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Methods: a Companion to Methods in Enzymology

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https://www.readbyqxmd.com/read/28434905/using-quantitative-intravital-multiphoton-microscopy-to-dissect-hepatic-transport-in-rats
#1
Kenneth W Dunn, Jennifer C Ryan
Hepatic solute transport is a complex process whose disruption is associated with liver disease and drug-induced liver injury. Intravital multiphoton fluorescence excitation microscopy provides the spatial and temporal resolution necessary to characterize hepatic transport at the level of individual hepatocytes in vivo and thus to identify the mechanisms and cellular consequences of cholestasis. Here we present an overview of the use of fluorescence microscopy for studies of hepatic transport in living animals, and describe how we have combined methods of intravital microscopy and digital image analysis to dissect the effects of drugs and pathological conditions on the function of hepatic transporters in vivo...
April 20, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28434904/deep-sequencing-approaches-for-the-analysis-of-prokaryotic-transcriptional-boundaries-and-dynamics
#2
REVIEW
Katherine James, Simon J Cockell, Nikolay Zenkin
The identification of the protein-coding regions of a genome is straightforward due to the universality of start and stop codons. However, the boundaries of the transcribed regions, conditional operon structures, non-coding RNAs and the dynamics of transcription, such as pausing of elongation, are non-trivial to identify, even in the comparatively simple genomes of prokaryotes. Traditional methods for the study of these areas, such as tiling arrays, are noisy, labour-intensive and lack the resolution required for densely-packed bacterial genomes...
April 20, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28434903/measuring-transcription-dynamics-in-living-cells-using-a-photobleaching-approach
#3
REVIEW
Hodaya Hochberg, Yehuda Brody, Yaron Shav-Tal
The transcriptional kinetics of RNA polymerase II, the enzyme responsible for mRNA transcription in the nucleoplasm, can be modulated by a variety of factors. It is therefore important to establish experimental systems that will enable the readout of transcription kinetics of specific genes as they occur in real time within individual cells. This can be performed by implementing fluorescent tagging of the mRNA under live-cell conditions. This chapter describes how to generate fluorescently tagged genes and mRNA, and how a photobleaching approach can produce information on mRNA transcription kinetics...
April 20, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28434902/intravital-microscopy-of-biosensor-activities-and-intrinsic-metabolic-states
#4
REVIEW
Seth Winfree, Takashi Hato, Richard N Day
Intravital microscopy (IVM) is an imaging tool that is capable of detecting subcellular signaling or metabolic events as they occur in tissues in the living animal. Imaging in highly scattering biological tissues, however, is challenging because of the attenuation of signal in images acquired at increasing depths. Depth-dependent signal attenuation is the major impediment to IVM, limiting the depth from which significant data can be obtained. Therefore, making quantitative measurements by IVM requires methods that use internal calibration, or alternatively, a completely different way of evaluating the signals...
April 20, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28433606/in-silico-methods-for-co-transcriptional-rna-secondary-structure-prediction-and-for-investigating-alternative-rna-structure-expression
#5
REVIEW
Irmtraud M Meyer
RNA transcripts are the primary products of active genes in any living organism, including many viruses. Their cellular destiny not only depends on primary sequence signals, but can also be determined by RNA structure. Recent experimental evidence shows that many transcripts can be assigned more than a single functional RNA structure throughout their cellular life and that structure formation happens co-transcriptionally, i.e. as the transcript is synthesised in the cell. Moreover, functional RNA structures are not limited to non-coding transcripts, but can also feature in coding transcripts...
