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https://www.readbyqxmd.com/read/29337374/pooled-lentiviral-delivery-genetic-screens
#1
Federica Piccioni, Scott T Younger, David E Root
Pooled cell-based screens of mammalian genetic perturbations enable systematic large-scale, even genome-scale, evaluation of gene function. Pooled screens introduce genetic perturbations into a cell population through viral transduction such that each cell integrates into its DNA a single or small number of library perturbations with barcodes identifying the perturbations. One then selects and physically isolates the subset of cells that exhibit the phenotype of interest. Sequencing the barcodes in the hit cells reveals which genes favored or inhibited the hit phenotype...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337373/making-use-of-cancer-genomic-databases
#2
Chad J Creighton
The vast amounts of genomic data now deposited in public repositories represent rich resources for cancer researchers. Large-scale genomics initiatives such as The Cancer Genome Atlas have made available data from multiple molecular profiling platforms (e.g., somatic mutation, RNA and protein expression, and DNA methylation) for the same set of over 10,000 human tumors. There has been much collective effort toward providing user-friendly software tools for biologists lacking computational skills to ask questions of large-scale genomic datasets...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337370/modulating-gene-expression-in-epstein-barr-virus-ebv-positive-b-cell-lines-with-crispra-and-crispri
#3
Liang Wei Wang, Stephen J Trudeau, Chong Wang, Catherine Gerdt, Sizun Jiang, Bo Zhao, Benjamin E Gewurz
Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29315667/collision-free-poisson-motion-planning-in-ultra-high-dimensional-molecular-conformation-spaces
#4
Rasmus Fonseca, Dominik Budday, Henry van den Bedem
The function of protein, RNA, and DNA is modulated by fast, dynamic exchanges between three-dimensional conformations. Conformational sampling of biomolecules with exact and nullspace inverse kinematics, using rotatable bonds as revolute joints and noncovalent interactions as holonomic constraints, can accurately characterize these native ensembles. However, sampling biomolecules remains challenging owing to their ultra-high dimensional configuration spaces, and the requirement to avoid (self-) collisions, which results in low acceptance rates...
January 5, 2018: Journal of Computational Chemistry
https://www.readbyqxmd.com/read/29286106/integrative-analysis-of-long-non-coding-rnas-and-messenger%C3%A2-rna-expression-profiles-in-systemic-lupus-erythematosus
#5
Qing Luo, Xue Li, Chuxin Xu, Lulu Zeng, Jianqing Ye, Yang Guo, Zikun Huang, Junming Li
Thousands of long noncoding RNAs (lncRNAs) have been reported and represent an important subset of pervasive genes associated with a broad range of biological functions. Abnormal expression levels of lncRNAs have been demonstrated in multiple types of human disease. However, the role of lncRNAs in systemic lupus erythematosus (SLE) remains poorly understood. In the present study, the expression patterns of lncRNAs and messenger RNAs (mRNAs) were investigated in peripheral blood mononuclear cells (PBMCs) in SLE using Human lncRNA Array v3...
December 22, 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/29208530/analytical-performances-of-simultaneous-detection-of-hiv-1-hiv-2-and-hepatitis-c-specific-antibodies-and-hepatitis-b-surface-antigen-hbsag-by-multiplex-immunochromatographic-rapid-test-with-serum-samples-a-cross-sectional-study
#6
Leman Robin, Ralph-Sydney Mboumba Bouassa, Zita Aleyo Nodjikouambaye, Laura Charmant, Mathieu Matta, Sylvie Simon, Mounir Filali, Souleymane Mboup, Laurent Bélec
BACKGROUND: The HIV/HCV/HBsAg Triplex consists in manually performed, visually interpreted, lateral flow, immunochromatographic rapid diagnostic test simultaneously detecting in 15min human immunodeficiency virus (HIV)-1 and HIV-2 and hepatitis C virus (HCV)- specific antibodies (Ab) (IgG and IgM) and hepatitis B virus (HBV) surface antigen (HBsAg) in serum, plasma and whole blood. METHODS: A hospital-based cross-sectional study was conducted on a prospective panel of serum samples from adult inpatients included from routine analysis irrespectively of age and sex, including 250 sera positive for HIV-1-specific Ab, 250 for HCV-specific Ab, 250 for HBsAg and 250 sera negative for HIV- and HCV- Ab and HBsAg, and from 110 HIV-2-infected patients living in Ivory Coast, according to the results obtained by the reference chemiluminiscent microparticle immunoassay (CMIA) Abbott Architect i2000SR analyzer (Abbott Diagnostic, Chicago, IL, USA)...
