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Methods: a Companion to Methods in Enzymology

Ana M Matia-González, Valentina Iadevaia, André P Gerber
We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins...
October 13, 2016: Methods: a Companion to Methods in Enzymology
Tzu-Fang Lou, Chase A Weidmann, Jordan Killingsworth, Traci M Tanaka Hall, Aaron C Goldstrohm, Zachary T Campbell
RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way...
October 8, 2016: Methods: a Companion to Methods in Enzymology
Abul Arif, Jie Jia, Dalia Halawani, Paul L Fox
Phosphorylation of many aminoacyl tRNA synthetases (AARSs) has been recognized for decades, but the contribution of post-translational modification to their primary role in tRNA charging and decryption of genetic code remains unclear. In contrast, phosphorylation is essential for performance of diverse noncanonical functions of AARSs unrelated to protein synthesis. Phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) has been investigated extensively in our laboratory for more than a decade, and has served as an archetype for studies of other AARSs...
October 8, 2016: Methods: a Companion to Methods in Enzymology
Régine Brielle, Marie-Laure Pinel-Marie, Sophie Chat, Reynald Gillet, Brice Felden
Polysomes are macromolecular complexes made up of multiple ribosomes simultaneously translating a single mRNA into polypeptide chains. Together, the cellular mRNAs translated in this way are referred to 'translatome.' Translation determines a cell's overall gene expression profile. Studying translatome leads to a better understanding of the translational machinery and of its complex regulatory pathways. Given its fundamental role in cell homeostasis and division, bacterial translation is an important target for antibiotics...
October 8, 2016: Methods: a Companion to Methods in Enzymology
Manuel Gunkel, Inn Chung, Stefan Wörz, Katharina I Deeg, Ronald Simon, Guido Sauter, David T W Jones, Andrey Korshunov, Karl Rohr, Holger Erfle, Karsten Rippe
The microscopic analysis of telomere features provides a wealth of information on the mechanism by which tumor cells maintain their unlimited proliferative potential. Accordingly, the analysis of telomeres in tissue sections of patient tumor samples can be exploited to obtain diagnostic information and to define tumor subgroups. In many instances, however, analysis of the image data is conducted by manual inspection of 2D images at relatively low resolution for only a small part of the sample. As the telomere feature signal distribution is frequently heterogeneous, this approach is prone to a biased selection of the information present in the image and lacks subcellular details...
October 7, 2016: Methods: a Companion to Methods in Enzymology
Sylvain Debard, Gaétan Bader, Johan-Owen De Craene, Ludovic Enkler, Séverine Bär, Daphné Laporte, Evelyne Myslinski, Bruno Senger, Sylvie Friant, Hubert Dominique Becker
By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species...
October 7, 2016: Methods: a Companion to Methods in Enzymology
Joanne Lannigan, Uta Erdbruegger
Extracellular Vesicles (EVs) are potent bio-activators and inter-cellular communicators that play an important role in both health and disease. It is for this reason there is a strong interest in understanding their composition and origin, with the hope of using them as important biomarkers or therapeutics. Due to their very small size, heterogeneity, and large numbers there has been a need for better tools to measure them in an accurate and high throughput manner. While traditional flow cytometry has been widely used for this purpose, there are inherent problems with this approach, as these instruments have traditionally been developed to measure whole cells, which are orders of magnitude larger and express many more molecules of identifying epitopes...
October 6, 2016: Methods: a Companion to Methods in Enzymology
Henry Hui, Kathy A Fuller, Wendy N Erber, Matthew D Linden
Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype...
October 5, 2016: Methods: a Companion to Methods in Enzymology
Jean-Yves Tinevez, Nick Perry, Johannes Schindelin, Genevieve M Hoopes, Gregory D Reynolds, Emmanuel Laplantine, Sebastian Y Bednarek, Spencer L Shorte, Kevin W Eliceiri
We present TrackMate, an open source Fiji plugin for the automated, semi-automated, and manual tracking of single-particles. It offers a versatile and modular solution that works out of the box for end users, through a simple and intuitive user interface. It is also easily scriptable and adaptable, operating equally well on 1D over time, 2D over time, 3D over time, or other single and multi-channel image variants. TrackMate provides several visualization and analysis tools that aid in assessing the relevance of results...
October 3, 2016: Methods: a Companion to Methods in Enzymology
Nevena Cvetesic, Ita Gruic-Sovulj
The covalent coupling of cognate amino acid-tRNA pairs by corresponding aminoacyl-tRNA synthetases (aaRS) defines the genetic code and provides aminoacylated tRNAs for ribosomal protein synthesis. Besides the cognate substrate, some non-cognate amino acids may also compete for tRNA aminoacylation. However, their participation in protein synthesis is generally prevented by an aaRS proofreading activity located in the synthetic site and in a separate editing domain. These mechanisms, coupled with the ability of certain aaRSs to discriminate well against non-cognate amino acids in the synthetic reaction alone, define the accuracy of the aminoacylation reaction...