April 19, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28435001/hi-c-2-0-an-optimized-hi-c-procedure-for-high-resolution-genome-wide-mapping-of-chromosome-conformation
#6
Houda Belaghzal, Job Dekker, Johan H Gibcus
Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (Kb) resolution...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28435000/context-dependent-intravital-imaging-of-therapeutic-response-using-intramolecular-fret-biosensors
#7
REVIEW
James R W Conway, Sean C Warren, Paul Timpson
Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug activity is assessed. Microenvironmental factors, such as hypoxia, pH, fibrosis, immune infiltration and stromal interactions have all been shown to have pronounced effects on drug activity in a more complex setting, which is often lost in simpler two- or three-dimensional assays...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28434999/smfret-experiments-of-the-rna-polymerase-ii-transcription-initiation-complex
#8
REVIEW
Nicole Malkusch, Thilo Dörfler, Julia Nagy, Tobias Eilert, Jens Michaelis
Single-molecule fluorescence and in particular single-molecule Förster Resonance Energy Transfer (smFRET(1)) is a powerful tool to provide real-time information on the dynamic architecture of large macromolecular structures such as eukaryotic transcription initiation complexes. In contrast to other structural biology methods, not only structural details, but dynamics transitions are revealed thus closing in on the underlying molecular mechanisms. Here, we describe a comprehensive quantitative biophysical toolbox which can be used for rigorous analysis of dynamic protein-nucleic acid complexes and is applied to the study of eukaryotic transcription initiation...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28434998/use-of-two-photon-microscopy-to-study-leishmania-major-infection-of-the-skin
#9
REVIEW
Matheus Batista Carneiro, Leah Shan Hohman, Jackson G Egen, Nathan C Peters
Intra-vital two-photon microscopy (2P-IVM) allows for in-situ investigation of tissue organization, cell behavior and the dynamic interactions between different cell types in their natural environment. This methodology has also expanded our understanding of the immune response against pathogens. Leishmania are protozoan intracellular parasites that have adapted to successfully establish infection within the context of an inflammatory response in the skin following transmission by the bite of an infected sand fly...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28434997/multi-photon-microscopy-in-cardiovascular-research
#10
REVIEW
Zhuojun Wu, Timo Rademakers, Fabian Kiessling, Michael Vogt, Erik Westein, Christian Weber, Remco Ta Megens, Marc van Zandvoort
Multiphoton laser scanning microscopy has proven profound value for ex vivo 3D histology and in vivo imaging of motionless tissue. The development of triggering systems and fast imaging methods, combined with advanced preparation procedures solved the challenging task of intravital imaging of the fast pulsating heart and major arteries in animals and further increased the popularity of intravital multiphoton imaging in cardiovascular research. This review article will highlight the potential of multiphoton microscopy for the visualization and characterization of dynamical and structural processes involved in cardiac and vascular diseases, both in an ex vivo and an intravital animal setting...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28434996/effect-of-heparin-and-heparan-sulphate-on-open-promoter-complex-formation-for-a-simple-tandem-gene-model-using-ex-situ-atomic-force-microscopy
#11
Oliver Chammas, William A Bonass, Neil H Thomson
The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RPo) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ(70)RNAP) on linear DNA using ex-situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ(70)RNAP and RNAP after RPo formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter...
April 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28414097/single-molecule-and-super-resolution-imaging-of-transcription-in-living-bacteria
#12
REVIEW
Mathew Stracy, Achillefs N Kapanidis
In vivo single-molecule and super-resolution techniques are transforming our ability to study transcription as it takes place in its native environment in living cells. This review will detail the methods for imaging single molecules in cells, and the data-analysis tools which can be used to extract quantitative information on the spatial organization, mobility, and kinetics of the transcription machinery from these experiments. Furthermore, we will highlight studies which have applied these techniques to shed new light on bacterial transcription...
April 13, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28412289/structural-studies-of-the-endogenous-spliceosome-the-supraspliceosome
#13
REVIEW
Joseph Sperling, Ruth Sperling
Pre-mRNA splicing is executed in mammalian cell nuclei within a huge (21 MDa) and highly dynamic molecular machine - the supraspliceosome - that individually package pre-mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature. Detailed structural analysis of this huge and complex structure requires a stepwise approach using hybrid methods. Structural studies of the supraspliceosome by room temperature electron tomography, cryo-electron tomography, and scanning transmission electron microscope mass measurements revealed that it is composed of four native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA...
