Preparation of HIV monoclonal antibody-conjugated pulchellin in order to study its intracellular trafficking pathway in HIV-infected cells by confocal microscopy

NASA Astrophysics Data System (ADS)

Sadraeian, M.; Tsutae, F. M.; Moreira, H. H. T.; Araujo, A. P. U.; Guimarães, F. E. G.; Pincus, S. H.

2015-06-01

Pulchellin is a type 2 of ribosome-inactivating proteins isolated from some seeds significantly growing in Brazil. It is a potent agent to inhibit the protein synthesis in cancer cells and also HIV-infected cells. Pulchellin can be conjugated to HIV monoclonal antibodies to specifically target the HIV-infected cells. To analyze the protein synthesis inhibition by Pulchellin, the intracellular localization of the immunoconjugate should be compared to Pulchellin. In this case, the intracellular trafficking of this protein in cells can be determined by confocal microscopy. In our study, we utilized Pulchellin to construct HIV monoclonal antibody-conjugated Pulchellin A chain in order to target HIV-infected lymphocyte cells. Afterward the conjugation was labeled with the superior Alexa Fluor 488 dye. As a subsequent step, we are interested in studying the intracellular trafficking pathway of this novel conjugation in HIV-infected cells by confocal microscopy. Moreover, possible quantitative methods for fluorescent labeling of the immunoconjugate during confocal microscopy will be investigated.

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

PubMed

Hsieh, Feng-Jen; Chen, Yen-Wei; Huang, Yao-Kuan; Lee, Hsien-Ming; Lin, Chun-Hung; Chang, Huan-Cheng

2018-02-06

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry.

PubMed

Woda, Marcia; Mathew, Anuja

2015-01-01

Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at -80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV(+) B cells from immune, but not naïve donors secreted antibodies that bound DENV after in vitro stimulation. Overall, Alexa Fluor dye-labeled DENVs are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

Affibody Molecules for In vivo Characterization of HER2-Positive Tumors by Near-Infrared Imaging

PubMed Central

Lee, Sang Bong; Hassan, Moinuddin; Fisher, Robert; Chertov, Oleg; Chernomordik, Victor; Kramer-Marek, Gabriela; Gandjbakhche, Amir; Capala, Jacek

2012-01-01

Purpose HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared optical imaging. The goal is to provide probes that will minimally interfere with the studied system, i.e., whose binding does not interfere with the binding of the therapeutic agents, and whose effect on the target cells is minimal. Experimental Design We used three different types of HER2-specific Affibody molecules [monomer ZHER2:342, dimer (ZHER2:477)2, and albumin-binding domain-fused-(ZHER2:342)2] as targeting agents, and labeled them with Alexa Fluor dyes. Trastuzumab was also conjugated, using commercially available kits, as a standard control. The resulting conjugates were characterized in vitro by toxicity assays, Biacore affinity measurements, flow cytometry, and confocal microscopy. Semi-uantitative in vivo near-infrared optical imaging studies were carried out using mice with subcutaneous xenografts of HER2-positive tumors. Results The HER2-specific Affibody molecules were not toxic to HER2-overexpressing cells and their binding to HER2 did interfere with neither binding nor effectives of trastuzumab. The binding affinities and specificities of the Affibody-Alexa Fluor fluorescent conjugates to HER2 were unchanged or minimally affected by the modifications. Pharmacokinetics and biodistribution studies showed the albumin-binding domain-fused-(ZHER2:342)2-Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo. Conclusion Our results suggest that Affibody-Alexa Fluor conjugates may be used as a specific near-infrared probe for the non-invasive semi-quantitative imaging of HER2 expression in vivo. PMID:18559604

Cell uptake, intracellular distribution, fate and reactive oxygen species generation of polymer brush engineered CeO2-x NPs

NASA Astrophysics Data System (ADS)

Qiu, Yuan; Rojas, Elena; Murray, Richard A.; Irigoyen, Joseba; Gregurec, Danijela; Castro-Hartmann, Pablo; Fledderman, Jana; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio E.

