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Find video protocols related to scientific articles indexed in Pubmed.
Identification of a selective G-quadruplex DNA binder using a multistep virtual screening approach.
Chem. Commun. (Camb.)
PUBLISHED: 11-13-2014
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To efficiently identify small molecules binding to a G-quadruplex structure while avoiding binding to duplex DNA, we performed a multistep structure-based virtual screening by simultaneously taking into account G-quadruplex DNA and duplex DNA. Among the 13 compounds selected, one outstanding ligand shows significant selectivity for G-quadruplex binding as determined using SPR, FRET-based competition and luciferase activity assay.
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An Integrated Imaging Probe Design: The Synthesis of (99m) Tc/Re-Containing Macrocyclic Peptide Scaffolds.
Chemistry
PUBLISHED: 08-07-2014
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?-Sheets account for over 30?% of all secondary structural conformations found in proteins. The intramolecular hydrogen bonding that exists between the two peptide strands is imperative in maintaining this secondary structure. With the proper design, cyclic peptides may act as scaffolds emulating active ?-sheet regions, enabling investigation of their importance in molecular recognition and protein aggregation. Starting from Fmoc-Lys(Fmoc)-OH, macrocyclic peptides were synthesized on a solid support, with peptide-chain elongation extending from both the alpha and epsilon amines of the lysine. The branching peptides were cyclized with a pyridyl tridentate chelation core followed by coordination using [(99m) Tc/Re(CO)3 (H2 O)3 ](+) . Variable temperature (1) H?NMR spectroscopy studies were performed, demonstrating that intramolecular hydrogen bonding exists between the two sides of the uncoordinated macrocyclic peptide scaffolds. Additionally, computational modelling and circular dichroism spectroscopic analysis revealed that the peptide backbone exists in a similar conformation both before and after metal coordination. The ability to seamlessly incorporate a tridentate chelation core into the backbone of a macrocyclic peptide, without disrupting the secondary structure, can greatly assist in the design of metal-centric peptidomimetic imaging agents. This novel integrated imaging probe approach may facilitate the investigation into protein-protein interactions using macrocyclic ?-sheet scaffolds.
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Molecular targeted viral nanoparticles as tools for imaging cancer.
Methods Mol. Biol.
PUBLISHED: 06-21-2014
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Viral nanoparticles (VNPs) are a novel class of bionanomaterials that harness the natural biocompatibility of viruses for the development of therapeutics, vaccines, and imaging tools. The plant virus, cowpea mosaic virus (CPMV), has been successfully engineered to create novel cancer-targeted imaging agents by incorporating fluorescent dyes, polyethylene glycol (PEG) polymers, and targeting moieties. Using straightforward conjugation strategies, VNPs with high selectivity for cancer-specific molecular targets can be synthesized for in vivo imaging of tumors. Here we describe the synthesis and purification of CPMV-based VNPs, the functionalization of these VNPs using click chemistry, and their use for imaging xenograft tumors in animal models. VNPs decorated with fluorescent dyes, PEG, and targeting ligands can be synthesized in one day, and imaging studies can be performed over hours, days, or weeks, depending on the application.
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Dual-modal magnetic resonance and fluorescence imaging of atherosclerotic plaques in vivo using VCAM-1 targeted tobacco mosaic virus.
Nano Lett.
PUBLISHED: 02-17-2014
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The underlying cause of major cardiovascular events, such as myocardial infarctions and strokes, is atherosclerosis. For accurate diagnosis of this inflammatory disease, molecular imaging is required. Toward this goal, we sought to develop a nanoparticle-based, high aspect ratio, molecularly targeted magnetic resonance (MR) imaging contrast agent. Specifically, we engineered the plant viral nanoparticle platform tobacco mosaic virus (TMV) to target vascular cell adhesion molecule (VCAM)-1, which is highly expressed on activated endothelial cells at atherosclerotic plaques. To achieve dual optical and MR imaging in an atherosclerotic ApoE(-/-) mouse model, TMV was modified to carry near-infrared dyes and chelated Gd ions. Our results indicate molecular targeting of atherosclerotic plaques. On the basis of the multivalency and multifunctionality, the targeted TMV-based MR probe increased the detection limit significantly; the injected dose of Gd ions could be further reduced 400x compared to the suggested clinical use, demonstrating the utility of targeted nanoparticle cargo delivery.
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Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes.
