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https://www.readbyqxmd.com/read/28641402/ec-tagging-allows-cell-type-specific-rna-analysis
#1
Naoki Hida, Mohamed Y Aboukilila, Dana A Burow, Rakesh Paul, Marc M Greenberg, Michael Fazio, Samantha Beasley, Robert C Spitale, Michael D Cleary
Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT...
June 21, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28639207/the-cell-biology-of-the-thyroid-disrupting-mechanism-of-dichlorodiphenyltrichloroethane-ddt
#2
REVIEW
M Rossi, A R Taddei, I Fasciani, R Maggio, F Giorgi
OBJECTIVE: Dichlorodiphenyltrichloroethane (DDT) is an organochlorine known for its pesticide properties and for its negative effects on human health. It was banned in most countries for its toxicity to the endocrine system, but due to its persistence at clinically relevant concentrations in both soil and animal tissues, DDT is still linked to several health and social problems. METHODS: We have previously shown that DDT exposure is causally related to the extracellular release of vesicular organelles such as microvesicles and/or exosomes by using immunocytochemistry with gold-tagged antibodies and various fluorescent membrane markers...
June 21, 2017: Journal of Endocrinological Investigation
https://www.readbyqxmd.com/read/28637755/seasonal-h3n2-and-2009-pandemic-h1n1-influenza-a-viruses-reassort-efficiently-but-produce-attenuated-progeny
#3
Kara L Phipps, Nicolle Marshall, Hui Tao, Shamika Danzy, Nina Onuoha, John Steel, Anice C Lowen
Reassortment of gene segments between co-infecting influenza A viruses (IAV) facilitates viral diversification and has significant epidemiological impact on seasonal and pandemic influenza. Since 1977, human IAVs of H1N1 and H3N2 subtypes have co-circulated with relatively few documented cases of reassortment. We evaluated the potential for viruses of the 2009 pandemic H1N1 (pH1N1) and seasonal H3N2 lineages to reassort under experimental conditions. Results of heterologous co-infections with pH1N1 and H3N2 viruses were compared to those obtained following co-infection with homologous, genetically tagged, pH1N1 viruses as a control...
June 21, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28635594/translation-repression-via-modulation-of-the-cytoplasmic-poly-a-binding-protein-in-the-inflammatory-response
#4
Xu Zhang, Xiaoli Chen, Qiuying Liu, Shaojie Zhang, Wenqian Hu
Gene expression is precisely regulated during the inflammatory response to control infection and limit the detrimental effects of inflammation. Here, we profiled global mRNA translation dynamics in the mouse primary macrophage-mediated inflammatory response and identified hundreds of differentially translated mRNAs. These mRNAs' 3'UTRs have enriched binding motifs for several RNA-binding proteins, which implies extensive translational regulatory networks. We characterized one such protein, Zfp36, as a translation repressor...
June 21, 2017: ELife
https://www.readbyqxmd.com/read/28629934/recent-advances-in-leishmania-reverse-genetics-manipulating-a-manipulative-parasite
#5
REVIEW
Samuel M Duncan, Nathaniel G Jones, Jeremy C Mottram
In this review we describe the expanding repertoire of molecular tools with which to study gene function in Leishmania. Specifically we review the tools available for studying functions of essential genes, such as plasmid shuffle and DiCre, as well as the rapidly expanding portfolio of available CRISPR/Cas9 approaches for large scale gene knockout and endogenous tagging. We include detail on approaches that allow the direct manipulation of RNA using RNAi and protein levels via Tet or DiCre induced overexpression and destabilization domain mediated degradation...
June 16, 2017: Molecular and Biochemical Parasitology
https://www.readbyqxmd.com/read/28628203/photoactivated-in-vivo-proximity-labeling
#6
David B Beck, Roberto Bonasio
Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28627442/the-association-of-rs710886-in-lncrna-pcat1-with-bladder-cancer-risk-in-a-chinese-population
#7
Yadi Lin, Yuqiu Ge, Yunyan Wang, Gaoxiang Ma, Xiaowei Wang, Hanting Liu, Meilin Wang, Zhengdong Zhang, Haiyan Chu
OBJECTIVE: The long noncoding RNA PCAT1 is an important gene involved in urinary tumors. In this study, we aimed to explore the association between polymorphisms in PCAT1 and bladder cancer susceptibility. METHODS: A two-stage case-control study was conducted to assess the association between four tagging SNPs (i.e., rs4871771, rs1902432, rs16901904 and rs710886) and bladder cancer risk. Odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated with unconditional univariate and multivariate logistic regression...
