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Methods: a Companion to Methods in Enzymology

Jean-François Gilles, Marc Dos Santos, Thomas Boudier, Susanne Bolte, Nicolas Heck
We present a new plugin for ImageJ called DiAna, for Distance Analysis, which comes with a user-friendly interface. DiAna proposes robust and accurate 3D segmentation for object extraction. The plugin performs automated object-based co-localization and distance analysis. DiAna offers an in-depth analysis of co-localization between objects and retrieves 3D measurements including co-localizing volumes and surfaces of contact. It also computes the distribution of distances between objects in 3D. With DiAna, we furthermore introduce an original method, which allows for estimating the statistical significance of object co-localization...
November 24, 2016: Methods: a Companion to Methods in Enzymology
Pengfei Fang, Min Guo
Aminoacyl-tRNA synthetases (aaRSs) are enzymes that function at the first step of translation, catalyzing the conjugation of amino acids to their cognate tRNAs for protein synthesis. While preserving this essential role, higher eukaryotic aaRSs, such as human cytoplasmic aaRSs, have developed other functions during evolution, including angiogenesis, inflammation, development, tumorigenesis, etc. These translational and non-translational functions of aaRSs are attractive targets for developing antibacterial, antifungal, anticancer agents and for treating other human diseases...
November 22, 2016: Methods: a Companion to Methods in Enzymology
Hanchao Zhao, Susan Martinis
Aminoacyl-tRNA synthetases (AARS) comprise an enzyme family that generates and maintains pools of aminoacylated tRNAs, which serve as essential substrates for protein synthesis. Many protein synthesis factors, including tRNA and AARS also have non-canonical functions. Particularly in mammalian cells, alternate functions of AARSs have been associated with re-distribution in the cell to sites that are removed from translation. Sub-fractionation methods for E. coli were designed and optimized to carefully investigate re-localization of bacterial AARSs and tRNA that might aid in conferring alternate activities...
November 22, 2016: Methods: a Companion to Methods in Enzymology
Stephanie N Oprescu, Laurie B Griffin, Asim A Beg, Anthony Antonellis
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes responsible for charging tRNA with cognate amino acids-the first step in protein synthesis. ARSs are required for protein translation in the cytoplasm and mitochondria of all cells. Surprisingly, mutations in 28 of the 37 nuclear-encoded human ARS genes have been linked to a variety of recessive and dominant tissue-specific disorders. Current data indicate that impaired enzyme function is a robust predictor of the pathogenicity of ARS mutations...
November 20, 2016: Methods: a Companion to Methods in Enzymology
Min Shen, James F Rusling, Chandra K Dixit
Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention...
November 19, 2016: Methods: a Companion to Methods in Enzymology
David Lalaouna, Karine Prévost, Alex Eyraud, Eric Massé
Recent advances in high-throughput sequencing have led to an explosion in the rate of small regulatory RNAs (sRNAs) discovery among bacteria. However, only a handful of them are functionally characterized. Most of the time, little to no targets are known. In Lalaouna et al. (2015), we proposed a new technology to uncover sRNAs targetome, which is based on the MS2-affinity purification (MAPS). We were able to prove its efficiency by applying it on well-characterized sRNAs of Escherichia coli. Thereafter, we adapted the procedure to other kind of RNA (mRNAs and tRNA-derived RNA fragments) and bacteria (pathogenic or Gram-positive strains)...
November 19, 2016: Methods: a Companion to Methods in Enzymology
Mélodie Migault, Emmanuelle Donnou-Fournet, Marie-Dominique Galibert, David Gilot
Targeting RNAs appears as an important opportunity to modulate biological processes. Here, we overviewed critical parameters implied in RNAs competition to bind small RNAs. These competitions influence small RNA availability and thereby gene expression and cell fate. We focused on the ability of RNAs to sequester small RNA, mainly the microRNAs (miRNAs) and proposed experimental workflows to demonstrate the existence and activity of RNA-sponge. From this basic science, we detailed tailored oligonucleotides, developed to challenge the binding of small RNA...
November 19, 2016: Methods: a Companion to Methods in Enzymology
Daniel Benhalevy, Hannah L McFarland, Aishe A Sarshad, Markus Hafner
The study of protein-RNA interactions is critical for our understanding of cellular processes and regulatory circuits controlled by RNA binding proteins (RBPs). Recent next generation sequencing-based approaches significantly promoted our understanding of RNA biology and its importance for cell function. We present a streamlined protocol for Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a technique that allows for the characterization of RBP binding sites on target RNAs at nucleotide resolution and transcriptome-wide scale...
November 18, 2016: Methods: a Companion to Methods in Enzymology
H Ma, R O'Kennedy
Recombinant antibodies are now very important in both therapeutics and diagnostics and offer significant advantages over conventional antibodies. The generation of a single-chain variable antibody fragment (scFv) (a common and important recombinant antibody format) is used to demonstrate the construction of a recombinant antibody library. An immunotube-based two-day panning approach, using Escherichia coli as an expression system, is utilised for antibody screening. The methods used for antibody selection and purification using immobilised metal affinity chromatography (IMAC) are described...
November 18, 2016: Methods: a Companion to Methods in Enzymology
Valerie Fitzgerald, Paul Leonard
Microtools that have been developed to allow in depth interrogation of individual cells in high throughput are improving our understanding of biological processes at the single cell level and are opening up new possibilities for biological research. In relation to antibody discovery, these tools are now helping to maximise the full potential of well-established methodologies for antibody generation. Being complementary to both recombinant and native antibody secreting cells, some of these tools are finding widespread use in the field...