Peptides
PUBLISHED: 01-16-2014
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Ghrelin and its receptor, the growth hormone secretagogue receptor (GHS-R), are expressed in the heart, and may function to promote cardiomyocyte survival, differentiation and contractility. Previously, we had generated a truncated analog of ghrelin conjugated to fluorescein isothiocyanate for the purposes of determining GHS-R expression in situ. We now report the generation and characterization of a far-red ghrelin analog, [Dpr(3)(octanoyl), Lys(19)(Cy5)]ghrelin (1-19), and show that it can be used to image changes in GHS-R in developing cardiomyocytes. We also generated the des-acyl analog, des-acyl [Lys(19)(Cy5)]ghrelin (1-19) and characterized its binding to mouse heart sections. Receptor binding affinity of Cy5-ghrelin as measured in HEK293 cells overexpressing GHS-R1a was within an order of magnitude of that of fluorescein-ghrelin and native human ghrelin, while the des-acyl Cy5-ghrelin did not bind GHS-R1a. Live cell imaging in HEK293/GHS-R1a cells showed cell surface labeling that was displaced by excess ghrelin. Interestingly, Cy5-ghrelin, but not the des-acyl analog, showed concentration-dependent binding in mouse heart tissue sections. We then used Cy5-ghrelin to track GHS-R expression in P19-derived cardiomyocytes. Live cell imaging at different time points after DMSO-induced differentiation showed that GHS-R expression preceded that of the differentiation marker aMHC and tracked with the contractility marker SERCA 2a. Our far-red analog of ghrelin adds to the tools we are developing to map GHS-R in developing and diseased cardiac tissues.
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High-Throughput Screening of One-Bead-One-Compound Peptide Libraries Using Intact Cells.
ACS Comb Sci
PUBLISHED: 07-19-2013
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Screening approaches based on one-bead-one-compound (OBOC) combinatorial libraries have facilitated the discovery of novel peptide ligands for cellular targeting in cancer and other diseases. Recognition of cell surface proteins is optimally achieved using live cells, yet screening intact cell populations is time-consuming and inefficient. Here, we evaluate the Complex Object Parametric Analyzer and Sorter (COPAS) large particle biosorter for high-throughput sorting of bead-bound human cell populations. When a library of RGD-containing peptides was screened against human cancer cells that express ?v?3 integrin, it was found that bead-associated cells are rapidly dissociated when sorted through the COPAS instrument. When the bound cells were reversibly cross-linked onto the beads, however, we demonstrated that cell/bead mixtures can be sorted quickly and accurately. This reversible cross-linking approach is compatible with matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based peptide sequence deconvolution. This approach should allow one to rapidly screen an OBOC library and identify novel peptide ligands against cell surface targets in their native conformation.
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Imaging of homeostatic, neoplastic, and injured tissues by HA-based probes.
Biomacromolecules
PUBLISHED: 12-12-2011
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An increase in hyaluronan (HA) synthesis, cellular uptake, and metabolism occurs during the remodeling of tissue microenvironments following injury and during disease processes such as cancer. We hypothesized that multimodality HA-based probes selectively target and detectably accumulate at sites of high HA metabolism, thus providing a flexible imaging strategy for monitoring disease and repair processes. Kinetic analyses confirmed favorable available serum levels of the probe following intravenous (i.v.) or subcutaneous (s.c.) injection. Nuclear (technetium-HA, (99m)Tc-HA, and iodine-HA, (125)I-HA), optical (fluorescent Texas Red-HA, TR-HA), and magnetic resonance (gadolinium-HA, Gd-HA) probes imaged liver ((99m)Tc-HA), breast cancer cells/xenografts (TR-HA, Gd-HA), and vascular injury ((125)I-HA, TR-HA). Targeting of HA probes to these sites appeared to result from selective HA receptor-dependent localization. Our results suggest that HA-based probes, which do not require polysaccharide backbone modification to achieve favorable half-life and distribution, can detect elevated HA metabolism in homeostatic, injured, and diseased tissues.
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Synthesis, radiometal labeling and in vitro evaluation of a targeted PPIX derivative.
Appl Radiat Isot
PUBLISHED: 09-08-2011
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In pursuit of a single molecule with potential applications for both in vitro fluorescence microscopy and in vivo PET imaging, novel targeted (69/71)Ga/(68)Ga-protoporphyrin IX (PPIX) probes were developed. Optical analysis, fluorescence microscopy and radiolabeling with gallium-68 were performed to confirm potential applications. The use of a targeted-PPIX ring, in conjunction with (69/71)Ga or (68)Ga, eliminates the need for the attachment of multiple imaging tags, allowing for either in vitro fluorescent-based evaluation or in vivo nuclear-based imaging.
