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RNA Imaging

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https://www.readbyqxmd.com/read/28334102/predation-by-bdellovibrio-bacteriovorus-significantly-reduces-viability-and-alters-the-microbial-community-composition-of-activated-sludge-flocs-and-granules
#1
Shugeng Feng, Chuan Hao Tan, Florentin Constancias, Gurjeet S Kohli, Yehuda Cohen, Scott A Rice
We recently isolated and characterized a predatory Bdellovibrio bacteriovorus strain from activated sludge (Ulu Pandan Water Reclamation Plant, Singapore), and this strain, B. bacteriovorus UP was able to prey upon a broad spectrum of bacterial isolates from the activated sludge when grown as planktonic cells or as biofilms. Here, we have tested the effect of Bdellovibrio predation on floccular and granular sludge to determine if the spatial organization, loosely or tightly aggregated communities, was protective from predation...
February 21, 2017: FEMS Microbiology Ecology
https://www.readbyqxmd.com/read/28333933/geometry-dependent-functional-changes-in-ipsc-derived-cardiomyocytes-probed-by-functional-imaging-and-rna-sequencing
#2
Christopher A Werley, Miao-Ping Chien, Jellert Gaublomme, Karthik Shekhar, Vincent Butty, B Alexander Yi, Joel M Kralj, William Bloxham, Laurie A Boyer, Aviv Regev, Adam E Cohen
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes...
2017: PloS One
https://www.readbyqxmd.com/read/28331082/kaposi-s-sarcoma-associated-herpesvirus-hijacks-rna-polymerase-ii-to-create-a-viral-transcriptional-factory
#3
Christopher Phillip Chen, Yuanzhi Lyu, Frank Chuang, Kazushi Nakano, Chie Izumiya, Di Jin, Mel Campbell, Yoshihiro Izumiya
Locally concentrated nuclear factors ensure efficient binding to the DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable pre-initiation complexes. Here we have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around KSHV genomes in the host cell nucleus. Using immunofluorescent labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate-early transcripts, we visualized active transcription of viral genomes in naturally infected cells...
March 22, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28329096/diffuse-white-matter-signal-abnormalities-on-magnetic-resonance-imaging-are-associated-with-human-immunodeficiency-virus-type-1-viral-escape-in-the-central-nervous-system-among-patients-with-neurological-symptoms
#4
Ruthiran Kugathasan, Dami A Collier, Lewis J Haddow, Kate El Bouzidi, Simon G Edwards, Jonathan D Cartledge, Robert F Miller, Ravindra K Gupta
Background.: Human immunodeficiency virus type 1 (HIV-1) can replicate independently in extravascular compartments such as the central nervous system, resulting in either cerebrospinal fluid (CSF) discordance (viral load [VL] in CSF 0.5 log10 copies HIV-1 RNA greater than plasma VL) or escape (detection of HIV VL >50 copies/mL in CSF in patients with suppressed plasma VL <50 copies/mL). Both discordance and escape may be associated with neurological symptoms. We explored risk factors for CSF discordance and escape in patients presenting with diverse neurological problems...
March 13, 2017: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America
https://www.readbyqxmd.com/read/28324612/quantitative-image-analysis-of-single-molecule-mrna-dynamics-in-living-cells
#5
José Rino, Ana C de Jesus, Maria Carmo-Fonseca
Single mRNA molecules can be imaged in living cells by a method that consists in genetically inserting binding sites for a bacteriophage protein in the gene of interest. The resulting reporter transgene is then integrated in the genome of cells that express the phage protein fused to a fluorescent tag. Upon transcription, binding of the fluorescent protein to its target sequence makes the RNA visible. With this approach it is possible to track, in real time, the life cycle of a precursor mRNA at the site of transcription in the nucleus and transport of mature mRNA to the cytoplasm...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28303387/caveolar-remodeling-is-a-critical-mechanotransduction-mechanism-of-the-stretch-induced-l-type-ca-2-channel-activation-in-vascular-myocytes
#6
Sang Woong Park, Kyung Chul Shin, Hyun Ji Park, Soon-Kyu Yoou, Jin-Yeon Park, Young-Sun Kang, Dong Jun Sung, Jae Gon Kim, Seung Hwa Park, BoKyung Kim, Hana Cho, Young Min Bae
Activation of L-type voltage-dependent Ca(2+) channels (VDCCL) by membrane stretch contributes to many biological responses such as myogenic contraction of arteries. However, mechanism for the stretch-induced VDCCL activation is unclear. In this study, we examined the hypothesis that caveolar remodeling and its related signaling cascade contribute to the stretch-induced activation of VDCCL in rat mesenteric arterial smooth muscle cells. The VDCCL currents were recorded with nystatin-perforated or with conventional whole-cell patch-clamp technique...
