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https://www.readbyqxmd.com/read/28730478/high-content-imaging-and-rnai-screens-for-investigating-kinase-network-plasticity
#1
Simon R Stockwell, Sibylle Mittnacht
High-content imaging connects the information-rich method of microscopy with the systematic objective principles of software-driven analysis. Suited to automation and, therefore, considerable scale-up of study size, this approach can deliver multiparametric data over cell populations or at the level of the individual cell and has found considerable utility in reverse genetic and pharmacological screens. Here we present a method to screen small interfering RNA (siRNA) libraries allowing subsequent observation of the impact of each knockdown on two interlinked, high-content, G1-/S-phase cell cycle transition assays related to cyclin-dependent kinase (CDK) 2 activity...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28730304/novel-ribonuclease-activity-of-cusativin-from-cucumis-sativus-for-mapping-nucleoside-modifications-in-rna
#2
Balasubrahmanyam Addepalli, Sarah Venus, Priti Thakur, Patrick A Limbach
A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein...
July 20, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28728963/structural-principles-of-fluorescent-rna-aptamers
#3
REVIEW
Robert J Trachman, Lynda Truong, Adrian R Ferré-D'Amaré
Several aptamer RNAs have been selected in vitro that bind to otherwise weakly fluorescent small molecules and enhance their fluorescence several thousand-fold. By genetically tagging cellular RNAs of interest with these aptamers and soaking cells in their cell-permeable cognate small-molecule fluorophores, it is possible to use them to study RNA localization and trafficking. These aptamers have also been fused to metabolite-binding RNAs to generate fluorescent biosensors. The 3D structures of three unrelated fluorogenic RNAs have been determined, and reveal a shared reliance on base quadruples (tetrads) to constrain the photo-excited chromophore...
July 17, 2017: Trends in Pharmacological Sciences
https://www.readbyqxmd.com/read/28726630/dissection-of-specific-binding-of-hiv-1-gag-to-the-packaging-signal-in-viral-rna
#4
Mauricio Comas-Garcia, Siddhartha Ak Datta, Laura Baker, Rajat Varma, Prabhakar R Gudla, Alan Rein
Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis-acting RNA element called the "packaging signal" (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength...
July 20, 2017: ELife
https://www.readbyqxmd.com/read/28724083/identification-of-candidate-genes-responsible-for-stem-pith-production-using-expression-analysis-in-solid-stemmed-wheat
#5
A J Oiestad, J M Martin, J Cook, A C Varella, M J Giroux
The wheat stem sawfly (WSS) is an economically important pest of wheat in the Northern Great Plains. The primary means of WSS control is resistance associated with the single quantitative trait locus (QTL) , which controls most stem solidness variation. The goal of this study was to identify stem solidness candidate genes via RNA-seq. This study made use of 28 single nucleotide polymorphism (SNP) makers derived from expressed sequence tags (ESTs) linked to contained within a 5.13 cM region. Allele specific expression of EST markers was examined in stem tissue for solid and hollow-stemmed pairs of two spring wheat near isogenic lines (NILs) differing for the QTL...
July 2017: Plant Genome
https://www.readbyqxmd.com/read/28723162/ultrasensitive-detection-of-cancer-prognostic-mirna-biomarkers-based-on-surface-plasmon-enhanced-light-scattering
#6
Chih-Tsung Yang, Mohammad Pourhassan-Moghaddam, Lin Wu, Ping Bai, Benjamin Thierry
The development of simple yet ultrasensitive biosensing approaches for the detection of cancer prognostic microRNA is an important step toward their successful clinical implementation. We demonstrate the relevance for the detection of circulating miRNA of a novel signal amplification scheme based on surface plasmon resonance enhanced light scattering (SP-LS). In addition to experimental optimization carried out using gold nanoparticle (AuNP) tags conjugated with a monoclonal antibody with high affinity for RNA*DNA hybrid duplexes, simulation modeling was conducted to obtain insights about SP-LS biosensing...
May 26, 2017: ACS Sensors
https://www.readbyqxmd.com/read/28722044/tripartite-polyionic-complex-pic-micelles-as-non-viral-vectors-for-mesenchymal-stem-cell-sirna-transfection
#7
Sophie Raisin, Marie Morille, Claire Bony, Danièle Noël, Jean-Marie Devoisselle, Emmanuel Belamie
In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA...
July 19, 2017: Biomaterials Science
https://www.readbyqxmd.com/read/28715370/inducible-t7-rna-polymerase-mediated-multigene-expression-system-pmgx
#8
Mohamed I Hassan, Fern R McSorley, Kinya Hotta, Christopher N Boddy
Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed...
