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Targeted Imaging of VCAM-1 mRNA in a Mouse Model of Laser-Induced Choroidal Neovascularization Using Antisense Hairpin-DNA-Functionalized Gold-Nanoparticles.
Uddin MI, Kilburn TC, Yang R, McCollum GW, Wright DW, Penn JS
(2018) Mol Pharm 15: 5514-5520
MeSH Terms: Animals, Biomarkers, Choroid, Choroidal Neovascularization, Disease Models, Animal, Fluorescent Dyes, Gold, Humans, Intravital Microscopy, Lasers, Male, Metal Nanoparticles, Mice, Mice, Inbred C57BL, Molecular Imaging, Molecular Probes, Oligodeoxyribonucleotides, Antisense, Optical Imaging, RNA, Messenger, Vascular Cell Adhesion Molecule-1, Wet Macular Degeneration
Show Abstract · Added April 10, 2019
Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.
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21 MeSH Terms
Photostable, hydrophilic, and near infrared quaterrylene-based dyes for photoacoustic imaging.
Yu J, Pin S, Lin X, Su M, Bai M, Kim K
(2018) Mater Sci Eng C Mater Biol Appl 93: 1012-1019
MeSH Terms: Cell Line, Tumor, Contrast Media, Dendrimers, Fluorescent Dyes, Humans, Infrared Rays, Optical Imaging, Photoacoustic Techniques
Show Abstract · Added April 2, 2019
Novel near-infrared contrast agents based on the quaterrylene structure were strategically developed and tested for high photo-stability. Both a dendrimeric quaterrylene molecule, QR-G2-COOH, and a small molecule cationic quaterrylene dye, QR-4PyC4, remain optically stable and continue to generate a competitive photoacoustic response when irradiated by short near-infrared laser pulses for a relatively long time in an in-vitro cell study, unlike indocyanine green that rapidly decreases photoacoustic signal amplitude. The small molecule dye, QR-4PyC4 exhibits not only significantly higher cellular uptake rate than QR-G2-COOH and indocyanine green, but also low toxicity at a concentration of up to 10 μM. The dendrimeric dye, QR-G2-COOH that has surface functional groups available for conjugation with targeting and therapeutic agents shows the highest photoacoustic amplitude with high optical stability. Therefore, QR-4PyC4 can be a promising universal, sensitive and reliable photoacoustic contrast agent and QR-G2-COOH has great potential as a nano-platform with stable photoacoustic imaging capability.
Copyright © 2018 Elsevier B.V. All rights reserved.
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Rhodol-based thallium sensors for cellular imaging of potassium channel activity.
Dutter BF, Ender A, Sulikowski GA, Weaver CD
(2018) Org Biomol Chem 16: 5575-5579
MeSH Terms: Fluorescent Dyes, HEK293 Cells, Humans, Methylation, Microscopy, Confocal, Optical Imaging, Potassium Channels, Spectrometry, Fluorescence, Thallium, Xanthones
Show Abstract · Added April 10, 2019
Thallium (Tl+) flux assays enable imaging of potassium (K+) channel activity in cells and tissues by exploiting the permeability of K+ channels to Tl+ coupled with a fluorescent Tl+ sensitive dye. Common Tl+ sensing dyes utilize fluorescein as the fluorophore though fluorescein exhibits certain undesirable properties in these assays including short excitation wavelengths and pH sensitivity. To overcome these drawbacks, the replacement of fluorescein with rhodols was investigated. A library of 13 rhodol-based Tl+ sensors was synthesized and their properties and performance in Tl+ flux assays evaluated. The dimethyl rhodol Tl+ sensor emerged as the best of the series and performed comparably to fluorescein-based sensors while demonstrating greater pH tolerance in the physiological range and excitation and emission spectra 30 nm red-shifted from fluorescein.
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In vivo bioluminescence imaging of labile iron accumulation in a murine model of infection.
