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Methods: a Companion to Methods in Enzymology

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https://www.readbyqxmd.com/read/28219745/interactive-exploration-for-continuously-expanding-neuron-databases
#1
Zhongyu Li, Dimitris N Metaxas, Aidong Lu, Shaoting Zhang
This paper proposes a novel framework to help biologists explore and analyze neurons based on retrieval of data from neuron morphological databases. In recent years, the continuously expanding neuron databases provide a rich source of information to associate neuronal morphologies with their functional properties. We design a coarse-to-fine framework for efficient and effective data retrieval from large-scale neuron databases. In the coarse-level, for efficiency in large-scale, we employ a binary coding method to compress morphological features into binary codes of tens of bits...
February 17, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28219744/metabolic-labeling-and-recovery-of-nascent-rna-to-accurately-quantify-mrna-stability
#2
Joseph Russo, Adam M Heck, Jeffrey Wilusz, Carol J Wilusz
Changes in the rate of mRNA decay are closely coordinated with transcriptional changes and together these events have profound effects on gene expression during development and disease. Traditional approaches to assess mRNA decay have relied on inhibition of transcription, which can alter mRNA decay rates and confound interpretation. More recently, metabolic labeling combined with chemical modification and fractionation of labeled RNAs has allowed the isolation of nascent transcripts and the subsequent calculation of mRNA decay rates...
February 17, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28215631/posttranscriptional-chemical-labeling-of-rna-by-using-bioorthogonal-chemistry
#3
REVIEW
Jerrin Thomas George, Seergazhi G Srivatsan
Recent developments in RNA labeling technology have provided viable tools to analyze RNA synthesis, processing and function in cell-free and cellular environments. Notably, emerging methodologies based on posttranscriptional chemical labeling by using bioorthogonal chemistry have enabled the visualization and profiling of exogenous and endogenous RNA transcripts. In this review, we first give an overview of different RNA labeling strategies based on chemical as well as genetically encoded systems. Subsequently, we provided a detailed discussion on methodologies that have been developed to introduce various bioorthogonal reactive groups into RNA transcripts, which are compatible for posttranscriptional functionalization...
February 16, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28213280/magnetic-lateral-flow-immunoassay-test-strip-development-considerations-for-proof-of-concept-evaluation
#4
R Connolly, R O' Kennedy
Lateral flow immunoassays (LFIA) have grown to become the predominant test device format for the diagnostics and point-of-care industries. The demand for robust and reproducible LFIAs has been facilitated through scale-up production methods using specialized and automated instruments. However, the feasibility of a LFIA device can still be evaluated in a small-scale laboratory setting through controlled manual preparation methods. The advent of super-paramagnetic (SPMP) labels for use in lateral flow has heralded the possibility of highly sensitive and stable LFIAs...
February 14, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28213279/tethering-rna-to-chromatin-for-fluorescence-microscopy-based-analysis-of-nuclear-organization
#5
Teresa Pankert, Thibaud Jegou, Maïwen Caudron-Herger, Karsten Rippe
Nuclear RNAs emerge as important factors to orchestrate the dynamic organization of the nucleus into functional subcompartments. By tethering RNAs to distinct genomic loci, the RNA-dependent chromatin changes can be dissected by fluorescence microscopic analysis. Here we describe how this approach is implemented in mammalian cells. It involves two high-affinity protein-nucleic acid interactions that can established with number of different protein domains and DNA and RNA sequences. A prototypic system is described here in detail: It consists of the binding of MS2 bacteriophage coat protein to its RNA recognition sequence and the interaction between the bacterial LacI repressor protein to its target lacO operator DNA sequences...
February 14, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28189773/mathematical-imaging-methods-for-mitosis-analysis-in-live-cell-phase-contrast-microscopy
#6
Joana Sarah Grah, Jennifer Alison Harrington, Siang Boon Koh, Jeremy Andrew Pike, Alexander Schreiner, Martin Burger, Carola-Bibiane Schönlieb, Stefanie Reichelt
In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets...
