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https://www.readbyqxmd.com/read/29236262/studying-nonsense-mediated-mrna-decay-in-mammalian-cells-using-a-multicolored-bioluminescence-based-reporter-system
#1
Andrew Nickless, Zhongsheng You
The nonsense-mediated mRNA decay (NMD) pathway degrades aberrant transcripts containing premature translation termination codons (PTCs) and also regulates the levels of many normal mRNAs containing NMD-inducing features. The activity of this pathway varies considerably in different cell types and can change in response to developmental and environmental cues. Modulating NMD activity represents a potential therapeutic avenue for certain genetic disorders and cancers. Simple reporter systems capable of faithfully assessing NMD activity in mammalian cells greatly facilitate both basic and translational research on NMD...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29235271/wide-field-of-view-common-path-lateral-shearing-digital-holographic-interference-microscope
#2
Priyanka Vora, Vismay Trivedi, Swapnil Mahajan, Nimit Patel, Mugdha Joglekar, Vani Chhaniwal, Ali-Reza Moradi, Bahram Javidi, Arun Anand
Quantitative three-dimensional (3-D) imaging of living cells provides important information about the cell morphology and its time variation. Off-axis, digital holographic interference microscopy is an ideal tool for 3-D imaging, parameter extraction, and classification of living cells. Two-beam digital holographic microscopes, which are usually employed, provide high-quality 3-D images of micro-objects, albeit with lower temporal stability. Common-path digital holographic geometries, in which the reference beam is derived from the object beam, provide higher temporal stability along with high-quality 3-D images...
December 2017: Journal of Biomedical Optics
https://www.readbyqxmd.com/read/29232627/the-phenomenology-of-cell-size-control
#3
REVIEW
Kurt M Schmoller
Cells control their size through an intricate balance of cell growth, cell division, and cell death. Extensive work on unicellular model organisms revealed that cell-size-dependent cell cycle progression accounts for major aspects of cell size regulation and provided insights into the underlying molecular mechanisms. Nevertheless, elaborate live-cell imaging approaches still reveal new phenomenological observations that challenge our simplified models of size regulation and raise the question of what determines optimal cell size...
December 9, 2017: Current Opinion in Cell Biology
https://www.readbyqxmd.com/read/29231816/cell-lineage-and-cell-cycling-analyses-of-the-4d%C3%A2-micromere-using-live-imaging-in-the-marine-annelid-platynereis-dumerilii
#4
B Duygu Özpolat, Mette Handberg-Thorsager, Michel Vervoort, Guillaume Balavoine
Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids...
December 12, 2017: ELife
https://www.readbyqxmd.com/read/29229997/splics-a-split-green-fluorescent-protein-based-contact-site-sensor-for-narrow-and-wide-heterotypic-organelle-juxtaposition
#5
Domenico Cieri, Mattia Vicario, Marta Giacomello, Francesca Vallese, Riccardo Filadi, Tina Wagner, Tullio Pozzan, Paola Pizzo, Luca Scorrano, Marisa Brini, Tito Calì
Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8-10 nm) and wide (40-50 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells...
December 11, 2017: Cell Death and Differentiation
https://www.readbyqxmd.com/read/29229552/caspase-6-is-a-dispensable-enabler-of-adult-mammalian-axonal-degeneration
#6
V Woo, C Cheng, A Duraikannu, A Chandrasekhar, K Purdy, J A Martinez, D W Zochodne
The progress of axonal degeneration (AxD) following injury or insult impacts both recovery from axonal transection and protection of axons from diverse insults, or axonopathy. Here we provide evidence that increases in capase-6 (Casp6) expression and action contribute to the progression of AxD. The expression of Casp6 protein and mRNA in distal branches of sensory axons undergoing AxD was confirmed. We developed and utilized a new model of axonopathy in live mice by serially visualizing the viability of cutaneous axons in the ear pinna that expressed an axonal YFP transgene, in response to capasaicin-induced AxD...
December 8, 2017: Neuroscience
https://www.readbyqxmd.com/read/29229224/studies-on-interactions-of-carbazole-derivatives-with-dna-cell-image-and-cytotoxicity
#7
Guoyan Hao, Jinyu Sun, Chunying Wei
DNA-binding agents have been considered as an established opportunity for the development of anticancer drugs and DNA fluorescence probes. This work reported the synthesis of two novel carbazole derivatives (1 and 2) and investigated their DNA binding properties, living cell image, and cytotoxicity. The results demonstrated that both compounds presented the higher binding affinity to G-quadruplex than to duplex DNA by means of UV-Vis absorption titration and fluorescent intercalator displacement. Continuous variation analysis indicated that their binding stoichiometries of the compound/G-quadruplex were near 2 except the compound/bcl-2...