Aron AT, Heffern MC, Lonergan ZR, Vander Wal MN, Blank BR, Spangler B, Zhang Y, Park HM, Stahl A, Renslo AR, Skaar EP, Chang CJ
(2017) Proc Natl Acad Sci U S A 114: 12669-12674
MeSH Terms: 2,2'-Dipyridyl, Acinetobacter Infections, Acinetobacter baumannii, Anemia, Iron-Deficiency, Animals, Cation Transport Proteins, Cations, Divalent, Disease Models, Animal, Ferric Compounds, Firefly Luciferin, Fluorescent Dyes, Gene Expression Regulation, Hepcidins, Homeostasis, Iron, Iron Overload, Iron Regulatory Protein 1, Iron Regulatory Protein 2, Luminescent Measurements, Mice, Mice, Transgenic, Quaternary Ammonium Compounds, Receptors, Transferrin, Signal Transduction, Transferrin
Show Abstract · Added March 15, 2018
Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.
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25 MeSH Terms
In Vivo Imaging of Retinal Hypoxia Using HYPOX-4-Dependent Fluorescence in a Mouse Model of Laser-Induced Retinal Vein Occlusion (RVO).
Uddin MI, Jayagopal A, McCollum GW, Yang R, Penn JS
(2017) Invest Ophthalmol Vis Sci 58: 3818-3824
MeSH Terms: Animals, Disease Models, Animal, Fluorescein Angiography, Fluoresceins, Fluorescent Dyes, Hypoxia, Image Processing, Computer-Assisted, Macular Edema, Male, Mice, Mice, Inbred C57BL, Nitroimidazoles, Radiation-Sensitizing Agents, Retinal Neovascularization, Retinal Vein, Retinal Vein Occlusion, Tomography, Optical Coherence
Show Abstract · Added December 21, 2017
Purpose - To demonstrate the utility of a novel in vivo molecular imaging probe, HYPOX-4, to detect and image retinal hypoxia in real time, in a mouse model of retinal vein occlusion (RVO).
Methods - Retinal vein occlusion was achieved in adult mice by photodynamic retinal vein thrombosis (PRVT). One or two major retinal vein(s) was/were occluded in close proximity to the optic nerve head (ONH). In vivo imaging of retinal hypoxia was performed using, HYPOX-4, an imaging probe developed by our laboratory. Pimonidazole-adduct immunostaining was performed and used as a standard ex vivo method for the detection of retinal hypoxia in this mouse RVO model. The retinal vasculature was imaged using fluorescein angiography (FA) and isolectin B4 staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) analysis.
Results - By application of the standard ex vivo pimonidazole-adduct immunostaining technique, retinal hypoxia was observed within 2 hours post-PRVT. The observed hypoxic retinal areas depended on whether one or two retinal vein(s) was/were occluded. Similar areas of hypoxia were imaged in vivo using HYPOX-4. Using OCT, retinal edema was observed immediately post-PRVT induction, resolving 8 days later. Nominal preretinal neovascularization was observed at 10 to 14 days post-RVO.
Conclusions - HYPOX-4 is an efficient probe capable of imaging retinal hypoxia in vivo, in RVO mice. Future studies will focus on its use in correlating retinal hypoxia to the onset and progression of ischemic vasculopathies.
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17 MeSH Terms
Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer.
Nakase H, Sakuma S, Fukuchi T, Yoshino T, Mohri K, Miyata K, Kumagai H, Hiwatari KI, Tsubaki K, Ikejima T, Tobita E, Zhu M, Wilson KJ, Washington K, Gore JC, Pham W
(2017) Int J Nanomedicine 12: 1747-1755
MeSH Terms: Adenocarcinoma, Adenoma, Antigens, Tumor-Associated, Carbohydrate, Colorectal Neoplasms, Fluorescent Dyes, Humans, Microscopy, Fluorescence, Molecular Probes, Nanoparticles, Optical Imaging, Peanut Agglutinin
Show Abstract · Added April 6, 2017
The Thomsen-Friedenreich (TF) antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident.
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11 MeSH Terms
Fluorescence-based measurement of cystine uptake through xCT shows requirement for ROS detoxification in activated lymphocytes.
