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https://www.readbyqxmd.com/read/28323041/quantitative-analyses-of-the-3d-nuclear-landscape-recorded-with-super-resolved-fluorescence-microscopy
#1
Volker J Schmid, Marion Cremer, Thomas Cremer
Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction...
March 18, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28256719/rna-whole-mount-in-situ-hybridization-proximity-ligation-assay-rish-pla-an-assay-for-detecting-rna-protein-complexes-in-intact-cells
#2
Ioannis M Roussis, Fiona A Myers, Garry P Scarlett
Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to the study of RNA regulation (rISH-PLA). The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single-cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogeneous cell populations from which conventional RNA-protein interaction detection techniques suffer...
March 3, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28252184/using-morpholinos-to-control-gene-expression
#3
Jon D Moulton
Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28252182/synthesis-of-geranyl-2-thiouridine-modified-rna
#4
Rui Wang, Phensinee Haruehanroengra, Jia Sheng
This unit describes the chemical synthesis of the S-geranyl-2-thiouridine (ges(2) U) phosphoramidite and its incorporation into RNA oligonucleotides through solid-phase synthesis. Starting from the 2-thiouracil nucleobase and the protected ribose, the 2-thiouridine is synthesized and the geranyl functionality is introduced into the 2-thio position by using geranyl bromide as the geranylating reagent before the conversion of this modified nucleoside into a phosphoramidite building block. The modified phosphoramidite is used to make the geranyl-RNA oligonucleotides with a solid-phase DNA synthesizer...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28252179/deformability-calculation-for-estimation-of-the-relative-stability-of-chemically-modified-rna-duplexes
#5
Yoshiaki Masaki, Mitsuo Sekine, Kohji Seio
Chemical modification of RNA duplexes alters their stability. We have attempted to develop a computational approach to estimate the thermal stability of chemically modified duplexes. These studies revealed that the deformability of chemically modified RNA duplexes, calculated from molecular dynamics simulations, could be used as a good indicator for estimating the effect of chemical modification on duplex thermal stability. This unit describes how deformability calculation can be applied to estimate the relative stability of chemically modified RNA duplexes...
March 2, 2017: Current Protocols in Nucleic Acid Chemistry
https://www.readbyqxmd.com/read/28244141/claudin-domain-containing-1-contributing-to-endothelial-cell-adhesion-decreases-in-presence-of-cerebellar-hemorrhage
#6
Masatoshi Ohnishi, Hiroyuki Ochiai, Kohei Matsuoka, Marina Akagi, Yuta Nakayama, Akiho Shima, Arisa Uda, Hiroshi Matsuoka, Jun Kamishikiryo, Akihiro Michihara, Atsuko Inoue
The claudin family comprises four-pass transmembrane proteins involved in the formation of tight junctions (TJs). Relatively recently, claudin domain containing (CLDND) 1, also known as claudin-25, was identified as a novel member of the claudin family. In the present study, we revealed that in the adult murine brain, CLDND1 is abundant in the cerebellum among common sites of intracerebral hemorrhage. Thus, the dynamics of CLDND1 after cerebellar hemorrhage were examined. Both CLDND1 mRNA and protein levels transiently decreased at 24 hr after hemorrhagic insult...
February 28, 2017: Journal of Neuroscience Research
https://www.readbyqxmd.com/read/28213929/exploration-of-the-brn4-regulated-genes-enhancing-adult-hippocampal-neurogenesis-by-rna-sequencing
#7
Jingjing Guo, Xiang Cheng, Lei Zhang, Linmei Wang, Yongxin Mao, Guixiang Tian, Wenhao Xu, Yuhao Wu, Zhi Ma, Jianbing Qin, Meiling Tian, Guohua Jin, Wei Shi, Xinhua Zhang
Adult hippocampal neurogenesis is essential for learning and memory, and its dysfunction is involved in neurodegenerative diseases. However, the molecular mechanisms underlying adult hippocampal neurogenesis are still largely unknown. Our previous studies indicated that the transcription factor Brn4 was upregulated and promoted neuronal differentiation of neural stem cells (NSCs) in the surgically denervated hippocampus in rats. In this study, we use high-throughput RNA sequencing to explore the molecular mechanisms underlying the enhancement of adult hippocampal neurogenesis induced by lentivirus-mediated Brn4 overexpression in vivo...
February 18, 2017: Journal of Neuroscience Research
https://www.readbyqxmd.com/read/28179023/optimization-of-dna-extraction-for-advancing-coral-microbiota-investigations
#8
Laura Weber, Emelia DeForce, Amy Apprill
BACKGROUND: DNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. However, extraction of microbial DNA from complex environmental samples like corals can be technically challenging, and extraction methods may impart biases on microbial community structure. METHODS: We designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition...
