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https://www.readbyqxmd.com/read/28967997/a-detailed-protocol-for-subcellular-rna-sequencing-subrna-seq
#1
Andreas Mayer, L Stirling Churchman
In eukaryotic cells, RNAs at various maturation and processing levels are distributed across cellular compartments. The standard approach to determine transcript abundance and identity in vivo is RNA sequencing (RNA-seq). RNA-seq relies on RNA isolation from whole-cell lysates and thus mainly captures fully processed, stable, and more abundant cytoplasmic RNAs over nascent, unstable, and nuclear RNAs. Here, we provide a step-by-step protocol for subcellular RNA-seq (subRNA-seq). subRNA-seq allows the quantitative measurement of RNA polymerase II-generated RNAs from the chromatin, nucleoplasm, and cytoplasm of mammalian cells...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28967993/production-of-purified-casrnps-for-efficacious-genome-editing
#2
Emily Lingeman, Chris Jeans, Jacob E Corn
CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28921497/quantitative-analysis-of-nucleic-acid-stability-with-ligands-under-high-pressure-to-design-novel-drugs-targeting-g-quadruplexes
#3
Shuntaro Takahashi, Naoki Sugimoto
Nucleic acids (DNA and RNA) can form various non-canonical structures. Because some serious diseases are caused by the conformational change of G-quadruplex DNA structures, the development of ligands that bind and stabilize G-quadruplex DNA is of interest to the field of nucleic acid chemistry. Volumetric changes (ΔV) in the biomolecular reaction include the structural change of biomolecules and hydration behaviors, which provide information about the tertiary interaction between G-quadruplex DNA and ligands...
September 18, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28921495/probing-rna-folding-pathways-by-rna-fingerprinting
#4
Sarah A Woodson
This unit provides protocols for using native polyacrylamide gel electrophoresis to distinguish folding and unfolding conformers of RNA. It is useful for studying conformers that can exchange in a period of minutes or seconds, and that are thus difficult to study by solution-based methods. Conformers that have been separated and immobilized in the gel matrix can be used to study catalytic activity with or without being eluted from the gel. The method can be applied to a wide variety of catalytic RNAs and RNA-protein complexes...
September 18, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28921494/solid-phase-synthesis-of-rna-analogs-containing-phosphorodithioate-linkages
#5
Xianbin Yang
The oligoribonucleotide phosphorodithioate (PS2-RNA) modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphorodiester backbone linkage. Like a natural phosphodiester RNA backbone linkage, a PS2-modified backbone linkage is achiral at phosphorus. PS2-RNAs are highly stable to nucleases and several in vitro assays have demonstrated their biological activity. For example, PS2-RNAs silenced mRNA in vitro and bound to protein targets in the form of PS2-aptamers (thioaptamers)...
September 18, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28918877/raltegravir-1200-mg-once-daily-versus-raltegravir-400-mg-twice-daily-with-tenofovir-disoproxil-fumarate-and-emtricitabine-for-previously-untreated-hiv-1-infection-a-randomised-double-blind-parallel-group-phase-3-non-inferiority-trial
#6
Pedro Cahn, Richard Kaplan, Paul E Sax, Kathleen Squires, Jean-Michel Molina, Anchalee Avihingsanon, Winai Ratanasuwan, Evelyn Rojas, Mohammed Rassool, Mark Bloch, Linos Vandekerckhove, Peter Ruane, Yazdan Yazdanpanah, Christine Katlama, Xia Xu, Anthony Rodgers, Lilly East, Larissa Wenning, Sandy Rawlins, Brenda Homony, Peter Sklar, Bach-Yen Nguyen, Randi Leavitt, Hedy Teppler
BACKGROUND: Once daily regimens are preferred for HIV-1 treatment, to facilitate adherence and improve quality of life. We compared a new once daily formulation of raltegravir to the currently marketed twice daily formulation. METHODS: In this randomised, double-blind, parallel-group, phase 3, non-inferiority study, we enrolled participants aged 18 years or older with HIV-1 RNA of 1000 or more copies per mL and no previous antiretroviral treatment at 139 sites worldwide...
September 11, 2017: Lancet HIV
https://www.readbyqxmd.com/read/28902396/data-analysis-pipeline-for-rna-seq-experiments-from-differential-expression-to-cryptic-splicing
#7
Hari Krishna Yalamanchili, Ying-Wooi Wan, Zhandong Liu
RNA sequencing (RNA-seq) is a high-throughput technology that provides unique insights into the transcriptome. It has a wide variety of applications in quantifying genes/isoforms and in detecting non-coding RNA, alternative splicing, and splice junctions. It is extremely important to comprehend the entire transcriptome for a thorough understanding of the cellular system. Several RNA-seq analysis pipelines have been proposed to date. However, no single analysis pipeline can capture dynamics of the entire transcriptome...