April 12, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28410977/intravital-imaging-of-the-kidney
#14
REVIEW
Takashi Hato, Seth Winfree, Pierre C Dagher
Two-photon intravital microscopy is a powerful tool that allows the examination of dynamic cellular processes in the live animal with unprecedented resolution. Indeed, it offers the ability to address unique biological questions that may not be solved by other means. While two-photon intravital microscopy has been successfully applied to study many organs, the kidney presents its own unique challenges that need to be overcome in order to optimize and validate imaging data. For kidney imaging, the complexity of renal architecture and salient autofluorescence merit special considerations as these elements directly impact image acquisition and data interpretation...
April 11, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28410976/genome-editing-via-delivery-of-cas9-ribonucleoprotein
#15
Mark DeWitt, Jacob E Corn, Dana Carroll
The CRISPR-Cas genome editing system is very powerful. The format of the CRISPR reagents and the means of delivery are often important factors in targeting efficiency. Delivery of recombinant Cas9 protein and guide RNA (gRNA) as a preformed ribonucleoprotein (RNP) complex has recently emerged as a powerful and general approach to genome editing. Here we outline methods to produce and deliver Cas9 RNPs. A donor DNA carrying desired sequence changes can also be included to program precise sequence introduction or replacement...
April 11, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28404480/2d-and-3d-fish-of-expanded-repeat-rnas-in-human-lymphoblasts
#16
Martyna O Urbanek, Michał Michalak, Wlodzimierz J Krzyzosiak
The first methods for visualizing RNAs within cells were designed for simple imaging of specific transcripts in cells or tissues and since then significant technical advances have been made in this field. Today, high-resolution images can be obtained, enabling visualization of single transcript molecules, quantitative analyses of images, and precise localization of RNAs within cells as well as co-localization of transcripts with specific proteins or other molecules. In addition, tracking of RNA dynamics within single cell has become possible...
April 9, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28392263/precision-genome-editing-using-crispr-cas9-and-linear-repair-templates-in-c-elegans
#17
Alexandre Paix, Andrew Folkmann, Geraldine Seydoux
The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates...
April 6, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28390838/super-resolution-microscopy-approaches-to-nuclear-nanostructure-imaging
#18
Christoph Cremer, Aleksander Szczurek, Florian Schock, Amine Gourram, Udo Birk
The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca...
April 5, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28385536/mining-the-topography-and-dynamics-of-the-4d-nucleome-to-identify-novel-cns-drug-pathways
#19
Gerald A Higgins, Ari Allyn-Feuer, Patrick Georgoff, Vahagn Nikolian, Hasan Alam, Brian D Athey
The pharmacoepigenome can be defined as the active, noncoding province of the genome including canonical spatial and temporal regulatory mechanisms of gene regulation that respond to xenobiotic stimuli. Many psychotropic drugs that have been in clinical use for decades have ill-defined mechanisms of action that are beginning to be resolved as we understand the transcriptional hierarchy and dynamics of the nucleus. In this review, we describe spatial, temporal and biomechanical mechanisms mediated by psychotropic medications...
April 3, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28366666/a-high-content-microscopy-assay-to-determine-drug-activity-against-intracellular-mycobacterium-tuberculosis
#20
Alyssa J Manning, Yulia Ovechkina, Amanda McGillivray, Lindsay Flint, David M Roberts, Tanya Parish
Tuberculosis is one of the infectious diseases with the greatest global burden, affecting millions of people. The rise of multi- and extensively-drug resistant forms of Mycobacterium tuberculosis over the last few decades has highlighted the urgent need for development of new drugs to treat the disease. Many drug development pipelines are based on in vitro assays examining a compound's effect on M. tuberculosis alone. These do not account for the effect of a compound on mammalian cells nor the interaction between host and pathogen...
March 31, 2017: Methods: a Companion to Methods in Enzymology
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