February 8, 2017: Microbiome
https://www.readbyqxmd.com/read/28166383/efficient-genome-editing-in-the-oomycete-phytophthora-sojae-using-crispr-cas9
#9
Yufeng Fang, Linkai Cui, Biao Gu, Felipe Arredondo, Brett M Tyler
Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community...
February 6, 2017: Current Protocols in Microbiology
https://www.readbyqxmd.com/read/28150861/quantitation-of-dna-and-rna-with-absorption-and-fluorescence-spectroscopy
#10
Sean R Gallagher
Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0...
February 2, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28139025/a-de-novo-splice-site-mutation-in-cask-causes-fg-syndrome-4-and-congenital-nystagmus
#11
P Dunn, G P Prigatano, S Szelinger, J Roth, A L Siniard, A M Claasen, R F Richholt, M De Both, J J Corneveaux, A M Moskowitz, C Balak, I S Piras, M Russell, A L Courtright, N Belnap, S Rangasamy, K Ramsey, J M Opitz, D W Craig, V Narayanan, M J Huentelman, I Schrauwen
Mutations in CASK cause X-linked intellectual disability, microcephaly with pontine and cerebellar hypoplasia, optic atrophy, nystagmus, feeding difficulties, GI hypomotility, and seizures. Here we present a patient with a de novo carboxyl-terminus splice site mutation in CASK (c.2521-2A>G) and clinical features of the rare FG syndrome-4 (FGS4). We provide further characterization of genotype-phenotype correlations in CASK mutations and the presentation of nystagmus and the FGS4 phenotype. There is considerable variability in clinical phenotype among patients with a CASK mutation, even among variants predicted to have similar functionality...
January 31, 2017: American Journal of Medical Genetics. Part A
https://www.readbyqxmd.com/read/28101966/the-free-energy-of-locking-a-ring-changing-a-deoxyribonucleoside-to-a-locked-nucleic-acid
#12
You Xu, Alessandra Villa, Lennart Nilsson
Locked nucleic acid (LNA), a modified nucleoside which contains a bridging group across the ribose ring, improves the stability of DNA/RNA duplexes significantly, and therefore is of interest in biotechnology and gene therapy applications. In this study, we investigate the free energy change between LNA and DNA nucleosides. The transformation requires the breaking of the bridging group across the ribose ring, a problematic transformation in free energy calculations. To address this, we have developed a 3-step (easy to implement) and a 1-step protocol (more efficient, but more complicated to setup), for single and dual topologies in classical molecular dynamics simulations, using the Bennett Acceptance Ratio method to calculate the free energy...
January 19, 2017: Journal of Computational Chemistry
https://www.readbyqxmd.com/read/28087069/safety-tolerability-and-antiviral-effect-of-rg-101-in-patients-with-chronic-hepatitis-c-a-phase-1b-double-blind-randomised-controlled-trial
#13
Meike H van der Ree, J Marleen de Vree, Femke Stelma, Sophie Willemse, Marc van der Valk, Svend Rietdijk, Richard Molenkamp, Janke Schinkel, Ad C van Nuenen, Ulrich Beuers, Salah Hadi, Marten Harbers, Eva van der Veer, Kai Liu, John Grundy, Amy K Patick, Adam Pavlicek, Jacqueline Blem, Michael Huang, Paul Grint, Steven Neben, Neil W Gibson, Neeltje A Kootstra, Hendrik W Reesink
BACKGROUND: miR-122 is an important host factor for hepatitis C virus (HCV) replication. The aim of this study was to assess the safety and tolerability, pharmacokinetics, and antiviral effect of a single dose of RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated oligonucleotide that antagonises miR-122, in patients with chronic HCV infection with various genotypes. METHODS: In this randomised, double-blind, placebo-controlled, multicentre, phase 1B study, patients were randomly assigned to RG-101 or placebo (7:1)...
January 10, 2017: Lancet
https://www.readbyqxmd.com/read/28060411/scarless-cas9-assisted-recombineering-no-scar-in-escherichia-coli-an-easy-to-use-system-for-genome-editing
#14
Christopher R Reisch, Kristala L J Prather
The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair...
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28055115/measurement-of-t-cell-telomere-length-using-amplified-signal-fish-staining-and-flow-cytometry
#15
Andrea L Henning, Danielle E Levitt, Jakob L Vingren, Brian K McFarlin
Exposure to pathogen-associated molecular patterns (PAMPS), damage-associated molecular patterns (DAMPS), and physiologically challenging stimuli either positively or negatively affect leukocyte maturity. Cellular maturity has implications for the effectiveness of host response to bacterial or viral infection and/or tissue injury. Thus, the ability to accurately assess cellular maturity and health is important to fully understand immune status and function. The most common technique for measuring cellular maturity is to measure telomere length; however, existing techniques are not optimized for single-cell measurements using flow cytometry...
