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Jochem Deen, Su Wang, Sven Van Snick, Volker Leen, Kris Janssen, Johan Hofkens, Robert K Neely
The methyltransferase enzymes can be applied to deliver a range of modifications to pre-determined sites on large DNA molecules with exceptional specificity and efficiency. To date, however, a limited number of modifications have been delivered in this way because of the complex chemical synthesis that is needed to produce a cofactor analogue carrying a specific function, such as a fluorophore. Here, we describe a method for the direct transfer of a series of functional compounds (seven fluorescent dyes, biotin and polyethylene glycol) to the DNA duplex...
March 13, 2018: Nucleic Acids Research
Henrik Lauridsen, Casper Bindzus Foldager, Line Hansen, Michael Pedersen
Non-invasive methods to track the progress of stem cell therapies are important in the development of future regenerative therapies. Super-paramagnetic iron oxide particles (SPIOs) have previously been applied to track cells using magnetic resonance imaging (MRI) in vivo in non-regenerative animal models. To the best of the author's knowledge, the present study investigated for the first time, the feasibility of tracking SPIO labeled cells in an intrinsic regenerative environment, the regenerating limb of the axolotl, and investigated the homing of stem cell-like blastema cells to the regenerative zone...
April 2018: Experimental and Therapeutic Medicine
Amy M Bittel, Ashley M Davis, Lei Wang, Michel A Nederlof, Jorge O Escobedo, Robert M Strongin, Summer L Gibbs
Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five...
March 15, 2018: Scientific Reports
Qianyi Zhang, Evan G R Davies, James R Bolton, Yang Liu
Monochloramine (NH2 Cl) is widely used for secondary disinfection by water utilities. However, Edmonton field stormwater sampling results have shown that NH2 Cl, because of its long-lasting property, can cause stormwater contamination through outdoor potable water uses during the summer season. To protect water sources, it is important to understand NH2 Cl dissipation mechanisms in stormwater. Natural organic matter (NOM) is the dominant species that contributes to NH2 Cl decay in stormwater. In this research, it is proposed that NOM reacted with both NH2 Cl and free chlorine through rapid and long-term reactions during NH2 Cl dissipation...
March 12, 2018: Science of the Total Environment
Nicole M Cordina, Nima Sayyadi, Lindsay M Parker, Arun Everest-Dass, Louise J Brown, Nicolle H Packer
Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules...
March 14, 2018: Scientific Reports
James L Kingsley, Jeffrey P Bibeau, S Iman Mousavi, Cem Unsal, Zhilu Chen, Xinming Huang, Luis Vidali, Erkan Tüzel
Fluorescence recovery after photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus, or cell membrane. Although many FRAP models have been developed over the past decades, the influence of the complex boundaries of 3D cellular geometries on the recovery curves, in conjunction with regions of interest and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied...
March 13, 2018: Biophysical Journal
David Parker, Edward Walter, Gareth Williams
A series of three europium(III) complexes has been created in which an APTRA moiety has been integrated into the sensitising chromophore (APTRA = o-aminophenol-N,N,N-triacetate). The constitutionally isomeric complexes EuL1 and EuL2 feature the APTRA unit linked to a metal-bound pyridine ring through an alkynyl unit, differing according to the disposition of the APTRA substituents relative to the C≡C unit (para-N and para-O respectively). In EuL3, the APTRA ring is directly bonded to the Eu-coordinated pyridine (para-O)...
March 13, 2018: Chemistry: a European Journal
John Quan Minh Nguyen, Melanie McWade, Giju Thomas, Bryce T Beddard, Jennifer L Herington, Bibhash C Paria, Herbert S Schwartz, Jennifer L Halpern, Ginger E Holt, Anita Mahadevan-Jansen
Fluorescence imaging is a well-established optical modality that has been used to localize and track fluorophores in vivo and has demonstrated great potential for surgical guidance. Despite the variety of fluorophores currently being researched, many existing intraoperative fluorescence imaging systems are specifically designed for a limited number of applications. We present a modular wide-field fluorescence overlay tissue imaging system for intraoperative surgical guidance that is comprised of commercially available standardized components...
