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Cancer Biology

Myeloid Progenitor Transformation Assay Authors:  Hiroshi Okuda and Akihiko Yokoyama, date: 12/05/2017, view: 151, Q&A: 0
Numerous oncogenes have been identified to cause leukemia. For example, chromosomal translocation generates various fusion genes of the mixed-lineage leukemia (MLL) gene and a partner gene in leukemia, whose gene products transform primary myeloid progenitors into an immortalized state. To characterize the transforming ability of leukemic ...
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In vivo Leukemogenesis Model Using Retrovirus Transduction Authors:  Hiroshi Okuda and Akihiko Yokoyama, date: 12/05/2017, view: 126, Q&A: 0
Various genetic alterations such as chromosomal translocation cause leukemia. For examples, gene rearrangements of the mixed-lineage leukemia (MLL) gene generate MLL fusion genes, whose products are potent oncogenic drivers in acute leukemia. To better understand the mechanism of disease onset, several murine leukemia models ...
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Developmental Biology

Notch Ligand Binding Assay Using Flow Cytometry Authors:  Shweta Varshney and Pamela Stanley, date: 12/05/2017, view: 189, Q&A: 0
Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most ...
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Mandibular Explant Assay for Investigating Extrinsic Stimuli on Bone and Cartilage Development Authors:  Jamie Jiang, Jessica W. Bertol and Walid D. Fakhouri, date: 12/05/2017, view: 168, Q&A: 0
A major issue in developmental biology is to determine how embryonic tissues respond to molecular signals in a timely manner and given the position-restricted instructions during morphogenesis, of which Meckel’s cartilage is a classical example. The ex-vivo explant model is a practical and convenient system that allows investigation of ...
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Immunology

Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells Authors:  Florent Ubelmann, Tatiana Burrinha and Claudia Guimas Almeida, date: 12/05/2017, view: 183, Q&A: 0
The established primary trigger of Alzheimer’s disease’s is β-amyloid (Aβ) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane (Koo et al., 1996; Ubelmann et ...
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Drosophila Model of Leishmania amazonensis Infection Authors:  Kendi Okuda and Neal Silverman, date: 12/05/2017, view: 155, Q&A: 0
This protocol describes how to generate and harvest antibody-free L. amazonensis amastigotes, and how to infect adult Drosophila melanogaster with these parasites. This model recapitulates key aspects of the interactions between Leishmania amastigotes and animal phagocytes.
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Microbiology

Brief Protocol for EDGE Bioinformatics: Analyzing Microbial and Metagenomic NGS Data Authors:  Casandra Philipson, Karen Davenport, Logan Voegtly, Chien-Chi Lo, Po-E Li, Yan Xu, Migun Shakya, Regina Z. Cer, Kimberly A. Bishop-Lilly, Theron Hamilton and Patrick S. G. Chain, date: 12/05/2017, view: 182, Q&A: 0
Next-generation sequencing (NGS) offers unparalleled resolution for untargeted organism detection and characterization. However, the majority of NGS analysis programs require users to be proficient in programming and command-line interfaces. EDGE bioinformatics was developed to offer scientists with little to no bioinformatics expertise a ...
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Determination of Mn Concentrations in Synechocystis sp. PCC6803 Using ICP-MS Authors:  Fabian Brandenburg, Hanan Schoffman, Nir Keren and Marion Eisenhut, date: 12/05/2017, view: 134, Q&A: 0
Manganese (Mn) is an essential micronutrient for all photoautotrophic organisms. Two distinct pools of Mn have been identified in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), with 80% of the Mn residing in the periplasm and 20% in cytoplasm and thylakoid lumen (Keren et al., 2002). In this protocol, we ...
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Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions Authors:  Amanda Hopes, Vladimir Nekrasov, Nigel Belshaw, Irina Grouneva, Sophien Kamoun and Thomas Mock, date: 12/05/2017, view: 201, Q&A: 0
Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include ...
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Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells Author:  Shubham Shrivastava, date: 12/05/2017, view: 126, Q&A: 0
Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with ...
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Stationary-phase Mutagenesis Soft-agar Overlay Assays in Bacillus subtilis Authors:  Karla Viridiana Castro-Cerritos, Norberto Villegas-Negrete, Norma Ramirez-Ramirez, Eduardo A. Robleto and Mario Pedraza-Reyes, date: 12/05/2017, view: 201, Q&A: 0
Elucidating how a population of non-growing bacteria generates mutations improves our understanding of phenomena like antibiotic resistance, bacterial pathogenesis, genetic diversity and evolution. To evaluate mutations that occur in nutritionally stressed non-growing bacteria, we have employed the strain B. subtilis YB955, which measures ...
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Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry Authors:  Antonio Lagares (Jr.) and Claudio Valverde, date: 12/05/2017, view: 129, Q&A: 0
We describe here a detailed protocol for the quantification of the intracellular content of polyhydroxybutyrate (PHB) in a population of bacterial cells by flow cytometry, which is based on a consensus of several previously reported works.
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Lentiviral Knockdown of Transcription Factor STAT1 in Peromyscus leucopus to Assess Its Role in the Restriction of Tick-borne Flaviviruses Authors:  Adaeze O. Izuogu and R. Travis Taylor, date: 12/05/2017, view: 126, Q&A: 0
Cellular infection with tick-borne flaviviruses (TBFVs) results in activation of the interferon (IFN) signaling pathway and subsequent upregulation of numerous genes termed IFN stimulated genes (ISGs) (Schoggins et al., 2011). Many ISGs function to prevent virus pathogenesis by acting in a broad or specific manner through protein-protein ...
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Molecular Biology

MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes Authors:  Maximilian G. Plach, Klaus Grasser and Thomas Schubert, date: 12/05/2017, view: 168, Q&A: 0
Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides ...
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Neuroscience

Proboscis Extension Reflex in Apis mellifera [Honeybee] with Only One Antenna Authors:  Yu Guo, Zilong Wang, Zhijiang Zeng, Shaowu Zhang and Runsheng Chen, date: 12/05/2017, view: 101, Q&A: 0
The proboscis extension reflex (PER) is a common classical conditioned reflex which is widely used in the neurology and ethology. In honeybees, PER experiments can train bees to associate an odor with a reward or punishment. Here we present a variation of the PER experiment in Apis mellifera that trains honeybees using only one antenna. ...
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Formalin Murine Model of Pain Authors:  Marc López-Cano, Víctor Fernández-Dueñas, Amadeu Llebaria and Francisco Ciruela, date: 12/05/2017, view: 152, Q&A: 0
Pain research is mostly based on experimental assays that use animal models, which may allow deciphering the physiopathology of this condition and to propel drug discovery. The formalin nociception test is considered one of the most predictive approaches to study acute pain in rodents. This test permits monitoring pain-related responses (i.e. ...
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A Novel Protocol to Quantitatively Measure the Endocytic Trafficking of Amyloid Precursor Protein (APP) in Polarized Primary Neurons with Sub-cellular Resolution Authors:  Florent Ubelmann, Tatiana Burrinha and Cláudia Guimas Almeida, date: 12/05/2017, view: 131, Q&A: 0
Alzheimer’s disease’s established primary trigger is β-amyloid (Aβ) (Mucke and Selkoe, 2012). The amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes depends on its sorting for degradation in lysosomes (Haass et al., 1992; Morel et al. ...
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Live Imaging of Axonal Transport in the Motor Neurons of Drosophila Larvae Authors:  Tsuyoshi Inoshita, Nobutaka Hattori and Yuzuru Imai, date: 12/05/2017, view: 109, Q&A: 0
Axonal transport, which is composed of microtubules, motor proteins and a variety of types of cargo, is a prominent feature of neurons. Monitoring these molecular dynamics is important to understand the biological processes of neurons as well as neurodegenerative disorders that are associated with axonal dysfunction. Here, we describe a protocol ...
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zPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain Authors:  Pierre Affaticati, Matthieu Simion, Elodie De Job, Laurie Rivière, Jean-Michel Hermel, Elodie Machado, Jean-Stéphane Joly and Arnim Jenett, date: 12/05/2017, view: 174, Q&A: 0
In studies of brain function, it is essential to understand the underlying neuro-architecture. Very young zebrafish larvae are widely used for neuroarchitecture studies, due to their size and natural transparency. However, this model system has several limitations, due to the immaturity, high rates of development and limited behavioral repertoire ...
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Plant Science

Automatic Leaf Epidermis Assessment Using Fourier Descriptors in Texture Images Authors:  Núbia Rosa da Silva, Marcos William da Silva Oliveira, Humberto Antunes de Almeida Filho, Luiz Felipe Souza Pinheiro, Rosana Marta Kolb and Odemir Martinez Bruno, date: 12/05/2017, view: 122, Q&A: 0
The identification of plant species is not a trivial task, since it is carried out by experts and depends on the presence of fruits, flowers and leaves. However, fruits and flowers are not available throughout the year, while leaves are accessible most of the year. In order to assist the specialized work of species identification, methods of ...
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Using Light and Electron Microscopy to Estimate Structural Variation in Thylakoid Membranes Authors:  Hatsumi Nozue and Kiyokazu Kametani, date: 12/05/2017, view: 113, Q&A: 0
The shapes of chloroplasts and the architectures of internal thylakoid membranes are altered by growth and environmental changes (Lichtenthaler et al., 1981; Kutik, 1985; Terashima and Hikosaka, 1995). These morphological alterations proceed via transitional intermediates, during which dynamic and heterogeneous thylakoid membranes are ...
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Investigating the Shape of the Shoot Apical Meristem in Bamboo Using a Superellipse Equation Authors:  Qiang Wei and Peijian Shi, date: 12/05/2017, view: 112, Q&A: 0
The shoot apical meristem is the origin of bamboo wood. Its structure and morphology are important for maintaining the normal development of bamboo wood. However, the traditional method to describe the morphology of the shoot apical meristem in bamboo or other plants only depends on qualitative approaches. Here we present a protocol for precisely ...
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Stem Cell

GC/MS-based Analysis of Volatile Metabolic Profile Along in vitro Differentiation of Human Induced Pluripotent Stem Cells Authors:  Rosamaria Capuano, Rosa Valentina Talarico, Paola Spitalieri, Roberto Paolesse, Giuseppe Novelli, Federica Sangiuolo and Corrado Di Natale, date: 12/05/2017, view: 123, Q&A: 0
Human induced pluripotent stem cells (hiPSCs) are a promising tool in cell-based therapies for degenerative diseases. A safe application of hiPSCs in vivo, requires the detection of the presence of residual undifferentiated pluripotent cells that can potentially cause the insurgence of teratomas. Several studies point out that metabolic ...
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Systems Biology

Low-input Capture-C: A Chromosome Conformation Capture Assay to Analyze Chromatin Architecture in Small Numbers of Cells Authors:  A. Marieke Oudelaar, Damien J. Downes, James O.J. Davies and Jim R. Hughes, date: 12/05/2017, view: 212, Q&A: 0
Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This protocol describes two new low-input Capture-C approaches that can generate high-quality 3C interaction profiles from 10,000-20,000 cells, depending on the ...
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