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Ester Jiménez-Moreno, Zijian Guo, Bruno L Oliveira, Inês S Albuquerque, Annabel Kitowski, Ana Guerreiro, Omar Boutureira, Tiago Rodrigues, Gonzalo Jiménez-Osés, Gonçalo J L Bernardes
The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol-containing molecules and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics...
December 8, 2016: Angewandte Chemie
Yilin Wang, Pakorn Kanchanawong
Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale...
December 1, 2016: Journal of Visualized Experiments: JoVE
David S Kittle, Fartash Vasefi, Chirag G Patil, Adam Mamelak, Keith L Black, Pramod V Butte
The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data...
December 8, 2016: Scientific Reports
Sergey Alyatkin, Ilya Asharchuk, Kirill Khaydukov, Andrey Nechaev, Oleg Lebedev, Yuri Vainer, Vladimir Semchishen, Evgeny Khaydukov
The mechanism of upconversion at the nanoscale is still under discussion. In this paper, we report on the experimental results of anti-Stokes luminescence kinetics in the upconversion nanoparticles of β-NaYF4: 20%Yb(3+); 0.6%Tm(3+). The parameters of the luminescence kinetics were found to be unambiguously dependent on the number of excitation quanta n, which are necessary for certain transitions between the energy states of thulium ions. The observed correlation has been explained by means of the long-lasting energy migration between the ytterbium ions...
December 8, 2016: Nanotechnology
Dan-Dan Tao, Qian Wang, Xiao-Sheng Yan, Na Chen, Zhao Li, Yun-Bao Jiang
We report that in the Ag(+) coordination polymers of a chiral thiol ligand containing an AIE fluorophore, tetraphenylethene (TPE), the TPE chromophores experience H-type aggregation, and yet a substantial enhancement of the fluorescence is observed, though to a lesser extent than that in the aggregates of the thiol ligand itself, which undergoes J-type aggregation. We show that this is not due to the difference in the freedom of the rotation of the fluorophore in the two types of aggregate, but is due to a small increase in the radiation rate constant in the coordination polymers while the much higher radiationless rate constant remains more or less unchanged...
December 8, 2016: Chemical Communications: Chem Comm
Huanyao Gao, Wei Sun, Zhiquan Song, Yanbao Yu, Li Wang, Xian Chen, Qisheng Zhang
Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motif coupled with bio-orthogonal chemistry has been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1)...
December 7, 2016: Chembiochem: a European Journal of Chemical Biology
Jia-Ren Lin, Mohammad Fallahi-Sichani, Jia-Yun Chen, Peter K Sorger
Cyclic Immunofluorescence (CycIF) is a public-domain method for performing highly multiplexed immunofluorescence imaging using a conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up to 30 channels by sequential 4- to 6-channel imaging followed by fluorophore inactivation. Three variant methods are described, the most generally useful of which involves staining fixed cells with antibodies directly conjugated to Alexa Fluor dyes and imaging in four colors, inactivating fluorophores using a mild base in the presence of hydrogen peroxide and light, and then performing another round of staining and imaging...
December 7, 2016: Current Protocols in Chemical Biology
Wenjuan Cai, Huizhi Guang, Chuangjian Cai, Jianwen Luo
Quantitative evaluation of hindlimb ischemia is essential for early diagnosis and therapy of peripheral arterial disease (PAD). Dynamic imaging using near-infrared (NIR) fluorophore indocyanine green (ICG) is a noninvasive and effective tool to monitor multiple vascular parameters including perfusion rate (PR), perfusion vascular density (PVD) and hemodynamics. It has been previously demonstrated that temperature changes could lead to significant variations of blood flow rate and vascular perfusion. In this paper, multiparametric evaluation of hindlimb ischemia was performed at different temperatures...
December 7, 2016: Journal of Biophotonics
Nobuo Yoshimoto, Shun'ichi Kuroda
Tyrosine phosphorylation is an essential posttranslational modification in intracellular signaling molecules. Since tyrosine phosphorylation occurs in less than 0.1 % of all phosphorylated amino acids in mammalian cells, it is difficult to detect the nascent phosphotyrosine at a high signal-to-noise ratio due to high intracellular backgrounds (i.e., unexpected crosstalks among endogenous signaling molecules). In order to address this issue, we reconstituted the mammalian signaling pathway involving an extracellular ligand and a receptor tyrosine kinase (RTK) in Saccharomyces cerevisiae, a lower eukaryote that lacks endogenous tyrosine kinases...
2017: Methods in Molecular Biology
Sarah Willkomm, Adrian Zander, Dina Grohmann
Deciphering the molecular mechanisms of eukaryotic Argonaute proteins is crucial for the understanding of RNA interference (RNAi), a posttranscriptional gene silencing process. Fluorescence-based single-molecule studies like single-molecule Förster resonance energy transfer (FRET) between a donor and acceptor dye represent a versatile tool to gain a mechanistic understanding of the structural dynamics of a biomolecular complex. Until today it was not possible to site-specifically introduce fluorophores into eukaryotic Argonaute...