April 2018: Journal of Medical Imaging
Kuo-Ting Chen, Christian Ieritano, Yann Seimbille
Herein, we describe a synthetic strategy for the regioselective labeling of peptides by using a bioorthogonal click reaction between 2-cyanobenzothiazole (CBT) and a 1,2-aminothiol moiety. This methodology allows for the facile and site-specific modification of peptides with various imaging agents, including fluorophores and radioisotope-containing prosthetic groups. We investigated the feasibility of an early-stage incorporation of dipeptide 1 into targeting vectors, such as c[RGDyK(C)] and HER2 pep , during solid-phase peptide synthesis...
March 2018: ChemistryOpen
Agnese Secondo, Alba Esposito, Tiziana Petrozziello, Francesca Boscia, Pasquale Molinaro, Valentina Tedeschi, Anna Pannaccione, Roselia Ciccone, Natascia Guida, Gianfranco Di Renzo, Lucio Annunziato
Nuclear envelope (NE) is a Ca2+ -storing organelle controlling neuronal differentiation through nuclear Ca2+ concentrations ([Ca2+ ]n ). However, how [Ca2+ ]n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na+ /Ca2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3'K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation...
December 2018: Cell Death Discovery
Jetty van Ginkel, Mike Filius, Malwina Szczepaniak, Pawel Tulinski, Anne S Meyer, Chirlmin Joo
Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides...
March 12, 2018: Proceedings of the National Academy of Sciences of the United States of America
Christina Boeck, Sophia Pfister, Alexander Bürkle, Valerie Vanhooren, Claude Libert, Juan Salinas-Manrique, Detlef E Dietrich, Iris-Tatjana Kolassa, Alexander Karabatsiakis
BACKGROUND: Glycans are short chains of saccharides linked to glycoproteins that are known to be involved in a wide range of inflammatory processes. As depression has been consistently associated with chronic low-grade inflammation, we asked whether patients with Major Depressive Disorder show alterations in the N-glycosylation pattern of serum proteins that might be linked to associated changes in inflammatory processes. METHODS: In a study cohort of 21 female patients with an acute depressive episode and 21 non-depressed female control subjects aged between 50 and 69 years, we analyzed the serum N-glycan profile by DNA Sequencer Adapted-Fluorophore Assisted Carbohydrate Electrophoresis (DSA-FACE) and assessed the serum levels of interleukin (IL)- 6, tumor necrosis factor (TNF)-α and C-reactive protein (CRP) by chemiluminescence immunoassays and nephelometry...
February 27, 2018: Journal of Affective Disorders
Katia Pane, Mariavittoria Verrillo, Angela Avitabile, Elio Pizzo, Mario Varcamonti, Anna Zanfardino, Antimo Di Maro, Camilla Rega, Angela Amoresano, Viviana Izzo, Alberto Di Donato, Valeria Cafaro, Eugenio Notomista
Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence...
March 12, 2018: Bioconjugate Chemistry
Finub James Shirley, Pieter Neutens, Rita Vos, Md Mahmud-Ul-Hasan, Liesbet Lagae, Niels Verellen, Pol Van Dorpe
Most fluorescent immunoassays require a wash step prior to read-out due to the otherwise overwhelming signal of the large number of unbound (bulk) fluorescent molecules, that dominate over the signal from the molecules of interest, usually bound to a substrate. Supercritical angle fluorescence (SAF) sensing is one of the most promising alternatives to total internal reflection fluorescence for fluorescence imaging and sensing. However, detailed experimental investigation of the influence of collection angle on the SAF surface sensitivity, i...
March 12, 2018: Analytical Chemistry
Qing Hu, Qing Chen, Xiuyun Yan, Bomei Ding, Dawei Chen, Lifang Cheng
AIM: To develop a nanocarrier for targeted delivery of agents to the cartilage. MATERIALS & METHODS: Chondrocyte affinity peptide modified PEGylated polyamidoamine conjugates (CAP-PEG-PAMAM) were prepared and rhodamine B isothiocyanate (RB) fluorophore was linked on them for comparative biological tracing and profiling. RESULTS: CAP4-PP-RB exhibited much more efficient cellular uptake in vitro than that of PEG-PAMAM-RB. Both the conjugates were likely internalized by chondrocytes via clathrin and caveolin co-mediated endocytosis, and delivered to lysosomes...