September 13, 2017: Current Protocols in Bioinformatics
https://www.readbyqxmd.com/read/28884792/profiling-of-single-cell-transcriptomes
#8
Wanze Chen, Vincent Gardeux, Antonio Meireles-Filho, Bart Deplancke
Complex biological systems are composed of multiple cell types whose transcriptional activity can vary due to differences in cell state, environmental stimulation, or intrinsic programs. Conventional bulk analysis methods capture the average transcriptional programs of the cell population, thus missing the unique cellular signature of each single cell. In recent years, the development of single-cell RNA-sequencing (scRNA-seq) technologies has provided a powerful approach to dissect the cellular heterogeneity of complex biological systems...
September 8, 2017: Current Protocols in Mouse Biology
https://www.readbyqxmd.com/read/28867499/coformulated-bictegravir-emtricitabine-and-tenofovir-alafenamide-versus-dolutegravir-with-emtricitabine-and-tenofovir-alafenamide-for-initial-treatment-of-hiv-1-infection-gs-us-380-1490-a-randomised-double-blind-multicentre-phase-3-non-inferiority-trial
#9
Paul E Sax, Anton Pozniak, M Luisa Montes, Ellen Koenig, Edwin DeJesus, Hans-Jürgen Stellbrink, Andrea Antinori, Kimberly Workowski, Jihad Slim, Jacques Reynes, Will Garner, Joseph Custodio, Kirsten White, Devi SenGupta, Andrew Cheng, Erin Quirk
BACKGROUND: Integrase strand transfer inhibitors (INSTIs) coadministered with two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) are recommended as first-line treatment for HIV, and coformulated fixed-dose combinations are preferred to facilitate adherence. We report 48-week results from a study comparing initial HIV-1 treatment with bictegravir-a novel INSTI with a high in-vitro barrier to resistance and low potential as a perpetrator or victim of clinically relevant drug interactions-coformulated with the NRTI combination emtricitabine and tenofovir alafenamide as a fixed-dose combination to dolutegravir administered with coformulated emtricitabine and tenofovir alafenamide...
August 31, 2017: Lancet
https://www.readbyqxmd.com/read/28862343/cellular-detection-of-g-quadruplexes-by-optical-imaging-methods
#10
Souheila Amor, Sunny Y Yang, Judy M Y Wong, David Monchaud
G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration)...
September 1, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28802816/safety-and-efficacy-of-an-8-week-regimen-of-grazoprevir-plus-ruzasvir-plus-uprifosbuvir-compared-with-grazoprevir-plus-elbasvir-plus-uprifosbuvir-in-participants-without-cirrhosis-infected-with-hepatitis-c-virus-genotypes-1-2-or-3-c-crest-1-and-c-crest-2-part
#11
Edward J Gane, Stephen Pianko, Stuart K Roberts, Alexander J Thompson, Stefan Zeuzem, Eli Zuckerman, Ziv Ben-Ari, Graham R Foster, Kosh Agarwal, Alex L Laursen, Jan Gerstoft, Wei Gao, Hsueh-Cheng Huang, Brian Fitzgerald, Doreen Fernsler, Jerry J Li, Anjana Grandhi, Hong Liu, Feng-Hsiu Su, Shuyan Wan, Zhen Zeng, Huei-Ling Chen, Frank J Dutko, Bach-Yen T Nguyen, Janice Wahl, Michael N Robertson, Eliav Barr, Wendy W Yeh, Rebeca M Plank, Joan R Butterton, Rafael Esteban
BACKGROUND: New hepatitis C virus (HCV) therapies with pan-genotypic efficacy are needed. The goals of part A of C-CREST-1 and C-CREST-2 were to compare the efficacies of two doses (300 mg or 450 mg once daily) of uprifosbuvir (MK-3682; NS5B inhibitor) in an 8-week regimen combined with grazoprevir (NS3/4A inhibitor; 100 mg once daily) and an NS5A inhibitor, either elbasvir (50 mg once daily) or ruzasvir (MK-8408; 60 mg once daily), and to evaluate the safety and tolerability of these combination regimens in individuals infected with genotypes 1, 2, or 3...