2015-04-01

Cerium Oxide nanoparticles (CeO2-x NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO2-x NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO2-x NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO2-x NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell endosomes and lysosomes after 24 h of incubation. They also show higher co-localisation with lipid bodies when compared to unmodified CeO2-x NPs. The brush coating does not prevent CeO2-x NPs from displaying antioxidant properties.Cerium Oxide nanoparticles (CeO2-x NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO2-x NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO2-x NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO2-x NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell endosomes and lysosomes after 24 h of incubation. They also show higher co-localisation with lipid bodies when compared to unmodified CeO2-x NPs. The brush coating does not prevent CeO2-x NPs from displaying antioxidant properties. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr00884k

Lateral resolution testing of a novel developed confocal microscopic imaging system

NASA Astrophysics Data System (ADS)

Zhang, Xin; Zhang, Yunhai; Chang, Jian; Huang, Wei; Xue, Xiaojun; Xiao, Yun

2015-10-01

Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths 405nm 488nm 561nm and 638nm which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.

Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

PubMed

Hayashi-Takanaka, Yoko; Stasevich, Timothy J; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

2014-01-01

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

Fis protein induced λF-DNA bending observed by single-pair fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Chi-Cheng, Fu; Wunshain, Fann; Yuan Hanna, S.

2006-03-01

Fis, a site-specific DNA binding protein, regulates many biological processes including recombination, transcription, and replication in E.coli. Fis induced DNA bending plays an important role in regulating these functions and bending angle range from ˜50 to 95 dependent on the DNA sequence. For instance, the average bending angle of λF-DNA (26 bp, 8.8nm long, contained λF binding site on the center) measured by gel mobility shift assays was ˜ 94 . But the traditional method cannot provide information about the dynamics and the angle distribution. In this study, λF-DNA was labeled with donor (Alexa Fluor 546) and acceptor (Alexa Fluor 647) dyes on its two 5' ends and the donor-acceptor distances were measured using single-pair fluorescence resonance energy transfer (sp-FRET) with and without the present of Fis protein. Combing with structure information of Fis-DNA complex, the sp-FRET results are used to estimate the protein induced DNA bending angle distribution and dynamics.

Imaging axonal transport in the rat visual pathway.

PubMed

Abbott, Carla J; Choe, Tiffany E; Lusardi, Theresa A; Burgoyne, Claude F; Wang, Lin; Fortune, Brad

2013-02-01

A technique was developed for assaying axonal transport in retinal ganglion cells using 2 µl injections of 1% cholera toxin b-subunit conjugated to AlexaFluor488 (CTB). In vivo retinal and post-mortem brain imaging by confocal scanning laser ophthalmoscopy and post-mortem microscopy were performed. The transport of CTB was sensitive to colchicine, which disrupts axonal microtubules. The bulk rates of transport were determined to be approximately 80-90 mm/day (anterograde) and 160 mm/day (retrograde). Results demonstrate that axonal transport of CTB can be monitored in vivo in the rodent anterior visual pathway, is dependent on intact microtubules, and occurs by active transport mechanisms.

Colocalization of cellular nanostructure using confocal fluorescence and partial wave spectroscopy.

PubMed

Chandler, John E; Stypula-Cyrus, Yolanda; Almassalha, Luay; Bauer, Greta; Bowen, Leah; Subramanian, Hariharan; Szleifer, Igal; Backman, Vadim