October 3, 2016: Methods: a Companion to Methods in Enzymology
Jonathan Jagodnik, Anaïs Brosse, Thao Nguyen Le Lam, Claude Chiaruttini, Maude Guillier
In all three kingdoms of life, RNA is not only involved in the expression of genetic information, but also carries out extremely diverse cellular functions. This versatility is essentially due to the fact that RNA molecules can exploit the power of base pairing to allow them to fold into a wide variety of structures through which they can perform diverse roles, but also to selectively target and bind to other nucleic acids. This is true in particular for bacterial small regulatory RNAs that act by imperfect base-pairing with target mRNAs, and thereby control their expression through different mechanisms...
September 28, 2016: Methods: a Companion to Methods in Enzymology
Emre Balta, Julian Stopp, Laura Castelletti, Henning Kirchgessner, Yvonne Samstag, Guido H Wabnitz
Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells). However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions...
September 27, 2016: Methods: a Companion to Methods in Enzymology
Xavier Descombes
The marked point process framework has been successfully developed in the field of image analysis to detect a configuration of predefined objects. The goal of this paper is to show how it can be particularly applied to biological imagery. We present a simple model that shows how some of the challenges specific to biological data are well addressed by the methodology. We further describe an extension to this first model to address other challenges due, for example, to the shape variability in biological material...
September 21, 2016: Methods: a Companion to Methods in Enzymology
Yi Shi, Na Wei, Xiang-Lei Yang
Aminoacyl tRNA synthetases (AARSs) are best known for their essential role in translation in the cytoplasm. The concept that AARSs also exist in the nucleus started to draw attention around the turn of the new millennium, when aminoacylated tRNAs were first found in the nuclei of Xenopus oocytes. It is now expected that all cytoplasmic AARSs are present in the nucleus. In addition to tRNA aminoacylation, nuclear AARSs were found to regulate a spectrum of biological processes and responses, with many AARSs functioning through regulation at the level of gene transcription...
September 21, 2016: Methods: a Companion to Methods in Enzymology
Cindy Meyer, Aitor Garzia, Thomas Tuschl
Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) are sensitive techniques for detecting nucleic acids and proteins in cultured cells. However, these techniques are rarely applied together, and standard protocols are not readily compatible for sequential application on the same specimen. Here, we provide a user-friendly step-by-step protocol to perform multicolor RNA-FISH in combination with IF to simultaneously detect the subcellular localization of distinct RNAs and proteins in cultured cells...
September 21, 2016: Methods: a Companion to Methods in Enzymology
Margery G H Pelletier, Klaudia Szymczak, Anna M Barbeau, Gianna N Prata, Kevin S O'Fallon, Peter Gaines
Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells...
September 20, 2016: Methods: a Companion to Methods in Enzymology
Gabriele Nübel, Frieda A Sorgenfrei, Andres Jäschke
RNA modifications are widely distributed in Nature, and their thorough analysis helps answering fundamental biological questions. Nowadays, mass spectrometry or deep-sequencing methods are often used for the analysis. With the raising number of newly discovered RNA modifications, such as the 5'-NAD cap in Escherichia coli, there is an important need for new, less complex and fast analytical tools to analyze the occurrence, amount, and distribution of modified RNAs in cells. To accomplish this task, we have revisited the previously developed affinity gel electrophoresis principles and copolymerized acryloylaminophenyl boronic acid (APB) in standard denaturing polyacrylamide gels to retard the NAD- or FAD-modified RNAs compared to the unmodified RNAs in the gels...
September 16, 2016: Methods: a Companion to Methods in Enzymology
Viraga Haridas, Shahin Ranjbar, Ivan A Vorobjev, Anne E Goldfeld, Natasha S Barteneva
Imaging flow cytometry has been applied to address questions in infection biology, in particular, infections induced by intracellular pathogens. This methodology, which utilizes specialized analytic software makes it possible to analyze hundreds of quantified features for hundreds of thousands of individual cellular or subcellular events in a single experiment. Imaging flow cytometry analysis of host cell-pathogen interaction can thus quantitatively addresses a variety of biological questions related to intracellular infection, including cell counting, internalization score, and subcellular patterns of co-localization...
September 15, 2016: Methods: a Companion to Methods in Enzymology
Nicolas R Chevalier, Elodie Gazquez, Sylvie Dufour, Vincent Fleury
No abstract text is available yet for this article.
September 15, 2016: Methods: a Companion to Methods in Enzymology
Michael H Schwartz, Tao Pan
The fidelity of tRNA aminoacylation is a critical determinant for the ultimate accuracy of protein synthesis. Although aminoacyl-tRNA synthetases are assumed to consistently maintain high tRNA charging fidelity, recent evidence has demonstrated that the fidelity of the aminoacylation reaction can be actively regulated and liable to change. Accordingly, the ability to conveniently assay the fidelity of tRNA charging is becoming increasingly relevant for studying mistranslation. Here we describe a combined radioactivity and microarray based method that can quantitatively elucidate which individual cognate or noncognate tRNA isoacceptors are charged with amino acid...
September 14, 2016: Methods: a Companion to Methods in Enzymology
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