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Biochemistry

Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor Authors:  Rajan Lamichhane, Jeffrey J. Liu, Raymond F. Pauszek III and David P. Millar, date: 06/20/2017, view: 1463, Q&A: 0
Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 ...
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Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method Authors:  Karin Ernits, Katrin Viigand, Triinu Visnapuu, Kristina Põšnograjeva and Tiina Alamäe, date: 06/20/2017, view: 1372, Q&A: 0
α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput differential scanning fluorometry method, also ...
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Biophysics

Modification and Application of a Commercial Whole-body Plethysmograph to Monitor Respiratory Abnormalities in Neonatal Mice Authors:  Yen-Ting Lai and Yi-Shuian Huang, date: 06/20/2017, view: 1208, Q&A: 0
Proper breathing is essential for mammals to acquire oxygen after birth and requires coordinated actions among several tissues, including diaphragm, intercostal muscles, trachea, bronchi, lung and respiration-regulating neurons located in the medulla oblongata. Genetically modified mice that die early postnatally may have respiratory defects ...
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Cancer Biology

Soft Agar Colony Formation Assay as a Hallmark of Carcinogenesis Authors:  Feng Du, Xiaodi Zhao and Daiming Fan, date: 06/20/2017, view: 2046, Q&A: 0
Soft agar colony formation assay is established to estimate the anchorage-independent growth ability of cells. In this assay, a bottom layer of agar with complete media is poured and solidified first, followed by an upper layer containing a specified number of cells suspended in medium-agar mixture. After two weeks of incubation, the number of ...
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Cell Biology

Functional ex-vivo Imaging of Arterial Cellular Recruitment and Lipid Extravasation Authors:  Emiel P.C. van der Vorst, Sanne L. Maas, Almudena Ortega-Gomez, Jeroen M.M. Hameleers, Mariaelvy Bianchini, Yaw Asare, Oliver Soehnlein, Yvonne Döring, Christian Weber and Remco T.A. Megens, date: 06/20/2017, view: 1361, Q&A: 0
The main purpose of this sophisticated and highly versatile method is to visualize and quantify structural vessel wall properties, cellular recruitment, and lipid/dextran extravasation under physiological conditions in living arteries. This will be of interest for a broad range of researchers within the field of inflammation, hypertension, ...
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Developmental Biology

Validating Candidate Congenital Heart Disease Genes in Drosophila Authors:  Jun-yi Zhu, Yulong Fu, Adam Richman and Zhe Han, date: 06/20/2017, view: 1331, Q&A: 0
Genomic sequencing efforts can implicate large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system to validate candidate gene association with pathology is therefore useful. We present such a system employing Drosophila to validate candidate congenital heart ...
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Microbiology

Producing GST-Cbx7 Fusion Proteins from Escherichia coli Authors:  Thao Ngoc Huynh and Xiaojun Ren , date: 06/20/2017, view: 1281, Q&A: 1
This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside ...
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Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes Authors:  David J. Nolan, Susanna L. Lamers, Rebecca Rose, James J. Dollar, Marco Salemi and Michael S. McGrath, date: 06/20/2017, view: 1380, Q&A: 0
The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al ...
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RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs Authors:  Jeremy G. Bird, Bryce E. Nickels and Richard H. Ebright, date: 06/20/2017, view: 1485, Q&A: 0
It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as ‘non-canonical initiating nucleotides’ (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA ...
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Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System Authors:  Reka A. Haraszti, Andrew Coles, Neil Aronin, Anastasia Khvorova and Marie-Cécile Didiot, date: 06/20/2017, view: 1534, Q&A: 0
Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. ...
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Culturing Bacteria from Caenorhabditis elegans Gut to Assess Colonization Proficiency Authors:  Facundo Rodriguez Ayala, Sebastián Cogliati, Carlos Bauman, Cecilia Leñini, Marco Bartolini, Juan Manuel Villalba, Federico Argañaraz and Roberto Grau, date: 06/20/2017, view: 1740, Q&A: 0
Determining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut ...
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Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins Authors:  Xu Zheng and David Pincus, date: 06/20/2017, view: 1459, Q&A: 0
This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen peptide. Next, the bait protein is recaptured ...
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Tagged Highly Degenerate Primer (THDP)-PCR for Community Analysis of Methane- and Ammonia-oxidizing Bacteria Based on Copper-containing Membrane-bound Monooxygenases (CuMMO) Authors:  Jian-Gong Wang, Fei Xia, Jemaneh Zeleke, Bin Zou and Zhe-Xue Quan, date: 06/20/2017, view: 1019, Q&A: 0
We describe a two-step PCR strategy using tagged highly degenerate primer (THDP-PCR) targeting copper-containing membrane-bound monooxygenases (CuMMO) genes for community analysis of methane- or ammonia-oxidizing bacteria. This strategy consists of a primary CuMMO gene-specific PCR followed by a secondary PCR with a tag as a single primer. This ...
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Molecular Biology

Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity Authors:  Trung M. Hoang, Tatiana Kent and Richard T. Pomerantz, date: 06/20/2017, view: 1247, Q&A: 0
DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since ...
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In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair Authors:  Melike Çağlayan and Samuel H. Wilson, date: 06/20/2017, view: 1720, Q&A: 0
We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan et al., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture including dGTP or 8-oxo-dGTP, pol β ...
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Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans Authors:  ChangHwan Lee, Hannah S. Seidel, Tina R. Lynch, Erika B. Sorensen, Sarah L. Crittenden and Judith Kimble, date: 06/20/2017, view: 2240, Q&A: 0
Single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA (Raj et al., 2008; Lee et al., 2016a). We adapted this technique to visualize RNAs in the C. elegans whole ...
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Dense sgRNA Library Construction Using a Molecular Chipper Approach Authors:  Jijun Cheng, Wen Pan and Jun Lu, date: 06/20/2017, view: 1587, Q&A: 0
Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for ...
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Neuroscience

