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Te-Sha Tsai, Sonika Tyagi, Justin C St John
STUDY QUESTION: What are the molecular differences between mitochondrial DNA (mtDNA)-deficient and mtDNA-normal oocytes and how does mitochondrial supplementation alter these? SUMMARY ANSWER: Changes to DNA methylation in a 5' cytosine-phosphate-guanine 3' (CpG) island in the mtDNA-specific replication factor (DNA polymerase gamma (POLG)) of mtDNA-deficient oocytes mediates an increase in mtDNA copy number by the 2-cell stage that positively modulates the expression of nuclear genes, which affect cellular and metabolic processes, following autologous mitochondrial supplementation...
March 13, 2018: Human Reproduction
Keyun Wang, Mingliang Ye
Protein methylation is receiving increasing attention for its important role in regulating diverse biological processes, including epigenetic regulation of gene transcription, RNA processing, DNA damage repair, and signal transduction. Proteome level analysis of protein methylation requires the enrichment of various forms of methylated peptides. Unfortunately, immunoaffinity purification can only enrich a subset of them due to the lack of pan-specific antibodies. Chromatography-based methods, however, can enrich methylated peptides in a global manner...
February 21, 2018: Current Protocols in Protein Science
Ekaterina G Viktorova, Sunil Khattar, Siba Samal, George A Belov
Poliovirus is a prototype member of the Enterovirus genus of the Picornaviridae family of small positive strand RNA viruses, which include important human and animal pathogens. Quantitative assessment of viral replication is very important for investigation of the virus biology and the development of anti-viral strategies. The poliovirus genome structure allows replacement of structural genes with a reporter protein, such as a luciferase or a fluorescent protein, whose signals can be detected and quantified in vivo, thus permitting observation of replication kinetics in live cells...
February 22, 2018: Current Protocols in Microbiology
Zexu Ma, Liping Yang, Yan-Jin Zhang
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirale. PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a genome around 15 kb in length. For propagation of PRRSV in vitro, the MARC-145 cell line is the most often used in a laboratory setting. Infectious cDNA clones of many PRRSV strains have been established, from which these viruses can be recovered. PRRSV titration is generally done in MARC-145 cells. PRRSV RNA copy numbers can be assessed by reverse transcription and real-time PCR...
February 22, 2018: Current Protocols in Microbiology
Alan Winston, Frank A Post, Edwin DeJesus, Daniel Podzamczer, Giovanni Di Perri, Vicente Estrada, François Raffi, Peter Ruane, Paula Peyrani, Gordon Crofoot, Patrick W G Mallon, Francesco Castelli, Mingjin Yan, Stephanie Cox, Moupali Das, Andrew Cheng, Martin S Rhee
BACKGROUND: Abacavir and tenofovir alafenamide offer reduced bone toxicity compared with tenofovir disoproxil fumarate. We aimed to compare safety and efficacy of tenofovir alafenamide plus emtricitabine with that of abacavir plus lamivudine. METHODS: In this randomised, double-blind, active-controlled, non-inferiority phase 3 trial, HIV-1-positive adults (≥18 years) were screened at 79 sites in 11 countries in North America and Europe. Eligible participants were virologically suppressed (HIV-1 RNA <50 copies per mL) and on a stable three-drug regimen containing abacavir plus lamivudine...
February 20, 2018: Lancet HIV
Alina Franzen, Timo J Vogt, Tim Müller, Jörn Dietrich, Andreas Schröck, Carsten Golletz, Peter Brossart, Friedrich Bootz, Jennifer Landsberg, Glen Kristiansen, Dimo Dietrich
Background: DNA methylation of the immune checkpoint gene PD-L1 has recently been shown to be associated with PD-L1 mRNA expression in various malignancies. This study aimed to investigate the association of PD-L1 and PD-L2 methylation with mRNA expression, immune cell infitration, protein expression and human papilloma virus (HPV) infection in head and neck squamous cell carcinoma (HNSCC) patients. Results: DNA methylation of PD-L1 and PD-L2 correlates inversely with mRNA expression ( PD-L1 : p ≤ 0...
January 2, 2018: Oncotarget
Qing-Lan Lyu, Bi-Mei Jiang, Bin Zhou, Li Sun, Zhong-Yi Tong, Yuan-Bin Li, Yu-Ting Tang, Hui Sun, Mei-Dong Liu, Xian-Zhong Xiao
BACKGROUND: Nucleolin (NCL) is the most abundant RNA-binding protein in the cell nucleolus and plays an important role in chromatin stability, ribosome assembly, ribosomal RNA maturation, ribosomal DNA transcription, nucleocytoplasmic transport, and regulation of RNA stability and translation efficiency. In addition to its anti-apoptotic properties, the underlying mechanisms associated with NCL-related roles in different cellular processes remain unclear. In this study, the effect of NCL on microRNA (miRNA) expression was evaluated by generating transgenic mice with myocardial overexpression of NCL and by analyzing microarrays of mature and precursor miRNAs from mice...
February 5, 2018: Chinese Medical Journal
W Scott Young, June Song, Éva Mezey
This unit presents protocols to locate RNA transcripts in tissues. Numerous approaches are detailed, including those that use radiolabeled or colorimetric probes. Also, the probes may be modified oligodeoxynucleotides, singly or in pairs, as well as ribonucleic acids. High sensitivity and specificity are obtained, especially with sets of oligodeoxynucleotide pairs. © 2018 by John Wiley & Sons, Inc.
January 22, 2018: Current Protocols in Neuroscience
Maja-Lisa Clausen, Tove Agner, Berit Lilje, Sofie M Edslev, Thor Bech Johannesen, Paal Skytt Andersen
Importance: Skin microbiome correlates with disease severity for lesional and nonlesional skin, indicating a global influence of atopic dermatitis (AD). A relation between skin microbiome and filaggrin gene (FLG) mutations proposes a possible association between skin microbiome and host genetics. Objectives: To assess skin and nasal microbiome diversity and composition in patients with AD and compare with healthy controls, and to investigate the microbiome in relation to disease severity and FLG mutations in patients with AD...
January 17, 2018: JAMA Dermatology
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
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
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
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
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
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)...
March 2018: Journal of Virological Methods
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...
March 2018: Environmental and Molecular Mutagenesis
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
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
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
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
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