February 9, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28179124/comparative-analysis-of-2d-and-3d-distance-measurements-to-study-spatial-genome-organization
#7
Elizabeth H Finn, Gianluca Pegoraro, Sigal Shachar, Tom Misteli
The spatial organization of genomes is non-random, cell-type specific, and has been linked to cellular function. The investigation of spatial organization has traditionally relied extensively on fluorescence microscopy. The validity of the imaging methods used to probe spatial genome organization often depends on the accuracy and precision of distance measurements. Imaging-based measurements may either use 2 dimensional datasets or 3D datasets which include the z-axis information in image stacks. Here we compare the suitability of 2D vs 3D distance measurements in the analysis of various features of spatial genome organization...
February 5, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28163103/optimizing-antibody-expression-the-nuts-and-bolts
#8
REVIEW
B Vijayalakshmi Ayyar, Sushrut Arora, Shiva Shankar Ravi
Antibodies are extensively utilized entities in biomedical research, diagnostics, and therapeutics. Many of these applications require high amounts of antibodies. However, meeting this ever-increasing demand of antibodies in the global market is one of the outstanding challenges. The need to maintain a balance between demand and supply of antibodies has led the researchers to discover better means and methods for optimizing their expression. These strategies aim to increase the volumetric productivity of the antibodies along with the reduction of associated manufacturing costs...
February 2, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28161540/genome-organization-in-the-nucleus-from-dynamic-measurements-to-a-functional-model
#9
REVIEW
Anat Vivante, Eugene Brozgol, Irena Bronshtein, Yuval Garini
A biological system is by definition a dynamic environment encompassing kinetic processes that occur at different length scales and time ranges. To explore this type of system, spatial information needs to be acquired at different time scales. This means overcoming significant hurdles, including the need for stable and precise labeling of the required probes and the use of state of the art optical methods. However, to interpret the acquired data, biophysical models that can account for these biological mechanisms need to be developed...
February 1, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28132896/genetics-and-biochemistry-remain-essential-in-the-structural-era-of-the-spliceosome
#10
REVIEW
Megan Mayerle, Christine Guthrie
The spliceosome is not a single macromolecular machine. Rather it is a collection of dynamic heterogeneous subcomplexes that rapidly interconvert throughout the course of a typical splicing cycle. Because of this, for many years the only high resolution structures of the spliceosome available were of smaller, isolated protein or RNA components. Consequently much of our current understanding of the spliceosome derives from biochemical and genetic techniques. Now with the publication of multiple, high resolution structures of the spliceosome, some question the relevance of traditional biochemical and genetic techniques to the splicing field...
January 26, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28131869/quantifying-receptor-trafficking-and-colocalization-with-confocal-microscopy
#11
Jeremy A Pike, Iain B Styles, Joshua Z Rappoport, John K Heath
Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocytosis. Unbiased and robust image analysis workflows are required for the identification, and study, of aberrant trafficking. After a brief review of related strategies, identifying both good and bad practice, custom workflows for the analysis of live cell 3D time-lapse data are presented. Strategies for data pre-processing, including denoising and background subtraction are considered. We use a 3D level set protocol to accurately segment cells using only the signal from fluorescently labelled receptor...
January 26, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28126557/fluorescently-labelled-multiplex-lateral-flow-immunoassay-based-on-cadmium-free-quantum-dots
#12
Natalia V Beloglazova, Aleksander M Sobolev, Mickael D Tessier, Zeger Hens, Irina Yu Goryacheva, Sarah De Saeger
A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures...
January 23, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28108198/ted-a-tolerant-edit-distance-for-segmentation-evaluation
#13
Jan Funke, Jonas Klein, Francesc Moreno-Noguer, Albert Cardona, Matthew Cook
In this paper, we present a novel error measure to compare a computer-generated segmentation of images or volumes against ground truth. This measure, which we call Tolerant Edit Distance (TED), is motivated by two observations that we usually encounter in biomedical image processing: (1) Some errors, like small boundary shifts, are tolerable in practice. Which errors are tolerable is application dependent and should be explicitly expressible in the measure. (2) Non-tolerable errors have to be corrected manually...