December 5, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/29205500/a-functional-snp-in-the-3-utr-of-tap2-gene-interacts-with-microrna-hsa-mir-1270-to-suppress-the-gene-expression
#7
Bridgett Knox, Yong Wang, Lora J Rogers, Jiekun Xuan, Dianke Yu, Huaijin Guan, Jiwei Chen, Tieliu Shi, Baitang Ning, Susan A Kadlubar
The transporter associated with antigen processing 2 (TAP2) is involved in the development of multidrug resistance and the etiology of immunological diseases. In this study, we investigated whether the expression of TAP2 can be perturbed by single nucleotide polymorphisms (SNPs) located in 3'-untranslated region (3'-UTR) of the gene via interactions with microRNAs. Using a series of in silico assays, we selected the candidate microRNAs (miRNAs) with the potential to interact with functional SNPs of TAP2. The SNP rs241456-located in the 3'-UTR of TAP2-resides in a potential binding site for hsa-miR-1270 and hsa-miR-620...
December 5, 2017: Environmental and Molecular Mutagenesis
https://www.readbyqxmd.com/read/29170628/targeted-genetic-screen-in-amyotrophic-lateral-sclerosis-reveals-novel-genetic-variants-with-synergistic-effect-on-clinical-phenotype
#8
Johnathan Cooper-Knock, Henry Robins, Isabell Niedermoser, Matthew Wyles, Paul R Heath, Adrian Higginbottom, Theresa Walsh, Mbombe Kazoka, Paul G Ince, Guillaume M Hautbergue, Christopher J McDermott, Janine Kirby, Pamela J Shaw
Amyotrophic lateral sclerosis (ALS) is underpinned by an oligogenic rare variant architecture. Identified genetic variants of ALS include RNA-binding proteins containing prion-like domains (PrLDs). We hypothesized that screening genes encoding additional similar proteins will yield novel genetic causes of ALS. The most common genetic variant of ALS patients is a G4C2-repeat expansion within C9ORF72. We have shown that G4C2-repeat RNA sequesters RNA-binding proteins. A logical consequence of this is that loss-of-function mutations in G4C2-binding partners might contribute to ALS pathogenesis independently of and/or synergistically with C9ORF72 expansions...
2017: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/29164646/b-factor-profile-prediction-for-rna-flexibility-using-support-vector-machines
#9
Ivantha Guruge, Ghazaleh Taherzadeh, Jian Zhan, Yaoqi Zhou, Yuedong Yang
Determining the flexibility of structured biomolecules is important for understanding their biological functions. One quantitative measurement of flexibility is the atomic Debye-Waller factor or temperature B-factor. Most existing studies are limited to temperature B-factors of proteins and their prediction. Only one method attempted to predict temperature B-factors of ribosomal RNA. Here, we developed and compared machine-learning techniques in prediction of temperature B-factors of RNAs. The best model based on Support Vector Machines yields Pearson's correction coefficient at 0...
November 21, 2017: Journal of Computational Chemistry
https://www.readbyqxmd.com/read/28967997/a-detailed-protocol-for-subcellular-rna-sequencing-subrna-seq
#10
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
#11
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
#12
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
#13
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
#14
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
#15
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...
November 2017: Lancet HIV
https://www.readbyqxmd.com/read/28902396/data-analysis-pipeline-for-rna-seq-experiments-from-differential-expression-to-cryptic-splicing
#16
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
#17
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
#18
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...
November 4, 2017: Lancet
https://www.readbyqxmd.com/read/28862343/cellular-detection-of-g-quadruplexes-by-optical-imaging-methods
#19
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
#20
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
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