Optogenetic Stimulation and Recording of Primary Cultured Neurons with Spatiotemporal Control Authors:  Jérémie Barral and Alex D Reyes, date: 06/20/2017, view: 1802, Q&A: 0
We studied a network of cortical neurons in culture and developed an innovative optical device to stimulate optogenetically a large neuronal population with both spatial and temporal precision. We first describe how to culture primary neurons expressing channelrhodopsin. We then detail the optogenetic setup based on the workings of a fast Digital ...
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Contusion Spinal Cord Injury Rat Model Authors:  Chuan-Wen Chiu, Henrich Cheng and Shie-Liang Hsieh, date: 06/20/2017, view: 1398, Q&A: 0
Spinal cord injury (SCI) can lead to severe disability, paralysis, neurological deficits and even death. In humans, most spinal cord injuries are caused by transient compression or contusion of the spinal cord associated with motor vehicle accidents. Animal models of contusion mimic the typical SCI’s found in humans and these models are key to the ...
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Stereotaxic Surgery for Suprachiasmatic Nucleus Lesions in Mice Authors:  Kimiko Shimizu and Yoshitaka Fukada, date: 06/20/2017, view: 2239, Q&A: 0
Site-specific lesions are invaluable methods for investigating the function of brain regions within the central nervous system and can be used to study neural mechanisms of behaviors. Precise stereotaxic surgery is required to lesion small regions of the brain such as the suprachiasmatic nucleus (SCN), which harbors the master circadian clock. In ...
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Representation-mediated Aversion as a Model to Study Psychotic-like States in Mice Authors:  Arnau Busquets-Garcia, Edgar Soria-Gómez, Guillaume Ferreira and Giovanni Marsicano, date: 06/20/2017, view: 1149, Q&A: 0
Several paradigms for rodent models of the cognitive and negative endophenotypes found in schizophrenic patients have been proposed. However, significant efforts are needed in order to study the pathophysiology of schizophrenia-related positive symptoms. Recently, it has been shown that these positive symptoms can be studied in rats by using ...
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Transplantation of Embryonic Cortical Tissue into Lesioned Adult Brain in Mice Authors:  Cong Wang, Hao Gao and Shengxiang Zhang, date: 06/20/2017, view: 1295, Q&A: 0
Transplantation of embryonic cortical tissue for repairing the damaged brain has provided a potential therapy for brain injury and diseases. The grafted tissue can successfully survive and participate in reestablishing the functional neural circuit of the host brain. Transplantation surgery can be combined with fluorescently labeled transgenic ...
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Plant Science

Root Aliphatic Suberin Analysis Using Non-extraction or Solvent-extraction Methods Authors:  Camille Delude, Sollapura J. Vishwanath, Owen Rowland and Frédéric Domergue, date: 06/20/2017, view: 1332, Q&A: 0
Here we describe both non-extraction and solvent-extraction methods for root aliphatic suberin analysis. The non-extraction method is fast as roots are directly depolymerized using acidic transmethylation. However, suberin aliphatic components are isolated together with all the other acyl chains making up the lipids (e.g., membranes) ...
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Extraction and Activity of O-acetylserine(thiol)lyase (OASTL) from Microalga Chlorella sorokiniana Authors:  Giovanna Salbitani and Simona Carfagna, date: 06/20/2017, view: 1399, Q&A: 0
O-acetylserine(thiol)lyase (OASTL) is an enzyme catalysing the reaction of inorganic sulphide with O-acetylserine to form the S-containing amino acid L-cysteine. Here we describe an improved protocol to evaluate the activity of this enzyme from the microalga Chlorella sorokiniana. It is a colorimetric assay based on the reaction between ...
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Estimation of Stomatal Aperture in Arabidopsis thaliana Using Silicone Rubber Imprints Authors:  Telma E. Scarpeci, María I. Zanor and Estela M. Valle, date: 06/20/2017, view: 1509, Q&A: 0
Estimation of stomatal aperture using low viscosity silicone-base impression material has the advantage of working with the whole leaf. The developmental stage and the environment strongly affect the stomatal aperture. Therefore, it is mandatory to have accurate estimations of the stomatal aperture of intact leaves under different situations. With ...
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Analysis of 3D Cellular Organization of Fixed Plant Tissues Using a User-guided Platform for Image Segmentation Authors:  Ethel Mendocilla Sato and Célia Baroux, date: 06/20/2017, view: 1570, Q&A: 0
The advent of non-invasive, high-resolution microscopy imaging techniques and computational pipelines for high-throughput image processing has contributed to gain insights in plant organ morphogenesis at the cellular level. Confocal scanning laser microscopy (CSLM) allows the generation of three dimensional images constituted of serial optical ...
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Phototaxis Assay for Chlamydomonas reinhardtii Authors:  Noriko Ueki and Ken-ichi Wakabayashi, date: 06/20/2017, view: 1785, Q&A: 0
Phototaxis is a behavior in which organisms move toward or away from the light source (positive or negative phototaxis, respectively). It is crucial for phototrophic microorganisms to inhabit under proper light conditions for phototaxis. The unicellular green alga Chlamydomonas reinhardtii rapidly changes its swimming direction upon light ...
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Targeted Mutagenesis Using RNA-guided Endonucleases in Mosses Authors:  Toshihisa Nomura and Hitoshi Sakakibara, date: 06/20/2017, view: 1907, Q&A: 0
RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a ...
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