Siska PJ, Kim B, Ji X, Hoeksema MD, Massion PP, Beckermann KE, Wu J, Chi JT, Hong J, Rathmell JC
(2016) J Immunol Methods 438: 51-58
MeSH Terms: Amino Acid Transport System y+, B-Lymphocytes, Cell Line, Tumor, Cellular Reprogramming, Cystine, Flow Cytometry, Fluorescein-5-isothiocyanate, Fluorescence, Fluorescent Dyes, Glutathione, Humans, Lymphocyte Activation, Microscopy, Fluorescence, Reactive Oxygen Species, Receptors, Antigen, T-Cell, Signal Transduction, T-Lymphocytes, Up-Regulation
Show Abstract · Added January 29, 2018
T and B lymphocytes undergo metabolic re-programming upon activation that is essential to allow bioenergetics, cell survival, and intermediates for cell proliferation and function. To support changes in the activity of signaling pathways and to provide sufficient and necessary intracellular metabolites, uptake of extracellular nutrients increases sharply with metabolic re-programming. One result of increased metabolic activity can be reactive oxygen species (ROS), which can be toxic when accumulated in excess. Uptake of cystine allows accumulation of cysteine that is necessary for glutathione synthesis and ROS detoxification. Cystine uptake is required for T cell activation and function but measurements based on radioactive labeling do not allow analysis on single cell level. Here we show the critical role for cystine uptake in T cells using a method for measurement of cystine uptake using a novel CystineFITC probe. T cell receptor stimulation lead to upregulation of the cystine transporter xCT (SLC7a11) and increased cystine uptake in CD4+ and CD8+ human T cells. Similarly, lipopolysaccharide stimulation increased cystine uptake in human B cells. The CystineFITC probe was not toxic and could be metabolized to prevent cystine starvation induced cell death. Furthermore, blockade of xCT or competition with natural cystine decreased uptake of CystineFITC. CystineFITC is thus a versatile tool that allows measurement of cystine uptake on single cell level and shows the critical role for cystine uptake for T cell ROS regulation and activation.
Copyright © 2016 Elsevier B.V. All rights reserved.
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18 MeSH Terms
In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy.
Uddin MI, Evans SM, Craft JR, Capozzi ME, McCollum GW, Yang R, Marnett LJ, Uddin MJ, Jayagopal A, Penn JS
(2016) Sci Rep 6: 31011
MeSH Terms: Animals, Disease Models, Animal, Fluorescent Dyes, Hypoxia, Intravital Microscopy, Mice, Neovascularization, Pathologic, Optical Imaging, Retina, Retinal Diseases
Show Abstract · Added October 14, 2016
Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.
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10 MeSH Terms
The use of fluorescently-tagged apoptolidins in cellular uptake and response studies.
Chong KM, Leelatian N, Deguire SM, Brockman AA, Earl D, Ihrie RA, Irish JM, Bachmann BO, Sulikowski GA
(2016) J Antibiot (Tokyo) 69: 327-30
MeSH Terms: A549 Cells, Biological Transport, Cell Line, Tumor, Flow Cytometry, Fluorescent Dyes, Humans, Macrolides, Staining and Labeling
Show Abstract · Added March 12, 2016
The apoptolidins are glycomacrolide microbial metabolites reported to be selectively cytotoxic against tumor cells. Using fluorescently tagged active derivatives we demonstrate selective uptake of these four tagged glycomacrolides in cancer cells over healthy human blood cells. We also demonstrate the utility of these five fluorescently tagged glycomacrolides in fluorescent flow cytometry to monitor cellular uptake of the six glycomacrolides and cellular response.
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8 MeSH Terms
Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.
Schlebach JP, Barrett PJ, Day CA, Kim JH, Kenworthy AK, Sanders CR
(2016) Biochemistry 55: 985-8
MeSH Terms: Amyloid beta-Protein Precursor, Caveolin 3, Cholesterol, Fluorescent Dyes, Humans, Hydrophobic and Hydrophilic Interactions, Membrane Microdomains, Microscopy, Confocal, Microscopy, Fluorescence, Models, Molecular, Myelin Proteins, Peptide Fragments, Phosphatidylcholines, Phosphatidylethanolamines, Protein Conformation, Protein Interaction Domains and Motifs, Recombinant Proteins, Rhodamines, Sphingomyelins, Unilamellar Liposomes
Show Abstract · Added February 12, 2016
The integration of membrane proteins into "lipid raft" membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.
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20 MeSH Terms