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Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrin-releasing Peptide receptors.
Small
PUBLISHED: 04-26-2011
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Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [?-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further development of this viral nanoparticle platform may open the door to potential clinical noninvasive molecular imaging strategies.
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Ghrelin receptor as a novel imaging target for prostatic neoplasms.
Prostate
PUBLISHED: 02-18-2011
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Ghrelin is a natural growth hormone secretagogue (GHS) that is co-expressed with its receptor GHSR in human prostate cancer (PCa) cells. Imaging probes that target receptors for ghrelin may delineate PCas from benign disease. The specificity of a novel ghrelin-imaging probe for PCa over normal tissue or benign disease was assessed.
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Design and characterization of a fluorescent ghrelin analog for imaging the growth hormone secretagogue receptor 1a.
Regul. Pept.
PUBLISHED: 01-20-2011
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Ghrelin is a 28-amino acid peptide hormone produced in the stomach. It binds to the growth hormone secretagogue receptor 1a (GHS-R1a), a class A G-protein-coupled receptor. In the present study, we describe the design, synthesis and characterization of a truncated, 18-amino acid analog of ghrelin conjugated to a fluorescent molecule, fluorocein isothiocyanate (FITC), through the addition of a lysine at its C terminus ([Dpr(octanoyl)(3), Lys(fluorescein)(19)]ghrelin(1-19)). Receptor binding affinity of this novel fluorescein-ghrelin(1-18) was similar to that of wild-type ghrelin and a synthetic GHS-R1a ligand, hexarelin. Live cell imaging in CHO/GHS-R1a cells demonstrated cell surface receptor labeling and internalization, and agonist activity of fluorescein-ghrelin(1-18) was confirmed by increased phosphorylation of ERK1/2. We also show that GHS-R1a protein is expressed primarily in the heart when compared to all other organs, suggesting high receptor density in the left ventricle. Finally, we demonstrate that fluorescein-ghrelin(1-18) binds specifically to heart tissue in situ, and its binding is displaced by both wt ghrelin and hexarelin. We have therefore developed a novel imaging probe, fluorescein-ghrelin(1-18), that can be used to image GHS-R1a in situ, for the purposes of investigating mechanisms of receptor trafficking or pharmacological agents that target GHS-R1a.
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Design, synthesis and in vitro characterization of Glucagon-Like Peptide-1 derivatives for pancreatic beta cell imaging by SPECT.
Bioorg. Med. Chem.
PUBLISHED: 09-13-2009
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Novel Glucagon-Like Peptide-1 (GLP-1) derivatives containing the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and naturally occurring Indium ((113/115)In) were prepared using solid-phase Fmoc methods. All synthesized peptides contained d-Ala-8, a modification known to improve resistance towards degradation by dipeptidyl peptidase-IV. The effect of increased distance between DOTA and the peptide chain was investigated using an (aminoethyl) ethoxy acetyl linker, in order to reduce steric effects imposed by DOTA. Placement of linker and DOTA moieties were also varied within the GLP-1 sequence to test for optimal metal-complex location. The binding affinity of the peptide derivatives was determined in vitro with Chinese hamster ovary cells stably transfected with a human GLP-1 receptor (CHO/GLP-1R) cell line and was shown to be in the nM range. Gamma camera imaging of an insulinoma cell line was carried out using (111)In-labeled peptides. Our results suggest that the prepared GLP-1 derivatives are suitable imaging probes for studying pancreatic islet function in vivo.
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Synthesis of bombesin-functionalized iron oxide nanoparticles and their specific uptake in prostate cancer cells.
J Nanopart Res
PUBLISHED: 06-23-2009
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The imaging of molecular markers associated with disease offers the possibility for earlier detection and improved treatment monitoring. Receptors for gastrin-releasing peptide are overexpressed on prostate cancer cells offering a promising imaging target, and analogs of bombesin, an amphibian tetradecapeptide have been previously demonstrated to target these receptors. Therefore, the pan-bombesin analog [?-Ala11, Phe13, Nle14]bombesin-(7-14) was conjugated through a linker to dye-functionalized superparamagnetic iron oxide nanoparticles for the development of a new potential magnetic resonance imaging probe. The peptide was conjugated via click chemistry, demonstrating a complementary alternative methodology to conventional peptide-nanoparticle conjugation strategies. The peptide-functionalized nanoparticles were then demonstrated to be selectively taken up by PC-3 prostate cancer cells relative to unfunctionalized nanoparticles and this uptake was inhibited by the presence of free peptide, confirming the specificity of the interaction. This study suggests that these nanoparticles have the potential to serve as magnetic resonance imaging probes for the detection of prostate cancer.