December 2, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/29228311/a-new-method-for-post-translationally-labeling-proteins-in-live-cells-for-fluorescence-imaging-and-tracking
#8
M Hinrichsen, M Lenz, J M Edwards, O K Miller, S G J Mochrie, P S Swain, U Schwarz-Linek, L Regan
We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP...
December 7, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/29227553/fluorescence-lifetime-imaging-of-a-caspase-3-apoptosis-reporter
#9
Johanna M Buschhaus, Anne E Gibbons, Kathryn E Luker, Gary D Luker
Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used. Changes in FRET by fluorescence lifetime imaging microscopy (FLIM) were measured. Unlike FRET measurements based on fluorescence intensity, lifetime measurements are independent of reporter concentration and scattering of light in tissue, making FLIM a robust method for imaging in 3D environments...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29227550/live-cell-visualization-of-calcium-flux-in-vibratome-cut-thick-sections-of-viable-tumor-tissue
#10
James Koh, Joyce A Hogue, Julie A Sosa
This unit outlines a live-cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium-sensing receptor (CASR), a class C G protein-coupled receptor (GPCR). The primary assay readout, intracellular calcium release induced by activation of the inositol triphosphate receptor, is potentially generalizable to multiple other GPCR signaling events that utilize this common downstream signal transduction pathway. Advantages of the approach include: (1) preservation of native tissue context and positional information, allowing direct visualization of intratumoral functional heterogeneity; (2) quantitative documentation of reactivity to a physiological stimulus in an experimentally tractable ex vivo system; and (3) generation of a dynamic, functional classifier of tumor biochemical behavior to augment static marker assessment...
December 11, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/29227184/curing-of-psi-by-hsp104-overexpression-clues-to-solving-the-puzzle
#11
Lois E Greene, Xiaohong Zhao, Evan Eisenberg
The yeast [PSI+] prion, which is the amyloid form of Sup35, has the unusual property of being cured not only by the inactivation of, but also by the overexpression of Hsp104. Even though this latter observation was made more than two decades ago, the mechanism of curing by Hsp104 overexpression has remained controversial. This question has been investigated in depth by our laboratory by combining live cell imaging of GFP-labeled Sup35 with standard plating assays of yeast overexpressing Hsp104. We will discuss why the curing of [PSI+] by Hsp104 overexpression is not compatible with a mechanism of either inhibition of severing of the prion seeds or asymmetric segregation of the seeds...
December 11, 2017: Prion
https://www.readbyqxmd.com/read/29227108/visualizing-single-cell-secretion-dynamics-with-single-protein-sensitivity
#12
Matthew McDonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Völkl, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar
Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT), and is demonstrated with Laz388 cells-an Epstein-Barr virus (EBV) transformed B cell line...
December 11, 2017: Nano Letters
https://www.readbyqxmd.com/read/29226684/quantum-dot-based-luminescent-nanoprobes-for-sigma-2-receptor-imaging
#13
Maria Laura Pati, Elisabetta Fanizza, Sonja Hager, Diana Groza, Petra Heffeter, Amelita Grazia Laurenza, Valentino Laquintana, Maria Lucia Curri, Nicoletta Depalo, Carmen Abate, Nunzio Denora
The increasing importance of sigma-2 receptor as target for the diagnosis and therapy of tumours paves the way for the development of innovative optically traceable fluorescent probes as tumour cell contrast and therapeutic agents. Here, a novel hybrid organic-inorganic nanostructure is developed by combining the superior fluorescent properties of inorganic quantum dots (QDs), coated with a hydrophilic silica shell (QD@SiO2 NPs), the versatility of the silica shell and the high selectivity for sigma-2 receptors of the two synthetic ligands, namely the 6-[(6-aminohexyl)oxy]-2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one (MLP66) and 6-[1-[3-(4-cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy]hexylamine (TA6)...
December 11, 2017: Molecular Pharmaceutics
https://www.readbyqxmd.com/read/29226666/fluorogenic-detection-of-monoamine-neurotransmitters-in-live-cells
#14
Kallol Bera, Anand Kant Das, Ananya Rakshit, Bidyut Sarkar, Anoop Rawat, Barun Kumar Maity, Sudipta Maiti
Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g. setrotonin, dopamine and nor-epinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λex = 401 nm and λem = 490 nm for serotonin; λex = 446 nm and λem = 557 nm for dopamine; and λex = 446 nm and λem = 544 nm for nor-epinephrine) are accessible to most modern confocal imaging instruments...