June 13, 2017: Gene
https://www.readbyqxmd.com/read/28623314/comparative-transcriptomic-and-proteomic-analyses-provide-insights-into-the-key-genes-involved-in-high-altitude-adaptation-in-the-tibetan-pig
#8
Bo Zhang, Yangzom Chamba, Peng Shang, Zhixiu Wang, Jun Ma, Liyuang Wang, Hao Zhang
Tibetan pigs that inhabit the Tibetan Plateau exhibit striking phenotypic and physiological differences from lowland pigs, and have adapted well to extreme conditions. However, the mechanisms involved in regulating gene expression at high altitude in these animals are not fully understood. In this study, we obtained transcriptomic and proteomic data from the heart tissues of Tibetan and Yorkshire pigs raised in the highlands (TH and YH) and lowlands (TL and YL) via RNA-seq and iTRAQ (isobaric tags for relative and absolute quantitation) analyses, respectively...
June 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28622431/loss-of-cdkc-2-increases-both-cell-division-and-drought-tolerance-in-arabidopsis-thaliana
#9
Lina Zhao, Yaqiong Li, Qi Xie, Yaorong Wu
Drought stress is one of abiotic stresses limiting plant growth and agricultural productivity. To further understand the mechanism of drought tolerance and identify the genes involved in this process, a genetic screen for altered drought response was conducted in Arabidopsis. One mutant with enhanced drought tolerance was isolated and named atdtm1 (Arabidopsis drought tolerance mutant 1), which has larger lateral organs, prolonged growth duration, increased relative water content and a reduced leaf stomatal density compared with the wild type...
June 16, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/28621907/role-of-cleavage-and-polyadenylation-specificity-factor-100-anchoring-poly-a-sites-and-modulating-transcription-termination
#10
Juncheng Lin, Ruqiang Xu, Xiaohui Wu, Yingjia Shen, Qingshun Q Li
CPSF100 is a core component of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex for the 3'-end formation of mRNA, but it still has no clear functional assignment. CPSF100 was reported to play a role in RNA silencing and promote flowering in Arabidopsis. However, the molecular mechanisms underlying these phenomena are not fully understood. Our genetics analyses indicate that plants with a hypomorphic mutant of CPSF100 (esp5) show defects in embryogenesis, reduced seed production, or altered root morphology...
June 16, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/28615101/-eukaryotic-expression-and-localization-of-human-mterf2-in-cervical-cancer-caski-cells
#11
Meitao Sun, Chenggui Zhang, Wen Mei, Yongqin Yang, Zefang Yang, Xiaojuan Zhang, Wei Xiong
Objective To construct the recombinant eukaryotic expression vector of human mitochondrial transcription termination factor 2 (MTERF2) gene and determine the cellular localization by overexpressing MTERF2 in human Caski cervical cancer cells. Methods The coding sequence of MTERF2 was amplified by reverse transcription-PCR using the total RNA extracted from human cervical cancer Caski cells, and then was inserted into p3×FLAG-CMV-14 vector. After being verified by PCR and DNA sequencing, the positive recombinant plasmid was transiently transfected into Caski cells...
June 2017: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
https://www.readbyqxmd.com/read/28613834/amino-functionalized-5-cap-analogs-as-tools-for-site-specific-sequence-independent-labeling-of-messenger-rna
#12
Marcin Warminski, Pawel J Sikorski, Zofia Warminska, Maciej Lukaszewicz, Anna Kropiwnicka, Joanna Zuberek, Edward Darzynkiewicz, Joanna Kowalska, Jacek Jemielity
mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labelling or affinity tagging of mRNA. We report new mRNA 5' cap analog-based tools that enable site-specific labelling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry. We explored two complementary methods: a co-transcriptional labelling method, in which the label is first attached to cap analog and then incorporated into RNA by transcription in vitro, and post-transcriptional labelling method, in which an amino-functionalized cap analog is incorporated into RNA followed by chemical labelling of the resulting transcript...
June 14, 2017: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/28611392/targeted-sequencing-of-both-dna-strands-barcoded-and-captured-individually-by-rna-probes-to-identify-genome-wide-ultra-rare-mutations
#13
Qing Wang, Xu Wang, Pheobe S Tang, Grace M O'leary, Ming Zhang
Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq)...