2017-03-01

A new multimodal confocal microscope has been developed, which includes a parallel Partial Wave Spectroscopic (PWS) microscopy path. This combination of modalities allows molecular-specific sensing of nanoscale intracellular structure using fluorescent labels. Combining molecular specificity and sensitivity to nanoscale structure allows localization of nanostructural intracellular changes, which is critical for understanding the mechanisms of diseases such as cancer. To demonstrate the capabilities of this multimodal instrument, we imaged HeLa cells treated with valinomycin, a potassium ionophore that uncouples oxidative phosphorylation. Colocalization of fluorescence images of the nuclei (Hoechst 33342) and mitochondria (anti-mitochondria conjugated to Alexa Fluor 488) with PWS measurements allowed us to detect a significant decrease in nuclear nanoscale heterogeneity (Σ), while no significant change in Σ was observed at mitochondrial sites. In addition, application of the new multimodal imaging approach was demonstrated on human buccal samples prepared using a cancer screening protocol. These images demonstrate that nanoscale intracellular structure can be studied in healthy and diseased cells at molecular-specific sites. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functionalization of Cellulose Nanocrystals with PEG-Metal-Chelating Diblock Copolymers via Controlled Conjugation in Aqueous Medium

NASA Astrophysics Data System (ADS)

Guo, Melinda

The surface of cellulose nanocrystals (CNCs) was successfully functionalized with metal chelating diblock copolymers via HyNic-4FB conjugation. Two types of PEG-metal-chelating block polymers with hydrazinonicotinate acetone hydrazine (HyNic) end groups were synthesized: mPEG-PGlu(DTPA) 18-HyNic and mPEG-PGlu(DTPA)25-HyNic. These two polymers both had a methoxy PEG (M ˜ 2000 Da) block that differed in the mean degree of polymerization of the metal-chelating block. They were characterized by 1H NMR spectroscopy and gel-permeation chromatography (GPC). 4-Formylbenzamide (4FB) groups were introduced onto the surface of CNCs and quantified through their reaction with 2-hydrazinopyridine. The polymers were grafted onto the surface of CNCs via bis-aryl hydrazone bond formation, and the kinetics of this reaction was explored by UV/Vis spectroscopy. The CNCs were also labeled with rhodamine and Alexa FluorRTM 488 dyes. Students in our collaborator's group in Pharmacy are examining applications of these materials as radiotherapeutic agents for cancer treatment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukundan, Harshini; Xei, Hongshi; Anderson, Aaron S

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyesmore » are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.« less

Toward photostable multiplex analyte detection on a single mode planar optical waveguide

NASA Astrophysics Data System (ADS)

Mukundan, Harshini; Xie, Hongzhi; Anderson, Aaron; Grace, W. Kevin; Martinez, Jennifer S.; Swanson, Basil

2009-02-01

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

PubMed Central

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E.

2018-01-01

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here. PMID:29541425

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.

PubMed

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E

2018-01-01

Titanium dioxide (TiO 2 ) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

Nanotechnology-Enabled Optical Molecular Imaging of Breast Cancer

DTIC Science & Technology

2010-07-01

Abbreviations Ab antibody AF-Ab Alexa Fluor labeled antibody CCD charge coupled device CTAB cetyltrimethylammonium bromide EDC 1-ethyl-[3-dimethylaminopropyl...mPEG-SH in figure 1. The carboxy-terminal nanorods were conjugated to antibodies using the zero-length crosslinker EDC stabilized by NHS [38]. Standard...multimode fiber coupler /positioner (Newport, model: F-915T) is utilized to mount the objective lens and a fiber chuck (Newport, model: FPH-DJ). With

New Fluorescent Macrolide Derivatives for Studying Interactions of Antibiotics and Their Analogs with the Ribosomal Exit Tunnel.

PubMed

Tereshchenkov, A G; Shishkina, A V; Karpenko, V V; Chertkov, V A; Konevega, A L; Kasatsky, P S; Bogdanov, A A; Sumbatyan, N V

2016-10-01

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Koshonna; Thurn, Ted; Xin, Lun

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE PAGES

Brown, Koshonna; Thurn, Ted; Xin, Lun; ...

2017-07-19

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer

PubMed Central

Mahalingam, Mohana; Girgenrath, Tanya; Svensson, Bengt; Thomas, David D.; Cornea, Razvan L.; Fessenden, James D.