December 11, 2017: ACS Chemical Neuroscience
https://www.readbyqxmd.com/read/29226432/multi-functional-peptide-microrna-nanocomplex-for-targeted-microrna-delivery-and-function-imaging
#15
Xiao Xiao, Xingxing Wang, Yuqi Wang, Tianren Yu, Lei Huang, Lei Chen, Jinbo Li, Chenyu Zhang, Yan Zhang
Targeted delivery of microRNA (miRNA) mimics into specific cells/tissues and real-time monitoring on the biological function of delivered miRNA mimics at molecular level represent two major challenges in the development of miRNA-based therapeutics. Here we report a highly efficient method to address these two challenges simultaneously by using the self-assembled nanocomplex formed by miRNA mimics with a multi-functional peptide conjugate. Using the nanocomplex formed by tumor-suppressive miR-34a and the multi-functional peptide conjugate FA-R9-FPcas3, we demonstrated the highly efficient and target-selective delivery of miR-34a into HeLa cells and tumors...
December 10, 2017: Chemistry: a European Journal
https://www.readbyqxmd.com/read/29226240/optical-computed-tomography-for-spatially-isotropic-four-dimensional-imaging-of-live-single-cells
#16
Laimonas Kelbauskas, Rishabh Shetty, Bin Cao, Kuo-Chen Wang, Dean Smith, Hong Wang, Shi-Hui Chao, Sandhya Gangaraju, Brian Ashcroft, Margaret Kritzer, Honor Glenn, Roger H Johnson, Deirdre R Meldrum
Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image...
December 2017: Science Advances
https://www.readbyqxmd.com/read/29225029/vagus-motor-neuron-topographic-map-determined-by-parallel-mechanisms-of-hox5-expression-and-time-of-axon-initiation
#17
Gabrielle R Barsh, Adam J Isabella, Cecilia B Moens
Many networks throughout the nervous system are organized into topographic maps, where the positions of neuron cell bodies in the projecting field correspond with the positions of their axons in the target field. Previous studies of topographic map development show evidence for spatial patterning mechanisms, in which molecular determinants expressed across the projecting and target fields are matched directly in a point-to-point mapping process. Here, we describe a novel temporal mechanism of topographic map formation that depends on spatially regulated differences in the timing of axon outgrowth and functions in parallel with spatial point-to-point mapping mechanisms...
December 1, 2017: Current Biology: CB
https://www.readbyqxmd.com/read/29223023/live-cell-imaging-reveals-different-modes-of-cytotoxic-action-of-extracts-derived-from-commonly-used-luting-cements
#18
Rita Trumpaitė-Vanagienė, Alina Čebatariūnienė, Virginijus Tunaitis, Alina Pūrienė, Augustas Pivoriūnas
OBJECTIVE: To compare cytotoxicity of extracts derived from commonly used luting cements: Hoffmann's Zinc Phosphate (ZPC), GC Fuji Plus Resin Modified Glass Ionomer (RMGIC) and 3M ESPE RelyX Unicem Resin Cement (RC) on primary human gingival fibroblasts (HGFs). DESIGN: HGFs were exposed to different concentrations of the ZPC, RMGIC and RC extracts. The cytotoxicity was assessed with the PrestoBlue Cell Viability Reagent and viable cells were counted by a haemocytometer using the trypan blue exclusion test...
November 27, 2017: Archives of Oral Biology
https://www.readbyqxmd.com/read/29222790/site-specific-direct-labeling-of-neurotrophins-and-their-receptors-from-biochemistry-to-advanced-imaging-applications
#19
Francesco Gobbo, Fulvio Bonsignore, Rosy Amodeo, Antonino Cattaneo, Laura Marchetti
We describe here a versatile methodological platform to achieve site-directed and stoichiometry-controlled labeling of neurotrophins and their receptors with various probes, ranging from biotin to small organic dyes. This labeling method works in vitro on purified neurotrophins as well as in a living cell context, where it achieves selective labeling of surface-exposed neurotrophin receptors. Here, we list all experimental details of our labeling protocols, along with examples of the wide range of applications in which these can be used...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29222784/methods-for-quantitative-analysis-of-axonal-cargo-transport
#20
Matias Alloatti, Luciana Bruno, Tomas L Falzone
Neurons rely on complex axonal transport mechanisms that mediate the intracellular dynamics of proteins, vesicles, and mitochondria along their high polarized structure. The fast improvement of live imaging techniques of fluorescent cargos allowed the identification of the diverse motion properties of different transported molecules. These properties arise as the result of molecular interactions between many players involved in axonal transport. Motor proteins, microtubule tracks, cargo association, and even axonal viscosity contribute to the proper axonal dynamics of different cargos...
2018: Methods in Molecular Biology
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