June 27, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28713413/identification-of-conserved-and-novel-micrornas-in-blueberry
#9
Junyang Yue, Xiaohui Lu, Huan Zhang, Jiao Ge, Xueling Gao, Yongsheng Liu
MicroRNAs (miRNAs) are a class of small endogenous RNAs that play important regulatory roles in cells by negatively affecting gene expression at both transcriptional and post-transcriptional levels. There have been extensive studies aiming to identify miRNAs and to elucidate their functions in various plant species. In the present study, we employed the high-throughput sequencing technology to profile miRNAs in blueberry fruits. A total of 9,992,446 small RNA tags with sizes ranged from 18 to 30 nt were obtained, indicating that blueberry fruits have a large and diverse small RNA population...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28713366/intratumoral-lentivector-mediated-tgf-%C3%AE-1-gene-downregulation-as-a-potent-strategy-for-enhancing-the-antitumor-effect-of-therapy-composed-of-cyclophosphamide-and-dendritic-cells
#10
Joanna Rossowska, Natalia Anger, Agnieszka Szczygieł, Jagoda Mierzejewska, Elżbieta Pajtasz-Piasecka
Vaccination with dendritic cells (DCs) stimulated with tumor antigens can induce specific cellular immune response that recognizes a high spectrum of tumor antigens. However, the ability of cancer cells to produce immunosuppressive factors drastically decreases the antitumor activity of DCs. The main purpose of the study was to improve the effectiveness of DC-based immunotherapy or chemoimmunotherapy composed of cyclophosphamide (CY) and DCs by application of lentivectors (LVs)-encoding short hairpin RNA specific for TGF-β1 (shTGFβ1 LVs)...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28707598/protein-tagging-destruction-and-infection
#11
Chetana Bhaskarla, Manoj Bhosale, Pip Banerjee, Nagasuma Chandra, Dipankar Nandi
Cells possess protein quality control mechanisms to maintain proper cellular homeostasis. In eukaryotes, the roles of the ubiquitination and proteasome-mediated degradation of cellular proteins is well established. Recent studies have elucidated protein tagging mechanisms in prokaryotes, involving transfer messenger RNA (tmRNA) and pupylation. In this review, newer insights and bioinformatics analysis of two distinct bacterial protein tagging machineries are discussed. The machinery for tmRNA-mediated tagging is present in several eubacterial representatives, e...
July 13, 2017: Current Protein & Peptide Science
https://www.readbyqxmd.com/read/28702783/origins-of-life-research-a-bibliometric-approach
#12
Arsev Umur Aydinoglu, Zehra Taşkın
This study explores the collaborative nature and interdisciplinarity of the origin(s) of life (OoL) research community. Although OoL research is one of the oldest topics in philosophy, religion, and science; to date there has been no review of the field utilizing bibliometric measures. A dataset of 5647 publications that are tagged as OoL, astrobiology, exobiology, and prebiotic chemistry is analyzed. The most prolific authors (Raulin, Ehrenfreund, McKay, Cleaves, Cockell, Lazcano, etc.), most cited scholars and their articles (Miller 1953, Gilbert 1986, Chyba & Sagan 1992, Wȁchtershȁuser 1988, etc...
July 13, 2017: Origins of Life and Evolution of the Biosphere
https://www.readbyqxmd.com/read/28701394/single-particle-detection-of-transcription-following-rotavirus-entry
#13
Eric N Salgado, Srigokul Upadhyayula, Stephen C Harrison
Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell-surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change...
July 12, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28698494/fluorogenic-labeling-strategies-for-biological-imaging
#14
REVIEW
Chenge Li, Alison G Tebo, Arnaud Gautier
The spatiotemporal fluorescence imaging of biological processes requires effective tools to label intracellular biomolecules in living systems. This review presents a brief overview of recent labeling strategies that permits one to make protein and RNA strongly fluorescent using synthetic fluorogenic probes. Genetically encoded tags selectively binding the exogenously applied molecules ensure high labeling selectivity, while high imaging contrast is achieved using fluorogenic chromophores that are fluorescent only when bound to their cognate tag, and are otherwise dark...