January 17, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28088364/antibodies-from-novel-repertoires-to-defining-and-refining-the-structure-of-biologically-important-targets
#14
REVIEW
Paul J Conroy, Ruby H P Law, Tom Caradoc-Davies, James C Whisstock
Antibodies represent a highly successful class of molecules that bind a wide-range of targets in therapeutic-, diagnostic- and research-based applications. The antibody repertoire is composed of the building blocks required to develop an effective adaptive immune response against foreign insults. A number of species have developed novel genetic and structural mechanisms from which they derive these antibody repertoires, however, traditionally antibodies are derived from human, and rodent sources. Due to their high-value therapeutic, diagnostic, biotechnological and research applications, much innovation has resulted in techniques and approaches to isolate novel antibodies...
January 11, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28087312/immunisation-choice-of-host-adjuvants-and-boosting-schedules-with-emphasis-on-polyclonal-antibody-production
#15
REVIEW
Philippe Delahaut
Polyclonal antibodies are frequently used as immunodiagnostic tools in fundamental research. They are also used for routine diagnostic purposes in human and veterinary medicine and for quality control procedures in the food-processing industry. The antibody is a major component of the detection system. It binds with the molecule to be identified. This conjugate is subsequently revealed by means of binding the antibody with a radio-isotope, a fluorescent substance, an enzyme inducing a color change, or a biosensor based analytical system...
January 10, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28057585/an-overview-of-data-science-uses-in-bioimage-informatics
#16
REVIEW
Anatole Chessel
This review aims at providing a practical overview of the use of statistical features and associated data science methods in bioimage informatics. To achieve a quantitative link between images and biological concepts, one typically replaces an object coming from an image (a segmented cell or intracellular object, a pattern of expression or localisation, even a whole image) by a vector of numbers. They range from carefully crafted biologically relevant measurements to features learnt through deep neural networks...
January 3, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28057586/deconvolutionlab2-an-open-source-software-for-deconvolution-microscopy
#17
REVIEW
Daniel Sage, Lauréne Donati, Ferréol Soulez, Denis Fortun, Guillaume Schmit, Arne Seitz, Romain Guiet, Cédric Vonesch, Michael Unser
Images in fluorescence microscopy are inherently blurred due to the limit of diffraction of light. The purpose of deconvolution microscopy is to compensate numerically for this degradation. Deconvolution is widely used to restore fine details of 3D biological samples. Unfortunately, dealing with deconvolution tools is not straightforward. Among others, end users have to select the appropriate algorithm, calibration and parametrization, while potentially facing demanding computational tasks. To make deconvolution more accessible, we have developed a practical platform for deconvolution microscopy called DeconvolutionLab...
January 2, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28027957/site-directed-chemical-probing-to-map-transient-rna-protein-interactions
#18
Mélodie Duval, Alessandra Marenna, Clément Chevalier, Stefano Marzi
RNA-protein interactions are at the bases of many biological processes, forming either tight and stable functional ribonucleoprotein (RNP) complexes (i.e. the ribosome) or transitory ones, such as the complexes involving RNA chaperone proteins. To localize the sites where a protein interacts on an RNA molecule, a common simple and inexpensive biochemical method is the footprinting technique. The protein leaves its footprint on the RNA acting as a shield to protect the regions of interaction from chemical modification or cleavages obtained with chemical or enzymatic nucleases...
December 24, 2016: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28012937/affinity-chromatography-a-versatile-technique-for-antibody-purification
#19
REVIEW
Sushrut Arora, Vikas Saxena, B Vijayalakshmi Ayyar
Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup...
December 22, 2016: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28003131/crispr-cas9-mediated-integration-enables-tag-eclip-of-endogenously-tagged-rna-binding-proteins
#20
Eric L Van Nostrand, Chelsea Gelboin-Burkhart, Ruth Wang, Gabriel A Pratt, Steven M Blue, Gene W Yeo
Identification of in vivo direct RNA targets for RNA binding proteins (RBPs) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus...
December 18, 2016: Methods: a Companion to Methods in Enzymology
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