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Fluorine and rhenium substituted ghrelin analogues as potential imaging probes for the growth hormone secretagogue receptor.
J. Med. Chem.
PUBLISHED: 03-28-2009
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In our effort to create imaging probes targeting the growth hormone secretagogue receptor (GHSR), we now report on the design and synthesis of fluorine and rhenium containing ghrelin analogues through modification of the n-octanoyl Ser-3 side chain. Fluorine analogues were designed whereby the fluorine atom is situated at the terminus of an aliphatic chain using diaminopropionic acid (Dpr) as residue-3. Truncated ghrelin(1-5) and ghrelin(1-14) fluorine-bearing analogues were prepared, the best of which had a 28 nM IC(50) for GHSR. Ghrelin(1-14) analogues were also prepared containing rhenium, as a surrogate metal for technetium-99m, with a cyclopentadienylrhenium tricarbonyl being situated at the terminus of the residue-3 side chain, yielding compounds the best of which had a 35 nM IC(50). This represents a rare case of incorporating rhenium into a peptide structure where the metal complex is required for biological activity. These fluorine and rhenium derivatives demonstrate the ability to modify the Ser-3 side chain of ghrelin in order to create imaging probes for the GHSR.
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Discovery of novel integrin ligands from combinatorial libraries using a multiplex "beads on a bead" approach.
Nano Lett.
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The development of screening approaches to identify novel affinity ligands has paved the way for a new generation of molecular targeted nanomedicines. Conventional methods typically bias the display of the target protein to ligands during the screening process. We have developed an unbiased multiplex "beads on a bead" strategy to isolate, characterize, and validate high affinity ligands from OBOC libraries. Novel non-RGD peptides that target ?(v)?(3) integrin were discovered that do not affect cancer or endothelial cell biology. The peptides identified here represent novel integrin-targeted agents that can be used to develop targeted nanomedicines without the risk of increased tumor invasion and metastasis.
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Synthesis of rhenium-centric reverse turn mimics.
Chemistry
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Molecular scaffolds have been shown to facilitate and stabilise secondary structural turn elements, with a central core-arranging functionality in a defined three-dimensional orientation. In a peptide-based molecular imaging probe, this approach is of particular value as it would essentially "hide" a metal radioisotope within the ligand framework, making the labelling element a critical component of the receptor-bound structure. Starting from a 1,2-diaminoethane loaded 2-chlorotrityl resin, a versatile set of triamine ligand systems were synthesised by using solid-phase Fmoc-based peptide chemistry. The resultant resin-bound peptides then underwent amide reduction by treatment with borane-THF at 65?°C. This provided complete conversion to the corresponding polyamine entities in high purity for the majority of the amino acids utilised. The triamines were then coordinated on solid support by using [NEt(4)](2)[Re(CO)(3)(Br)(3)] followed by resin cleavage and HPLC purification, to give the desired rhenium coordinated species. We have shown that amino acid sequences can be assembled, reduced and coordinated on-resin, resulting in a versatile set of metal-ligand constructs. These studies could be expanded to generate libraries of turn-based peptidomimetics containing Re/Tc(I) organometallic scaffolds, with the intention of developing an improved approach for finding new diagnostic and therapeutic radiopharmaceutical entities.
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Investigation of isomer formation upon coordination of bifunctional histidine analogues with 99mTc/Re(CO)3.
Dalton Trans
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Histidine is a convenient tridentate chelator used in the synthesis of technetium-99m radiopharmaceuticals, as it can be pendantly attached to a biomolecule for molecular imaging applications. Once coordinated, it forms a neutral complex that is capable of forming diastereomers at the alpha amine of the histidine. This is demonstrated through the synthesis and characterization of four different histidine chelators; three small molecule chelators, which consist of a benzylated histidine at the alpha amine, and one modified dipeptide, containing a phenylalanine derivative at the C-terminus and a histidine at the N-terminus. Upon rhenium coordination, two products are observed, each having the desired exact mass of the metal-containing species. The two products have been characterized through LC-MS, (1)H, gCOSY, NOESY and ROESY NMR experiments, and the relative stereochemistry determined. The implications of diastereomer formation when using this chelation system for creating molecular imaging agents is also discussed.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.