March 16, 2017: Pflügers Archiv: European Journal of Physiology
https://www.readbyqxmd.com/read/28303166/single-molecule-studies-of-high-mobility-group-b-architectural-dna-bending-proteins
#7
REVIEW
Divakaran Murugesapillai, Micah J McCauley, L James Maher, Mark C Williams
Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function...
February 2017: Biophysical Reviews
https://www.readbyqxmd.com/read/28301688/multiplex-single-cell-quantification-of-rare-rna-transcripts-from-protoplasts-in-a-model-plant-system
#8
Ulhas S Kadam, Burkhard Schulz, Joseph M K Irudayaraj
Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface enhanced Raman spectroscopy and gold nanoprobes at single cell resolution. We show the applicability of nanoparticle-based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold-nanoparticles decorate with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by TEM, ICP-MS, and fluorescence imaging...
March 16, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/28299722/a-versatile-method-for-the-preparation-of-particle-loaded-microbubbles-for-multimodality-imaging-and-targeted-drug-delivery
#9
Joshua Owen, Calum Crake, Jeong Yu Lee, Dario Carugo, Estelle Beguin, Alexandre A Khrapitchev, Richard J Browning, Nicola Sibson, Eleanor Stride
Microbubbles are currently in clinical use as ultrasound contrast agents and under active investigation as mediators of ultrasound therapy. To improve the theranostic potential of microbubbles, nanoparticles can be attached to the bubble shell for imaging, targeting and/or enhancement of acoustic response. Existing methods for fabricating particle-loaded bubbles, however, require the use of polymers, oil layers or chemical reactions for particle incorporation; embed/attach the particles that can reduce echogenicity; impair biocompatibility; and/or involve multiple processing steps...
March 15, 2017: Drug Delivery and Translational Research
https://www.readbyqxmd.com/read/28296575/live-cell-imaging-reveals-the-relocation-of-dsrna-binding-proteins-upon-viral-infection
#10
Deborah Barton, Elke Roovers, Quentin Gouil, Guilherme C da Fonseca, Rodrigo S Reis, Craig Jackson, Robyn Overall, Adriana Fusaro, Peter Waterhouse
Viral infection triggers a range of plant responses such as the activation of the RNA interference (RNAi) pathway. The double-stranded RNA binding (DRB) proteins, DRB3 and DRB4, are part of this pathway and aid in defending against DNA and RNA viruses, respectively. Using live cell imaging, we show that DRB2, DRB3 and DRB5 relocate from their uniform cytoplasmic distribution to concentrated accumulation in nascent viral replication complexes (VRCs) that develop following cell invasion by viral RNA. Inactivation of the DRB3 gene in Arabidopsis, by T-DNA insertion, rendered these plants less able to repress RNA viral replication...
March 15, 2017: Molecular Plant-microbe Interactions: MPMI
https://www.readbyqxmd.com/read/28295806/proteins-mediating-dna-loops-effectively-block-transcription
#11
Zsuzsanna Vörös, Yan Yan, Daniel T Kovari, Laura Finzi, David Dunlap
Loops are ubiquitous topological elements formed when proteins simultaneously bind to two non-contiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM)...
March 14, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28290446/live-cell-imaging-of-low-and-non-repetitive-chromosome-loci-using-crispr-cas9
#12
Peiwu Qin, Mahmut Parlak, Cem Kuscu, Jigar Bandaria, Mustafa Mir, Karol Szlachta, Ritambhara Singh, Xavier Darzacq, Ahmet Yildiz, Mazhar Adli
Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification...
March 14, 2017: Nature Communications
https://www.readbyqxmd.com/read/28278327/chorioretinal-lesions-presumed-secondary-to-zika-virus-infection-in-an-immunocompromised-adult
#13
Christopher R Henry, Luma Al-Attar, Alexis M Cruz-Chacón, Janet L Davis
Importance: Zika virus has spread rapidly throughout the Americas since 2015. The public health implications of Zika virus infection lend special importance to identifying the virus in unsuspected hosts. Objective: To describe relevant imaging studies and clinical features of chorioretinal lesions that are presumably associated with Zika virus and that share analogous features with chorioretinal lesions reported in cases of Dengue fever and West Nile virus. Design, Setting, and Participants: This is a case report from an academic referral center in Miami, Florida, of a woman in her 60s from Guaynabo, Puerto Rico, who presented with reduced visual acuity and bilateral diffuse, subretinal, confluent, placoid, and multifocal chorioretinal lesions...
March 9, 2017: JAMA Ophthalmology
https://www.readbyqxmd.com/read/28278199/monitoring-integrity-and-localization-of-modified-single-stranded-rna-oligonucleotides-using-ultrasensitive-fluorescence-methods
#14
Philipp Heissig, Waldemar Schrimpf, Philipp Hadwiger, Ernst Wagner, Don C Lamb
Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone...