January 5, 2017: Current Protocols in Cytometry
https://www.readbyqxmd.com/read/28036033/mec1-atr-the-program-manager-of-nucleic-acids-inc
#16
REVIEW
Wenyi Feng
Eukaryotic cells are equipped with surveillance mechanisms called checkpoints to ensure proper execution of cell cycle events. Among these are the checkpoints that detect DNA damage or replication perturbations and coordinate cellular activities to maintain genome stability. At the forefront of damage sensing is an evolutionarily conserved molecule, known respectively in budding yeast and humans as Mec1 (Mitosis entry checkpoint 1) and ATR (Ataxia telangiectasia and Rad3-related protein). Through phosphorylation, Mec1/ATR activates downstream components of a signaling cascade to maintain nucleotide pool balance, protect replication fork integrity, regulate activation of origins of replication, coordinate DNA repair, and implement cell cycle delay...
December 28, 2016: Genes
https://www.readbyqxmd.com/read/28026860/pioglitazone-together-with-imatinib-in-chronic-myeloid-leukemia-a-proof-of-concept-study
#17
Philippe Rousselot, Stéphane Prost, Joelle Guilhot, Lydia Roy, Gabriel Etienne, Laurence Legros, Aude Charbonnier, Valérie Coiteux, Pascale Cony-Makhoul, Francoise Huguet, Emilie Cayssials, Jean-Michel Cayuela, Francis Relouzat, Marc Delord, Heriberto Bruzzoni-Giovanelli, Laure Morisset, François-Xavier Mahon, François Guilhot, Philippe Leboulch
BACKGROUND: We recently reported that peroxisome proliferator-activated receptor γ agonists target chronic myeloid leukemia (CML) quiescent stem cells in vitro by decreasing transcription of STAT5. Here in the ACTIM phase 2 clinical trial, we asked whether pioglitazone add-on therapy to imatinib would impact CML residual disease, as assessed by BCR-ABL1 transcript quantification. METHODS: CML patients were eligible if treated with imatinib for at least 2 years at a stable daily dose, having yielded major molecular response (MMR) but not having achieved molecular response 4...
December 27, 2016: Cancer
https://www.readbyqxmd.com/read/27991686/co-expression-changes-of-lncrnas-and-mrnas-in-the-cervical-sympathetic-ganglia-in-diabetic-cardiac-autonomic-neuropathic-rats
#18
Guilin Li, Xuan Sheng, Yurong Xu, Huaide Jiang, Chaoran Zheng, Jingjing Guo, Shanshan Sun, Zhihua Yi, Shulan Qin, Shuangmei Liu, Yun Gao, Chunping Zhang, Hong Xu, Bing Wu, Lifang Zou, Shangdong Liang, Gaochun Zhu
Cardiac autonomic neuropathy in Type 2 diabetes (T2D) is often a devastating complication. Long non-coding RNAs (lncRNAs) have important effects on both normal development and disease pathogenesis. In this study, we explored the expression profiles of some lncRNAs involved in inflammation which may be co-expressed with messenger RNA (mRNA) in superior cervical and stellate ganglia after type 2 diabetic injuries. Total RNA isolated from 10 pairs of superior cervical and stellate ganglia in diabetic and normal male rats was hybridized to lncRNA arrays for detections...
December 19, 2016: Journal of Neuroscience Research
https://www.readbyqxmd.com/read/27976414/low-testosterone-alters-the-activity-of-mouse-prostate-stem-cells
#19
Ye Zhou, Ben Copeland, Maya Otto-Duessel, Miaoling He, Susan Markel, Tim W Synold, Jeremy O Jones
BACKGROUND: Low serum testosterone (low T) has been repeatedly linked to worse outcomes in men with newly diagnosed prostate cancer (PC). How low T contributes to these outcomes is unknown. Here we demonstrate that exposure to low T causes significant changes in the mouse prostate and prostate stem cells. METHODS: Mice were castrated and implanted with capsules to achieve castrate, normal, or sub-physiological levels of T. After 6 weeks of treatment, LC-MS/MS was used to quantify the levels of T and dihydrotestosterone (DHT) in serum and prostate tissue...
December 14, 2016: Prostate
https://www.readbyqxmd.com/read/27933721/shared-molecular-networks-in-orofacial-and-neural-tube-development
#20
Youssef A Kousa, Tamer A Mansour, Haitham Seada, Samaneh Matoo, Brian C Schutte
BACKGROUND: Single genetic variants can affect multiple tissues during development. Thus it is possible that disruption of shared gene regulatory networks might underlie syndromic presentations. In this study, we explore this idea through examination of two critical developmental programs that control orofacial and neural tube development and identify shared regulatory factors and networks. Identification of these networks has the potential to yield additional candidate genes for poorly understood developmental disorders and assist in modeling and perhaps managing risk factors to prevent morbidly and mortality...
December 9, 2016: Birth Defects Research. Part A, Clinical and Molecular Teratology
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