July 9, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28693298/chicken-rna-binding-protein-tia-1-contributes-to-stress-granule-formation-in-chicken-cells-and-tissues
#15
Yingjie Sun, Pin Zhang, Hang Zheng, Luna Dong, Lei Tan, Cuiping Song, Xusheng Qiu, Ying Liao, Chunchun Meng, Shengqing Yu, Chan Ding
T-cell internal antigen-1 (TIA-1) plays roles in regulating alternative pre-mRNA splicing, mRNA translation, and stress granule (SG) formation in human cells. As an evolutionarily conserved response to environmental stress, SG has been reported to exist in various species. However, SG formation in chicken cells and the role of chicken TIA-1 (cTIA-1) in SG assembly has not been elucidated. In the present study, we cloned cTIA-1 and showed that cTIA-1 facilitates in the assembly of canonical SGs in both human and chicken cells...
July 10, 2017: Journal of Veterinary Science
https://www.readbyqxmd.com/read/28689323/a-sensitive-colorimetric-assay-system-for-nucleic-acid-detection-based-on-isothermal-signal-amplification-technology
#16
Bo Hu, Jing Guo, Ying Xu, Hua Wei, Guojie Zhao, Yifu Guan
Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences...
July 8, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28683768/extrapolative-microrna-precursor-based-ssr-mining-from-tea-est-database-in-respect-to-agronomic-traits
#17
Anjan Hazra, Nirjhar Dasgupta, Chandan Sengupta, Sauren Das
Tea (Camellia sinensis, (L.) Kuntze) is considered as most popular drink across the world and it is widely consumed beverage for its several health-benefit characteristics. These positive traits primarily rely on its regulatory networks of different metabolic pathways. Development of microsatellite markers from the conserved genomic regions are being worthwhile for reviewing the genetic diversity of closely related species or self-pollinated species. Although several SSR markers have been reported, in tea, the trait-specific Simple Sequence Repeat (SSR) markers, leading to be useful in marker assisted breeding technique, are yet to be identified...
July 6, 2017: BMC Research Notes
https://www.readbyqxmd.com/read/28679406/the-common-promoter-polymorphism-rs11666254-downregulates-fpr2-alx-expression-and-increases-risk-of-sepsis-in-patients-with-severe-trauma
#18
Han Zhang, Yao Lu, Guixiang Sun, Fang Teng, Nian Luo, Jianxin Jiang, Aiqing Wen
BACKGROUND: Formyl peptide receptor 2-lipoxin receptor (FPR2/ALX) modulates the anti-inflammatory response and therefore may be a target for treating sepsis. The purpose of this study was to investigate the association between genetic variants of the FPR2/ALX gene and sepsis after severe trauma as well as to further analyze the functions of sepsis-related genetic polymorphisms. METHODS: Three tag single-nucleotide polymorphisms (tag SNPs) that captured all common alleles across the FPR2/ALX genomic region were genotyped using pyrosequencing in an initial sample consisting of 275 patients with severe trauma...
July 6, 2017: Critical Care: the Official Journal of the Critical Care Forum
https://www.readbyqxmd.com/read/28667016/an-rna-recognition-motif-containing-protein-functions-in-meiotic-silencing-by-unpaired-dna
#19
Dilini A Samarajeewa, Pennapa Manitchotpisit, Miranda Henderson, Hua Xiao, David G Rehard, Kevin A Edwards, Patrick K T Shiu, Thomas M Hammond
Meiotic silencing by unpaired DNA (MSUD) is a biological process that searches pairs of homologous chromosomes (homologs) for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of Neurospora crassa sad-7, a gene that encodes a protein with an RNA recognition motif. Orthologs of sad-7 are found in a wide range of ascomycete fungi. In N. crassa, sad-7 is required for a fully-efficient MSUD response to unpaired genes...
June 30, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28666732/a-functional-variant-of-smad4-enhances-thoracic-aortic-aneurysm-and-dissection-risk-through-promoting-smooth-muscle-cell-apoptosis-and-proteoglycan-degradation
#20
Ying Wang, Hao-Yue Huang, Guang-Liang Bian, Yun-Sheng Yu, Wen-Xue Ye, Fei Hua, Yi-Huan Chen, Zhen-Ya Shen
Recent studies indicate important roles for SMAD4 in SMCs proliferation, extracellular matrix maintenance, and blood vessel remodeling. However, the genetic effects of SMAD4 in the pathogenesis of thoracic aortic aneurysm and dissection (TAAD) are still largely unknown. Here we identified a functional variant of SMAD4 which might be involved in the pathological progression of TAAD. Five tagging SNPs of SMAD4 were genotyped in 202 TAAD cases and 400 controls using MALDI-TOF. rs12455792 CT or TT variant genotypes was associated with an significantly elevated TAAD risk (adjusted OR=1...
June 22, 2017: EBioMedicine
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