahashi, Naoki Sugimoto
Nucleic acids (DNA and RNA) can form various non‐canonical structures. Because some serious diseases are caused by the conformational change of G‐quadruplex DNA structures, the development of ligands that bind and stabilize G‐quadruplex DNA is of interest to the field of nucleic acid chemistry.
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Detecting the Interaction of Peptide Ligands with Plant Membrane Receptors
Sarah Refi Hind, Jason S. Hoki, Joshua A. Baccile, Patrick C. Boyle, Frank C. Schroeder, Gregory B. Martin
The field of plant receptor biology has rapidly expanded in recent years, however the demonstration of direct interaction between receptor‐ligand pairs remains a challenge. Click chemistry has revolutionized small molecule research but lacks popularity in plant research. Here we describe a method
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Plant Growth–Promoting Bacteria (PGPB): Isolation and Screening of PGP Activities
Adriana Ambrosini, Luciane M. P. Passaglia
Plant roots are associated with numerous and diverse types of beneficial and pathogenic microorganisms. Plant growth–promoting (rhizo)bacteria (PGPB or PGPR) are isolated from plants crops worldwide, and many of them are used as agricultural inoculants. Agricultural biofertilization and biocontrol
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Improved Genetic Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens
Marcos Fernando Basso, Bárbara Andrade Dias Brito da Cunha, Ana Paula Ribeiro, Polyana Kelly Martins, Wagner Rodrigo de Souza, Nelson Geraldo de Oliveira, Thiago Jonas Nakayama, Raphael Augusto das Chagas Noqueli Casari, Thais Ribeiro Santiago, Felipe Vinecky, Letícia Jungmann Cançado, Carlos Antônio Ferreira de Sousa, Patricia Abrão de Oliveira, Silvana Aparecida Creste Dias de Souza, Geraldo Magela de Almeida Cançado, Adilson Kenji Kobayashi, Hugo Bruno Correa Molinari
Sugarcane ( Saccharum spp.) is a monocotyledonous semi‐perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several
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Isolation of Novel MAMP‐like Activities and Identification of Cognate Pattern Recognition Receptors in Arabidopsis thaliana Using Next‐Generation Sequencing (NGS)–Based Mapping
Li Fan, Eunyoung Chae, Andrea A. Gust, Thorsten Nürnberger
Activation of pattern‐triggered plant immunity requires recognition of microbe‐derived molecular patterns (MAMPs) by plant‐encoded pattern recognition receptors (PRRs). Many plant PRRs are found in selected plant genera only. Transfer of single PRRs or of cassettes expressing several PRRs (PRR
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Extracellular Alkalinization Assay for the Detection of Early Defense Response
Natalia Moroz, Alisa Huffaker, Kiwamu Tanaka
Plant recognition of invading organisms occurs through identification of foreign molecules associated with attackers and of self‐derived, damage‐associated molecules. Perception of these molecules activates signaling processes including dynamic changes in ion balance, production of second messengers
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Rodent Test of Attention and Impulsivity: The 5‐Choice Serial Reaction Time Task
Guy A. Higgins, Leo B. Silenieks
The 5‐choice serial reaction time task (5‐CSRTT) is employed extensively to measure attention in rodents. The assay involves animals trained to respond to a brief, unpredictable visual stimulus presented in one of five locations. The effects of experimental manipulations on response speed and choice
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All‐Optical Electrophysiology for Disease Modeling and Pharmacological Characterization of Neurons
Christopher A. Werley, Ted Brookings, Hansini Upadhyay, Luis A. Williams, Owen B. McManus, Graham T. Dempsey
A key challenge for establishing a phenotypic screen for neuronal excitability is measurement of membrane potential changes with high throughput and accuracy. Most approaches for probing excitability rely on low‐throughput, invasive methods or lack cell‐specific information. These limitations
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Development and Applications of Patient‐Derived Xenograft Models in Humanized Mice for Oncology and Immune‐Oncology Drug Discovery
Bhavna Verma, Michael Ritchie, Maria Mancini
With the recent approval of four novel immune oncology agents for the treatment of various cancers, the emerging power of this drug class has been substantiated. However, the full potential of such agents is yet to be realized, with only a fraction of the patient population responding to these
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Murine Retrovirally‐Transduced Bone Marrow Engraftment Models of MLL‐Fusion‐Driven Acute Myelogenous Leukemias (AML)
Matthew C. Stubbs, Andrei V. Krivtsov
MLL ‐rearranged leukemia represents approximately 5% to 10% of adult acute myelogenous leukemia (AML) and nearly half of all infant/pediatric acute leukemia cases. These leukemias have a poor prognosis, and there are no approved therapeutic options. The rearrangement in the MLL gene leads to
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