2017: PloS One
https://www.readbyqxmd.com/read/28277931/antibodies-specific-for-nucleic-acid-modifications
#15
Regina Feederle, Aloys Schepers
Nucleotide modifications constitute marks in RNA and DNA that contribute to gene regulation, development and other cellular processes. The understanding of their intricate molecular roles has been hampered by the high number of different modifications, the lack of effective methods and tools for their detection and quantification as well as by their complex structure-function relationship. The recent development of RNA and DNA immunoprecipitation followed by high-throughput sequencing (RIP- and DIP-seq) initiated detailed transcriptome- and genome-wide studies...
February 23, 2017: RNA Biology
https://www.readbyqxmd.com/read/28276570/the-role-of-rna-uptake-in-platelet-heterogeneity
#16
Lauren Clancy, Lea M Beaulieu, Kahraman Tanriverdi, Jane E Freedman
The role of platelets in regulating vascular homeostasis has expanded beyond mediation of haemostasis and thrombosis. The discovery of platelet RNA and the presence of subpopulations of platelets containing varying amounts of RNA suggest a role for platelet transcripts in vascular function. As the RNA in anucleated platelets is biologically functional and may transfer to other vascular cells, we hypothesised that platelet RNA diminishes over the lifespan of the platelet with diminishing platelet size due to horizontal cellular transfer...
March 9, 2017: Thrombosis and Haemostasis
https://www.readbyqxmd.com/read/28273117/activation-of-odorant-receptor-in-colorectal-cancer-cells-leads-to-inhibition-of-cell-proliferation-and-apoptosis
#17
Lea Weber, Klaudia Al-Refae, Juliane Ebbert, Peter Jägers, Janine Altmüller, Christian Becker, Stephan Hahn, Günter Gisselmann, Hanns Hatt
The analysis and functional characterization of ectopically expressed human olfactory receptors (ORs) is becoming increasingly important, as many ORs have been identified in several healthy and cancerous tissues. OR activation has been demonstrated to have influence on cancer cell growth and progression. Here, ORs were identified using RNA-Seq analyses and RT-PCR. We demonstrated the OR protein localization in HCT116 cells using immunocytochemistry (IHC). In order to analyze the physiological role of OR51B4, we deorphanized the receptor by the use of CRE-Luciferase assays, conducted calcium imaging experiments as well as scratch- and proliferation assays...
2017: PloS One
https://www.readbyqxmd.com/read/28270749/identifying-candidate-genes-that-underlie-cellular-ph-sensitivity-in-serotonin-neurons-using-transcriptomics-a-potential-role-for-kir5-1-channels
#18
Madeleine M Puissant, Gary C Mouradian, Pengyuan Liu, Matthew R Hodges
Ventilation is continuously adjusted by a neural network to maintain blood gases and pH. Acute CO2 and/or pH regulation requires neural feedback from brainstem cells that encode CO2/pH to modulate ventilation, including but not limited to brainstem serotonin (5-HT) neurons. Brainstem 5-HT neurons modulate ventilation and are stimulated by hypercapnic acidosis, the sensitivity of which increases with increasing postnatal age. The proper function of brainstem 5-HT neurons, particularly during post-natal development is critical given that multiple abnormalities in the 5-HT system have been identified in victims of Sudden Infant Death Syndrome...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/28262838/galectin-3-is-a-non-classic-rna-binding-protein-that-stabilizes-the-mucin-muc4-mrna-in-the-cytoplasm-of-cancer-cells
#19
Lucie Coppin, Audrey Vincent, Frédéric Frénois, Belinda Duchêne, Fatima Lahdaoui, Laurence Stechly, Florence Renaud, Céline Villenet, Isabelle Van Seuningen, Emmanuelle Leteurtre, Johann Dion, Cyrille Grandjean, Françoise Poirier, Martin Figeac, Delphine Delacour, Nicole Porchet, Pascal Pigny
Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing...
March 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28261803/difference-in-release-kinetics-of-unwashed-and-washed-platelet-released-supernatants-from-bone-substitute-materials-the-impact-of-platelet-preparation-modalities
#20
C Knoop, M Edelmayer, K Janjić, M Pensch, M B Fischer, R Gruber, H Agis
BACKGROUND AND OBJECTIVE: In regenerative dentistry, platelet preparations are applied to stimulate bone healing and periodontal regeneration. Here, we pursue a strategy where bone substitutes are used as carriers for platelet-released supernatants. The mitogenic capacity and release kinetics of loaded bone substitutes were assessed. MATERIAL AND METHODS: Platelet-released supernatants of washed platelets (washed PRS) and platelet-released supernatants of unwashed platelets (unwashed PRS) were lyophilized onto the bone substitutes deproteinized bovine bone mineral, hydroxyapatite and β-tricalcium phosphate...
March 6, 2017: Journal of Periodontal Research
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