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Methods: a Companion to Methods in Enzymology

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https://read.qxmd.com/read/30776405/real-time-monitoring-of-protein-induced-dna-conformational-changes-using-single-molecule-fret
#1
Leonard Schärfen, Michael Schlierf
Apart from being storage devices for genetic information, nucleic acids can provide regulatory structures through evolutionarily optimized sequences. The interaction of proteins binding specifically to such sequences and resulting secondary structures, or the exposure of single-stranded DNA add a versatile regulatory framework for cells. Biochemical and structural biology experiments have revealed important underlying concepts of protein-DNA interactions but are often limited by ensemble averaging or static information...
February 15, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30772464/deep-resp-forest-a-deep-forest-model-to-predict-anti-cancer-drug-response
#2
Ran Su, Xinyi Liu, Leyi Wei, Quan Zou
The identification of therapeutic biomarkers predictive of drug response is crucial in personalized medicine. A number of computational models to predict response of anti-cancer drugs have been developed as the establishment of several pharmacogenomics screening databases. In our study, we proposed a deep cascaded forest model, Deep-Resp-Forest, to classify the anti-cancer drug response as "sensitive" or "resistant". We made three contributions in this study. Firstly, diverse molecular data could be effectively integrated to provide more information than single type of data for the classification...
February 14, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30771491/use-and-reliability-of-multiplex-bead-based-assays-luminex-at-containment-level-4
#3
Stuart D Dowall, Victoria A Graham, Tom Fletcher, Roger Hewson
In the UK, research on hazard group 4 (HG4) pathogens requires specialised Containment Level 4 (CL4) facilities. These differ from Biosafety Level 4 (BSL4) conditions in that work is conducted in class III microbiological safety cabinets for primary containment instead of using positive pressure suits. This presents unique challenges associated with the physical restrictions of working in a limited space, and prohibits the use of many techniques and specialist equipment. In consequence, detailed studies on the biology of HG4 pathogens and in particular their immunological relationships with the host are understudied in the UK; for example, the majority of immunological assays with which the immune system is interrogated require specialist equipment that is unsuitable for CL4...
February 13, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30771490/computer-aided-detection-of-mass-in-digital-breast-tomosynthesis-using-a-faster-region-based-convolutional-neural-network
#4
Ming Fan, Yuanzhe Li, Shuo Zheng, Weijun Peng, Wei Tang, Lihua Li
Digital breast tomosynthesis (DBT) is a newly developed three-dimensional tomographic imaging modality in the field of breast cancer screening designed to alleviate the limitations of conventional digital mammography-based breast screening methods. A computer-aided detection (CAD) system was designed for masses in DBT using a faster region-based convolutional neural network (faster-RCNN). To this end, a data set was collected, including 89 patients with 105 masses. An efficient detection architecture of convolution neural network with a region proposal network (RPN) was used for each slice to generate region proposals (i...
February 13, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30769100/analysis-of-rna-polymerase-ii-ubiquitylation-and-proteasomal-degradation
#5
Ana Tufegdzic Vidakovic, Michelle Harreman, A Barbara Dirac-Svejstrup, Stefan Boeing, Anindya Roy, Vesela Encheva, Michelle Neumann, Marcus Wilson, Ambrosius P Snijders, Jesper Q Svejstrup
Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation...
February 12, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30743000/use-of-the-nuclear-walk-on-methodology-to-determine-sites-of-rna-polymerase-ii-initiation-and-pausing-and-quantify-nascent-rnas-in-cells
#6
REVIEW
Christopher B Ball, Kyle A Nilson, David H Price
Transcription by RNA polymerase II (Pol II) is controlled during initiation, elongation, and termination by a large variety of transcription factors, the state of chromatin modifications, and environmental conditions. Herein we describe experimental approaches for the examination of Pol II transcription at semi-global and genome-wide scales through analysis of nascent Pol II transcripts. We begin with a description of the nuclear walk-on (NWO) assay, which involves rapid isolation of nuclei in the presence of EDTA, followed by extension of about a quarter of the nascent transcripts with 32 P-CTP...