2017: Methods in Molecular Biology
Pierre Adumeau, Kathryn E Carnazza, Christian Brand, Sean D Carlin, Thomas Reiner, Brian J Agnew, Jason S Lewis, Brian M Zeglis
The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of strategies for the multimodal PET/NIRF imaging of cancer a very enticing prospect. Indeed, in the context of colorectal cancer, a single multimodal PET/NIRF imaging agent could be used to stage the disease, identify candidates for surgical intervention, and facilitate the image-guided resection of the disease. While antibodies have proven to be highly effective vectors for the delivery of radioisotopes and fluorophores to malignant tissues, the use of radioimmunoconjugates labeled with long-lived nuclides such as (89)Zr poses two important clinical complications: high radiation doses to the patient and the need for significant lag time between imaging and surgery...
2016: Theranostics
Dirk N H Meineke, Mariano L Bossi, Haisen Ta, Vladimir N Belov, Stefan W Hell
Electronic energy transfer (EET) between chromophores is of fundamental importance for many biological processes and optoelectronic devices. However, common models fall short in fully describing the process, especially in bichromophoric model systems with a donor and acceptor connected by a rigid linker providing perpendicular geometries. Herein, we report a novel strategy to prepare bichromophores containing adamantane or 2-(2-adamantylidene)-adamantane as rigid spacers, providing a fixed distance between chromophores, and their parallel or perpendicular arrangement without chromophore rotation...
December 6, 2016: Chemistry: a European Journal
Manu Ben-Johny, Daniel N Yue, David T Yue
The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex...
December 6, 2016: Nature Communications
Anusuya Banerjee, Thomas Pons, Nicolas Lequeux, Benoit Dubertret
Semiconductor nanoparticles particularly quantum dots (QDs) are interesting alternatives to organic fluorophores for a range of applications such as biosensing, imaging and therapeutics. Addition of a programmable scaffold such as DNA to QDs further expands the scope and applicability of these hybrid nanomaterials in biology. In this review, the most important stages of preparation of QD-DNA conjugates for specific applications in biology are discussed. Special emphasis is laid on (i) the most successful strategies to disperse QDs in aqueous media, (ii) the range of different conjugation with detailed discussion about specific merits and demerits in each case, and (iii) typical applications of these conjugates in the context of biology...
December 6, 2016: Interface Focus
Emilie Allard-Vannier, Katel Hervé-Aubert, Karine Kaaki, Thibaut Blondy, Anastasia Shebanova, Konstantin V Shaitan, Anastasia A Ignatova, Marie-Louise Saboungi, Alexey V Feofanov, Igor Chourpa
BACKGROUND: This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. METHODS: SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, LSCM with single-photon and two-photon excitation...
December 2, 2016: Biochimica et Biophysica Acta
Debabrata Maity, Mao Li, Martin Ehlers, Carsten Schmuck
We report a fluorescence probe 1, which contains a naphthalimide fluorophore with two symmetric peptidic arms equipped with a tailor-made anion-binding motif, the guanidiniocarbonyl pyrrole moiety, for the detection of nucleoside triphosphates. Upon binding to nucleoside triphosphates, especially ATP, 1 shows significant turn-on fluorescence response. Probe 1 can also be applied for the imaging of ATP in cells.
December 5, 2016: Chemical Communications: Chem Comm
Mahmoud Razavian, Thomas Bordenave, Dimitris Georgiadis, Fabrice Beau, Jiasheng Zhang, Reza Golestani, Jakub Toczek, Jae-Joon Jung, Yunpeng Ye, Hye-Yeong Kim, Jinah Han, Vincent Dive, Laurent Devel, Mehran M Sadeghi
Matrix metalloproteinase (MMP)-12 plays a key role in the development of aneurysm. Like other members of MMP family, MMP-12 is produced as a proenzyme, mainly by macrophages, and undergoes proteolytic activation to generate an active form. Accordingly, molecular imaging of the MMP-12 active form can inform of the pathogenic process in aneurysm. Here, we developed a novel family of fluorescent probes based on a selective MMP-12 inhibitor, RXP470.1 to target the active form of MMP-12. These probes were stable in complex media and retained the high affinity and selectivity of RXP470...
December 5, 2016: Scientific Reports
F Belal, F Ibrahim, Z A Sheribah, H Alaa
In this paper, a simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of entacapone (ETC). The proposed method is based on forming a highly fluorescent product through the reduction of ETC with Zn/HCl. The produced fluorophore exhibits strong fluorescence at λem 345 nm after excitation at λex 240 nm. The use of fluorescence enhancers such as Tween-80 and carboxy methyl cellulose (CMC) greatly enhanced the fluorescence of the produced fluorophore by 150% and 200%, respectively...
December 5, 2016: Luminescence: the Journal of Biological and Chemical Luminescence
Marcel von der Haar, Christopher Heuer, Martin Pähler, Kathrin von der Haar, Patrick Lindner, Thomas Scheper, Frank Stahl
The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores' susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS)...
November 30, 2016: Biology
Brandon D Burch, Carolina Garrido, David M Margolis
Many techniques currently used to measure HIV RNA production in cells suffer from limitations that include high background signal or the potential to destroy cellular context. Fluorophore-binding RNA aptamers offer the potential for visualizing RNAs directly in living cells with minimal cellular perturbation. We inserted a sequence encoding a fluorophore-binding RNA aptamer, known as Spinach, into the HIV genome such that predicted RNA secondary structures in both Spinach and HIV were preserved. Chimeric HIV-Spinach RNAs were functionally validated in vitro by testing their ability to enhance the fluorescence of a conditional fluorophore (DFHBI), which specifically binds Spinach...
November 30, 2016: Virus Research
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