November 2017: Lancet. Gastroenterology & Hepatology
https://www.readbyqxmd.com/read/28802814/safety-and-efficacy-of-a-fixed-dose-combination-regimen-of-grazoprevir-ruzasvir-and-uprifosbuvir-with-or-without-ribavirin-in-participants-with-and-without-cirrhosis-with-chronic-hepatitis-c-virus-genotype-1-2-or-3-infection-c-crest-1-and-c-crest-2-part-b-two
#12
Eric Lawitz, Maria Buti, John M Vierling, Piero L Almasio, Savino Bruno, Peter J Ruane, Tarek I Hassanein, Beat Muellhaupt, Brian Pearlman, Ligita Jancoriene, Wei Gao, Hsueh-Cheng Huang, Aimee Shepherd, Brynne Tannenbaum, Doreen Fernsler, Jerry J Li, Anjana Grandhi, Hong Liu, Feng-Hsiu Su, Shuyan Wan, Frank J Dutko, Bach-Yen T Nguyen, Janice Wahl, Michael N Robertson, Eliav Barr, Wendy W Yeh, Rebeca M Plank, Joan R Butterton, Eric M Yoshida
BACKGROUND: There is a need for hepatitis C virus (HCV) therapies with excellent efficacy across genotypes and in diverse populations. Part A of the C-CREST-1 and C-CREST-2 trials led to the selection of a three-drug regimen of grazoprevir (MK-5172; an HCV NS3/4A protease inhibitor; 100 mg/day) plus ruzasvir (MK-8408; an NS5A inhibitor; 60 mg/day) plus uprifosbuvir (MK-3682; an HCV NS5B polymerase inhibitor; 450 mg/day). Part B of the studies tested this combination as a single formulation in different treatment durations in a broader population...
November 2017: Lancet. Gastroenterology & Hepatology
https://www.readbyqxmd.com/read/28758359/aggregation-of-snd1-in-stress-granules-is-associated-with-the-microtubule-cytoskeleton-during-heat-shock-stimulus
#13
Jie Shao, Fei Gao, Bingbing Zhang, Meng Zhao, Yunli Zhou, Jinyan He, Li Ren, Zhi Yao, Jie Yang, Chao Su, Xingjie Gao
Stress granules (SGs) are dynamic dense structures in the cytoplasm that form in response to a variety of environmental stress stimuli. Staphylococcal nuclease and Tudor domain containing 1 (SND1) is a type of RNA-binding protein and has been identified as a transcriptional co-activator. Our previous studies have shown that SND1 is a component of the stress granule, which forms under stress conditions. Here, we observed that SND1 granules were often surrounded by ɑ-tubulin-microtubules in 45°C-treated HeLa cells at 15 min or colocalized with microtubules at 30 or 45 min...
July 31, 2017: Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology
https://www.readbyqxmd.com/read/28752682/impairment-of-retinoic-acid-signaling-in-cornelia-de-lange-syndrome-fibroblasts
#14
Grazia Fazio, Laura Rachele Bettini, Silvia Rigamonti, Dorela Meta, Andrea Biondi, Giovanni Cazzaniga, Angelo Selicorni, Valentina Massa
BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare genetic disorder affecting the neurodevelopment, gastrointestinal, musculoskeletal systems. CdLS is caused by mutations within NIPBL, SMC1A, SMC3, RAD21, and HDAC8 genes. These genes codify for the "cohesin complex" playing a role in chromatid adhesion, DNA repair and gene expression regulation. The aim of this study was to investigate retinoic acid (RA) signaling pathway, a master developmental regulator, in CdLS cells. METHODS: Skin biopsies from CdLS patients and healthy controls were cultured and derived primary fibroblast cells were treated with RA or dimethyl sulfoxide (vehicle)...
October 2, 2017: Birth defects research
https://www.readbyqxmd.com/read/28736996/phytochemical-investigation-antitumor-activity-and-hepatoprotective-effects-of-acrocarpus-fraxinifolius-leaf-extract
#15
Heba A S El-Nashar, Omayma A Eldahshan, Omama E Elshawi, Abdel Nasser B Singab
Preclinical Research Nine known phenolic compounds were isolated from an aqueous methanolic extract of Acrocarpus fraxinifolius Weight and Arn leaves (AFL) family Fabaceae. This extract of AFL contained approximately 169 mg gallic acid/g as assessed by HPLC. The AFL extract had marginal antitumor activity (IC50  > 200 µL/mL) but showed a concentration-dependent hepatoprotective effect against CCl4 -induced hepatotoxicity in vitro. Cell viability was increased, ALT and AST activity declined and reduced GSH concentration and SOD activity were restored as compared with silymarin...