February 8, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30742999/tracking-of-single-trnas-for-translation-kinetics-measurements-in-chloramphenicol-treated-bacteria
#7
Ivan L Volkov, A Carolin Seefeldt, Magnus Johansson
Chloramphenicol is a broad-spectrum antibiotic targeting the protein synthesis machinery by binding to the bacterial ribosome. Chloramphenicol has been considered a classic general inhibitor of translation, blocking the accommodation of aa-tRNA into the A site of the large ribosomal subunit. However, recent studies suggest that this proposed mechanism is a simplification and that the effect of chloramphenicol on mRNA translation is much more dynamic. By tracking single dye-labelled elongator and initiator tRNAs in Escherichia coli cells treated with chloramphenicol, we observe the direct effect of chloramphenicol on translation kinetics...
February 8, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30742998/genome-wide-analysis-of-rna-and-protein-localization-and-local-translation-in-mesc-derived-neurons
#8
Katarzyna A Ludwik, Nicolai von Kuegelgen, Marina Chekulaeva
The subcellular localization and translation of mRNAs are fundamental biological processes. In neurons, they underlie cell growth and synaptic plasticity, which serves as a foundation of learning and memory. Multiple approaches have been developed to separate neurons on subcellular compartments - cell bodies (soma) and cell extensions (axons and dendrites) - for further biochemical analyses. Here we describe neurite/soma separation approach in combination with RNA sequencing and proteomic analyses to identify localized and locally translated RNAs and proteins...
February 8, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30742997/method-to-quantify-cytokines-and-chemokines-in-mouse-brain-tissue-using-bio-plex-multiplex-immunoassays
#9
Monica Manglani, Rejane Rua, Amy Hendricksen, Daniel Braunschweig, Qian Gao, Woei Tan, Brett Houser, Dorian B McGavern, Kenneth Oh
This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample. This assay has been used extensively to investigate analytes in plasma and serum samples as well as cultured and primary cells...
February 8, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30742996/multiplexing-protein-and-gene-level-measurements-on-a-single-luminex-platform
#10
Damon B Cook, Brian C McLucas, Leticia A Montoya, Chris M Brotski, Shelley Das, Markus Miholits, Thao H Sebata
BACKGROUND: The ability to simultaneously measure multiple secreted proteins and the corresponding gene expression levels from a single sample is valuable for comprehensive analysis. Bottlenecks to traditional immunoassays and gene expression assays include large sample consumption, time consuming experimental procedures, and complex data analysis. METHOD AND RESULTS: Here, we demonstrate two high-throughput assays measuring both messenger RNA (mRNA) expression and proteins in a single sample run on a Luminex platform...
February 8, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30742995/deriving-a-sub-nanomolar-affinity-peptide-from-tap-to-enable-smfret-analysis-of-rna-polymerase-ii-complexes
#11
Jheng-Syong Wu, Tzu-Yun Chen, Sam Song-Yao Lin, Shu-Yu Lin, Cheng-Yu Hung, I-Ping Tu, Hung-Ta Chen, Wei-Hau Chang
Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modified the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and used dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex...
February 8, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30738128/applications-of-high-throughput-sequencing-to-analyze-and-engineer-ribozymes
#12
REVIEW
Yohei Yokobayashi
A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such as regulation of gene expression and viral replication, but biological roles of many ribozymes still remain unknown. Ribozymes have also inspired researchers to engineer synthetic ribozymes that function as sensors or gene switches. To gain deeper understanding of the sequence-function relationship of ribozymes and to efficiently engineer synthetic ribozymes, a large number of ribozyme variants need to be examined which was limited to hundreds of sequences by Sanger sequencing...
February 6, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30716396/measuring-rna-polymerase-activity-genome-wide-with-high-resolution-run-on-based-methods
#13
REVIEW
Antonio Jordán-Pla, Maria E Pérez-Martínez, José E Pérez-Ortín
The biogenesis of RNAs is a multi-layered and highly regulated process that involves a diverse set of players acting in an orchestrated manner throughout the transcription cycle. Transcription initiation, elongation and termination factors act on RNA polymerases to modulate their movement along the DNA template in a very precise manner, more complex than previously anticipated. Genome-scale run-on-based methodologies have been developed to study in detail the position of transcriptionally-engaged RNA polymerases...
February 1, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30707952/time-resolved-analysis-of-transcription-through-chromatin
#14
Han-Wen Chang, Fu-Kai Hsieh, Smita S Patel, Vasily M Studitsky
During transcription along nucleosomal DNA, RNA polymerase II (Pol II) pauses at multiple positions and induces formation of multiple intermediates that aid in maintaining proper chromatin structure. To describe the kinetics of this multiple-step reaction, we utilized a computational model-based approach and KinTek Explorer software to analyze the time courses. Here we describe the stepwise protocol for analysis of the kinetics of transcription through a nucleosome that provides the rate constants for each step of this complex process...