November 15, 2016: Methods: a Companion to Methods in Enzymology
Adam C Mirando, Khadar Abdi, Peibin Wo, Karen M Lounsbury
Several recent reports have found a connection between specific aminoacyl-tRNA synthetases and the regulation of angiogenesis. As this new area of research is explored, it is important to have reliable assays to assess the specific angiogenesis functions of these enzymes. This review provides information about specific in vitro and in vivo methods that were used to assess the angiogenic functions of threonyl-tRNA synthetase including endothelial cell migration and tube assays as well as chorioallantoic membrane and tumor vascularization assays...
November 12, 2016: Methods: a Companion to Methods in Enzymology
Cindo O Nicholson, Matthew B Friedersdorf, Laura S Bisogno, Jack D Keene
Post-transcriptional processes orchestrate gene expression through dynamic protein-RNA interactions. These interactions occur at specific sites determined by RNA sequence, secondary structure, or nucleotide modifications. Methods have been developed either to quantify binding of whole transcripts or to identify the binding sites, but there is none proven to quantify binding at both the whole transcript and binding site levels. Here we describe digestion optimized RNA immunoprecipitation with deep sequencing (DO-RIP-seq) as a method that quantitates at the whole transcript target (RIP-Seq-Like or RSL) level and at the binding site level (BSL) using continuous metrics...
November 10, 2016: Methods: a Companion to Methods in Enzymology
Adela D Staszowska, Patrick Fox-Roberts, Elizabeth Foxall, Gareth E Jones, Susan Cox
Podosomes are adhesive structures formed on the plasma membrane abutting the extracellular matrix of macrophages, osteoclasts, and dendritic cells. They consist of an f-actin core and a ring structure composed of integrins and integrin-associated proteins. The podosome ring plays a major role in adhesion to the underlying extracellular matrix, but its detailed structure is poorly understood. Recently, it has become possible to study the nano-scale structure of podosome rings using localization microscopy. Unlike traditional microscopy images, localization microscopy images are reconstructed using discrete points, meaning that standard image analysis methods cannot be applied...
November 10, 2016: Methods: a Companion to Methods in Enzymology
Radka Saldova, Michelle Kilcoyne, Henning Stöckmann, Silvia Millán Martín, Amanda M Lewis, Catherine M E Tuite, Jared Q Gerlach, Marie Le Berre, Michael C Borys, Zheng Jian Li, Nicholas R Abu-Absi, Kirk Leister, Lokesh Joshi, Pauline M Rudd
This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same six time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays...
November 7, 2016: Methods: a Companion to Methods in Enzymology
France Lam, Damien Cladière, Cyndélia Guillaume, Katja Wassmann, Susanne Bolte
In the presented work we aimed at improving confocal imaging to obtain highest possible resolution in thick biological samples, such as the mouse oocyte. We therefore developed an image processing workflow that allows improving the lateral and axial resolution of a standard confocal microscope. Our workflow comprises refractive index matching, the optimization of microscope hardware parameters and image restoration by deconvolution. We compare two different deconvolution algorithms, evaluate the necessity of denoising and establish the optimal image restoration procedure...
November 5, 2016: Methods: a Companion to Methods in Enzymology
Laura Pérez-Cano, Miguel Romero-Durana, Juan Fernández-Recio
Deciphering the structural and energetic determinants of protein-RNA interactions harbors the potential to understand key cell processes at molecular level, such as gene expression and regulation. With this purpose, computational methods like docking aim to complement current biophysical and structural biology efforts. However, the few reported docking algorithms for protein-RNA interactions show limited predictive success rates, mainly due to incomplete sampling of the conformational space of both the protein and the RNA molecules, as well as to the difficulties of the scoring function in identifying the correct docking models...
November 2, 2016: Methods: a Companion to Methods in Enzymology
Jamie A Abbott, Nathan M Livingston, Shawn B Egri, Ethan Guth, Christopher S Francklyn
Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori...
October 26, 2016: Methods: a Companion to Methods in Enzymology
Christine Carapito, Lauriane Kuhn, Loukmane Karim, Magali Rompais, Thierry Rabilloud, Hagen Schwenzer, Marie Sissler
Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria...
October 25, 2016: Methods: a Companion to Methods in Enzymology
Hye Young Cho, Sunghoon Kim, Young Ho Jeon
Lysyl-tRNA synthetase (KRS) is an enzyme that conjugates lysine to its cognate tRNAs in the process of protein synthesis. In addition to its catalytic function, KRS binds to the 67-kDa laminin receptor (LR) on the cell membrane and facilitates cell migration and metastasis. Modulation of this interaction by small-molecule inhibitors can be exploited to suppress cancer metastasis. In this study, we present fragment-based methods for the identification of inhibitors and monitoring protein-protein interactions between KRS and LR...
October 24, 2016: Methods: a Companion to Methods in Enzymology
Eric A First, Charles J Richardson
Aminoacyl-tRNA synthetases play a central role in protein synthesis, catalyzing the attachment of amino acids to their cognate tRNAs. Here, we describe a spectrophotometric assay for tyrosyl-tRNA synthetase in which the Tyr-tRNA product is cleaved, regenerating the tRNA substrate. As tRNA is the limiting substrate in the assay, recycling it substantially increases the sensitivity of the assay while simultaneously reducing its cost. The tRNA aminoacylation reaction is monitored spectrophotometrically by coupling the production of AMP to the conversion of NAD(+) to NADH...
October 22, 2016: Methods: a Companion to Methods in Enzymology
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