March 12, 2018: Nanomedicine
Beatriz Lagunas, Liam Walker, Rana Muhammad Fraz Hussain, Ian Hands-Portman, Kate Woolley-Allen, Miriam L Gifford
Cell type-specific marker lines expressing fluorophores such as GFP or GUS can be used as starting material from which single cell types can be isolated by fluorescence-activated cell sorting (FACS) and/or for the study of root development. Establishing the stability of these lines is an essential step prior to further study to ensure that marker expression and localization is stable over time and during environmental perturbations of interest to researchers applying these lines as treatments. Here, we detail the use of root cross sectioning to investigate marker expression throughout the length and width of the root using the model legume Medicago truncatula as an example...
2018: Methods in Molecular Biology
Lauren E Jamieson, Jennifer Greaves, Jayde A McLellan, Kevin R Munro, Nicholas C O Tomkinson, Luke H Chamberlain, Karen Faulds, Duncan Graham
Intracellular uptake, distribution and metabolism of lipids are tightly regulated characteristics in healthy cells. An analytical technique capable of understanding these characteristics with a high level of species specificity in a minimally invasive manner is highly desirable in order to understand better how these become disrupted during disease. In this study, the uptake and distribution of three different alkyne tagged fatty acids in single cells were monitored and compared, highlighting the ability of Raman spectroscopy combined with alkyne tags for better understanding of the fine details with regard to uptake, distribution and metabolism of very chemically specific lipid species...
March 7, 2018: Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
Ali Bavali, Parviz Parvin, Mohammad Tavassoli, Mohammad Reza Mohebbifar
Angular dependence of the intensity and the emissive wavelength of the laser-induced fluorescence emission in hybrid media (fluorophores+nanoparticles) are investigated using various TiO2 densities as guest nanoscatterers in the ethanolic solutions of the host Rd6G and coumarin 4 (C4) molecules. It is shown that the intensity of the scattered photons varies in terms of the detection angle. When the nanoscatterer density increases at a certain excitation energy, the angular anisotropy enhances. While the emissive wavelength exhibits the spectral shift in terms of the angular variation for Rd6G fluorophores, it remains invariant for C4-based suspension...
March 1, 2018: Applied Optics
Stephen Donaldson, Hilton B de Aguiar
Over recent decades, lipid membranes have become standard models for examining the biophysics and biochemistry of cell membranes. Interrogation of lipid domains within biomembranes is generally done with fluorescence microscopy via exogenous chemical probes. However, fluorophores have limited partitioning tunability, with the majority segregating into the liquid-disordered phase, and fluorescence only strictly reports on the small percentage of tagged lipids. Here, we present simple, label-free imaging of domain formation in lipid monolayers, with chemical selectivity in unraveling lipid and cholesterol composition in different domain types...
March 9, 2018: Journal of Physical Chemistry Letters
Lifang Guo, Minggang Tian, Ruiqing Feng, Ge Zhang, Ruoyao Zhang, Xuechen Li, Zhiqiang Liu, Xiuquan He, Jing Zhi Sun, Xiaoqiang Yu
Lipid droplets (LDs) with own interface architecture not only play crucial roles in protecting cell from lipotoxicity and lipoapoptosis but also closely relate with many diseases such as fatty liver and diabetes. Thus, as one of important applied biomaterials, fluorescent probes with ultrahigh selectivity for in-situ and high-fidelity imaging LDs in living cells and tissues are critical to elucidate relevant physiological and pathological events as well as detect related diseases. However, available LDs probes only utilizing their waterless neutral cores but ignoring the unique phospholipid monolayer interfaces exhibit low selectivity...
March 9, 2018: ACS Applied Materials & Interfaces
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