January 29, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30707951/the-development-of-a-multiplex-serological-assay-for-avian-influenza-based-on-luminex-technology
#15
E A Evelien Germeraad, R P René Achterberg, S Sandra Venema, J Jacob Post, O Olav de Leeuw, G Guus Koch, F J Fimme Jan van der Wal, N Nancy Beerens
Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Traditional serological methods for the subtyping of AI antibodies are labour-intensive and have to be performed for each HA and NA subtype separately. This study describes the development of a multiplex serological assay for subtyping AI antibodies in poultry sera using Luminex xMAP technology...
January 29, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30707950/a-universal-method-for-the-functionalization-of-dyed-magnetic-microspheres-with-peptides
#16
Matthew B Coppock, Dimitra N Stratis-Cullum
The need for the functionalization of magnetic, water-soluble dyed microspheres with peptides is apparent with the ever-growing biointeraction capabilities and the increased use of dyed microspheres in multiplex, microsphere-based detection assays. This method describes the attachment of any peptide to dyed magnetic microspheres regardless of peptide length, size, or sequence. The method exploits 'click' chemistry with short reaction times in a mixed organic/water system for simultaneous selective surface functionalization and reduction of microsphere dye leaching...
January 29, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30703462/validation-of-monoplex-assays-detecting-antibodies-against-corynebacterium-diphtheriae-and-clostridium-tetani-toxins-rubella-virus-and-parvovirus-b19-for-incorporation-into-multiplex-serology
#17
Nicole Brenner, Julia Butt, Izaura Lima Bomfim, Julia Tabatabai, Michael Pawlita, Paul Schnitzler, Tim Waterboer
Serological assays detecting antibodies in serum or plasma samples are useful and versatile instruments to investigate an individual's infection and vaccination history, e.g. for clinical diagnosis, personal risk evaluation, and seroepidemiological studies. Multiplex Serology is a suspension bead array-based high-throughput methodology for simultaneous measurement of antibodies against multiple pathogens in a single reaction vessel, thus economizing sample volume, measurement time, and costs. We developed and validated bead-based pathogen-specific Monoplex Serology assays, i...
January 28, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30690077/development-and-validation-of-magnetic-bead-pentaplex-immunoassay-for-simultaneous-quantification-of-murine-serum-igg-antibodies-to-acellular-pertussis-diphtheria-and-tetanus-antigens-used-in-combination-vaccines
#18
Laxmikant Kadam, Krunal Patel, Manish Gautam, Shrikant Thorat, Prathamesh Kale, Arvind Kumar Ghule, Harish Rao, Yojana Shinde, Umesh Shaligram, Sunil Gairola
We describe here a magnetic bead-based multiplex (pentaplex) immunoassay (MIA) platform developed as an alternative to enzyme-linked immunosorbent assays (ELISA) used in immunogenicity testing of DTaP/TdaP vaccine in animals. MIA simultaneously measures the concentration of serum (IgG) antibodies against B. Pertussis antigens; pertussis toxin, , filamentous hemagglutinin (FHA), pertactin (PRN) and tetanus (T) and diphtheria (D) toxoid in the Tdap vaccine immunized animals. Assay validation experiments were done using a panel of serum samples...
January 25, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30684537/genetic-analysis-of-the-rna-polymerase-ii-ctd-in-drosophila
#19
Feiyue Lu, David S Gilmour
The Carboxy-terminal Domain (CTD) of RNA polymerase II (Pol II) plays essential roles in regulating gene expression in eukaryotes. Here, we describe multiple genetic approaches for studying the CTD in Drosophila that complement pre-existing molecular analyses of the Pol II CTD in other experimental models. These approaches will allow one to assess the effects of any CTD mutations in a developmentally complex organism. The approaches discussed in this work can in principle, be applied to analyze other transcription components in eukaryotes...
January 23, 2019: Methods: a Companion to Methods in Enzymology
https://read.qxmd.com/read/30684536/biochemical-methods-to-characterize-rna-polymerase-ii-elongation-complexes
#20
J Brooks Crickard, Joseph C Reese
Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins...
January 23, 2019: Methods: a Companion to Methods in Enzymology
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