June 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28610174/the-complete-mitochondrial-genome-of-the-styloperlid-stonefly-species-i-styloperla-i-i-spinicercia-i-wu-insecta-plecoptera-with-family-level-phylogenetic-analyses-of-the-pteronarcyoidea
#14
Ying Wang, Jinjun Cao, Weihai Li
We present the complete mitochondrial (mt) genome sequence of the stonefly, Styloperla spinicercia Wu, 1935 (Plecoptera: Styloperlidae), the type species of the genus Styloperla and the first complete mt genome for the family Styloperlidae. The genome is circular, 16,129 base pairs long, has an A+T content of 70.7%, and contains 37 genes including the large and small ribosomal RNA (rRNA) subunits, 13 protein coding genes (PCGs), 22 tRNA genes and a large non-coding region (CR). All of the PCGs use the standard initiation codon ATN except ND1 and ND5, which start with TTG and GTG...
March 13, 2017: Zootaxa
https://www.readbyqxmd.com/read/28608336/immunoprecipitation-and-high-throughput-sequencing-of-argonaute-bound-target-rnas-from-plants
#15
Alberto Carbonell
ARGONAUTE (AGO) proteins function in small RNA (sRNA)-based RNA silencing pathways to regulate gene expression and control invading nucleic acids. In posttranscriptional RNA silencing pathways, plant AGOs associate with sRNAs to interact with highly sequence-complementary target RNAs. Once the AGO-sRNA-target RNA ternary complex is formed, target RNA is typically repressed through AGO-mediated cleavage or through other cleavage-independent mechanisms. The universe of sRNAs associating with diverse plant AGOs has been determined though AGO immunoprecipitation (IP) and high-throughput sequencing of co-immunoprecipitated sRNAs...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28607052/efficient-ultra-high-affinity-chromatography-in-a-one-step-purification-of-complex-proteins
#16
Marina N Vassylyeva, Sergiy Klyuyev, Alexey D Vassylyev, Hunter Wesson, Zhuo Zhang, Matthew B Renfrow, Hengbin Wang, N Patrick Higgins, Louise T Chow, Dmitry G Vassylyev
Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10(-14)-10(-17) M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7)...
June 12, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28599709/electrochemical-biosensing-of-influenza-a-subtype-genome-based-on-meso-macroporous-cobalt-ii-oxide-nanoflakes-applied-to-human-samples
#17
J Mohammadi, A Moattari, N Sattarahmady, N Pirbonyeh, H Yadegari, H Heli
Meso/macroporous cobalt (II) oxide nanoflakes were electrodeposited in a one-step process in the presence of N-methylpyrrolidone. On the surface of nanoflakes, a specific single stranded DNA sequence from the genome of influenza A subtype was then immobilized to fabricate an electrochemical biosensor. Hybridization of the biosensor with complementary, non-complementary and base-mismatch sequences was electrochemically detected. The biosensor was also employed to detect complementary DNA of viral RNA in culture and human samples...
August 1, 2017: Analytica Chimica Acta
https://www.readbyqxmd.com/read/28589512/molecular-insights-into-rna-binding-properties-of-escherichia-coli-expressed-rna-dependent-rna-polymerase-of-antheraea-mylitta-cytoplasmic-polyhedrosis-virus
#18
Anirban Kundu, Madhuparna Bose, Madhurima Roy, Soumita Dutta, Poulomi Biswas, Pradeep Gautam, Amit Kumar Das, Ananta Kumar Ghosh
Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified...
June 6, 2017: Archives of Virology
https://www.readbyqxmd.com/read/28580901/prosurvival-long-noncoding-rna-pincr-regulates-a-subset-of-p53-targets-in-human-colorectal-cancer-cells-by-binding-to-matrin-3
#19
Ritu Chaudhary, Berkley Gryder, Wendy S Woods, Murugan Subramanian, Matthew F Jones, Xiao Ling Li, Lisa M Jenkins, Svetlana A Shabalina, Min Mo, Mary Dasso, Yuan Yang, Lalage M Wakefield, Yuelin Zhu, Susan M Frier, Branden S Moriarity, Kannanganattu V Prasanth, Pablo Perez-Pinera, Ashish Lal
Thousands of long noncoding RNAs (lncRNAs) have been discovered, yet the function of the vast majority remains unclear. Here, we show that a p53-regulated lncRNA which we named PINCR (p53-induced noncoding RNA), is induced ~100-fold after DNA damage and exerts a prosurvival function in human colorectal cancer cells (CRC) in vitro and tumor growth in vivo. Targeted deletion of PINCR in CRC cells significantly impaired G1 arrest and induced hypersensitivity to chemotherapeutic drugs. PINCR regulates the induction of a subset of p53 targets involved in G1 arrest and apoptosis, including BTG2, RRM2B and GPX1...
June 5, 2017: ELife
https://www.readbyqxmd.com/read/28573017/a-crispr-cas9-high-throughput-genome-editing-toolkit-for-kinetoplastids
#20
Tom Beneke, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter, Eva Gluenz
Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit...
May 2017: Royal Society Open Science
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