July 24, 2017: Drug Development Research
https://www.readbyqxmd.com/read/28678443/mitochondrial-ribosome-mitoribosome-profiling-for-monitoring-mitochondrial-translation-in-vivo
#16
Mary T Couvillion, L Stirling Churchman
Translation in the mitochondria is regulated by mechanisms distinct from those acting in the cytosol and in bacteria, yet precise methods for investigating it have lagged behind. This unit describes an approach, mitochondrial ribosome (mitoribosome) profiling, to quantitatively monitor mitochondrial translation with high temporal and spatial resolution in Saccharomyces cerevisiae. Mitoribosomes are immunoprecipitated from whole-cell lysate and the protected mRNA fragments are isolated. These fragments are then converted to sequencing libraries or analyzed by northern blot hybridization to reveal the distribution of mitoribosomes across the mitochondrial transcriptome...
July 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28654728/finding-similar-nucleotide-sequences-using-network-blast-searches
#17
Istvan Ladunga
The Basic Local Alignment Search Tool (BLAST) is the first tool in the annotation of nucleotide or amino acid sequences. BLAST is a flagship of bioinformatics due to its performance and user-friendliness. Beginners and intermediate users will learn how to design and submit blastn and Megablast searches on the Web pages at the National Center for Biotechnology Information. We map nucleic acid sequences to genomes, find identical or similar mRNAs, expressed sequence tag, and noncoding RNA sequences, and run Megablast searches, which are much faster than blastn...
June 27, 2017: Current Protocols in Bioinformatics
https://www.readbyqxmd.com/read/28654727/using-the-noncode-database-resource
#18
Li Xiyuan, Bu Dechao, Sun Liang, Wu Yang, Fang Shuangsang, Li Hui, Luo Haitao, Luo Chunlong, Fang Wenzheng, Chen Runsheng, Zhao Yi
NONCODE is a comprehensive database that aims to present the most complete collection and annotation of non-coding RNAs, especially long non-coding RNAs (lncRNA genes), and thus NONCODE is essential to modern biological and medical research. Scientists are producing a flood of new data from which new lncRNA genes and lncRNA-disease relationships are continually being identified. NONCODE assimilates such information from a wide variety of sources including published articles, RNA-seq data, micro-array data and databases on genetic variation (dbSNP) and genome-wide associations (GWAS)...
June 27, 2017: Current Protocols in Bioinformatics
https://www.readbyqxmd.com/read/28639574/mir-551b-5p-contributes-to-pathogenesis-of-vein-graft-failure-via-upregulating-early-growth-response-1-expression
#19
Ran Dong, Kui Zhang, Yue-Li Wang, Feng Zhang, Jian Cao, Ju-Bing Zheng, Hong-Jia Zhang
BACKGROUND: Vein graft failure (VGF) is a serious complication of coronary artery bypass graft, although the mechanism remains unclear. The study aimed to investigate the effects of microRNAs (miRNAs) on the endothelial dysfunction involved in VGF. METHODS: Human umbilical vein endothelial cells (HUVECs) were subjected to mechanical stretch stimulation to induce endothelial dysfunction. Genome-wide transcriptome profiling was performed using the Human miRNA OneArray® V4 (PhalanxBio Inc...
July 5, 2017: Chinese Medical Journal
https://www.readbyqxmd.com/read/28628208/carbocyclic-c-c-bond-formation-intramolecular-radical-ring-closure-to-yield-diastereomerically-pure-7-s-me-or-7-r-me-carba-lna-nucleotide-analogs
#20
Oleksandr Plashkevych, Ram Shankar Upadhayaya, Jyoti Chattopadhyaya
In light of the impressive gene-silencing properties of carba-LNA modified oligo DNA and RNA, both in antisense RNA and siRNA approaches, which have been confirmed as proof-of-concept for biochemical applications in post-transcriptional gene silencing, we envision the true potential of carba-LNA modifications to be revealed soon. Herein we provide detailed protocols for synthesis of carba-LNA-A, -G, -(5-Me) C, and -T nucleosides on a medium/large scale (gram scale), as well as important guidelines for incorporation of these modified carba-LNAs into DNA or RNA oligonucleotides...
June 19, 2017: Current Protocols in Nucleic Acid Chemistry
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