2014-01-01

Summary Ryanodine receptors (RyR) release Ca2+ to initiate striated muscle contraction. Three highly divergent regions in the RyR protein sequence (DR1, DR2, DR3) are proposed to confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryo-electron microscopic (EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to Cy3NTA or Cy5NTA, FRET acceptors targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the “clamp” region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the “handle” region, near the regulatory calmodulin binding site on the RyR. These localizations provide new insights into the roles of DRs in RyR allosteric regulation during excitation-contraction coupling. PMID:25132084

New method for estimating clustering of DNA lesions induced by physical/chemical mutagens using fluorescence anisotropy.

PubMed

Akamatsu, Ken; Shikazono, Naoya; Saito, Takeshi

2017-11-01

We have developed a new method for estimating the localization of DNA damage such as apurinic/apyrimidinic sites (APs) on DNA using fluorescence anisotropy. This method is aimed at characterizing clustered DNA damage produced by DNA-damaging agents such as ionizing radiation and genotoxic chemicals. A fluorescent probe with an aminooxy group (AlexaFluor488) was used to label APs. We prepared a pUC19 plasmid with APs by heating under acidic conditions as a model for damaged DNA, and subsequently labeled the APs. We found that the observed fluorescence anisotropy (r obs ) decreases as averaged AP density (λ AP : number of APs per base pair) increases due to homo-FRET, and that the APs were randomly distributed. We applied this method to three DNA-damaging agents, 60 Co γ-rays, methyl methanesulfonate (MMS), and neocarzinostatin (NCS). We found that r obs -λ AP relationships differed significantly between MMS and NCS. At low AP density (λ AP  < 0.001), the APs induced by MMS seemed to not be closely distributed, whereas those induced by NCS were remarkably clustered. In contrast, the AP clustering induced by 60 Co γ-rays was similar to, but potentially more likely to occur than, random distribution. This simple method can be used to estimate mutagenicity of ionizing radiation and genotoxic chemicals. Copyright © 2017 Elsevier Inc. All rights reserved.

Multifunctional porous silicon nanoparticles for cancer theranostics.

PubMed

Wang, Chang-Fang; Sarparanta, Mirkka P; Mäkilä, Ermei M; Hyvönen, Maija L K; Laakkonen, Pirjo M; Salonen, Jarno J; Hirvonen, Jouni T; Airaksinen, Anu J; Santos, Hélder A

2015-04-01

Nanomaterials provide a unique platform for the development of theranostic systems that combine diagnostic imaging modalities with a therapeutic payload in a single probe. In this work, dual-labeled iRGD-modified multifunctional porous silicon nanoparticles (PSi NPs) were prepared from dibenzocyclooctyl (DBCO) modified PSi NPs by strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. Hydrophobic antiangiogenic drug, sorafenib, was loaded into the modified PSi NPs to enhance the drug dissolution rate and improve cancer therapy. Radiolabeling of the developed system with (111)In enabled the monitoring of the in vivo biodistribution of the nanocarrier by single photon emission computed tomography (SPECT) in an ectopic PC3-MM2 mouse xenograft model. Fluorescent labeling with Alexa Fluor 488 was used to determine the long-term biodistribution of the nanocarrier by immunofluorescence at the tissue level ex vivo. Modification of the PSi NPs with an iRGD peptide enhanced the tumor uptake of the NPs when administered intravenously. After intratumoral delivery the NPs were retained in the tumor, resulting in efficient tumor growth suppression with particle-loaded sorafenib compared to the free drug. The presented multifunctional PSi NPs highlight the utility of constructing a theranostic nanosystems for simultaneous investigations of the in vivo behavior of the nanocarriers and their drug delivery efficiency, facilitating the selection of the most promising materials for further NP development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nano-bio assemblies for artificial light harvesting systems

NASA Astrophysics Data System (ADS)

Bain, Dipankar; Maity, Subarna; Patra, Amitava

2018-02-01

Ultrasmall fluorescent gold nanoclusters (Au NCs) have drawn considerable research interest owing to their molecular like properties such as d-sp and sp-sp transitions, and intense fluorescence. Fluorescent Au NCs have especial attraction in biological system owing to their biocompatibility and high photostability. Recently, several strategies have been adapted to design an artificial light-harvesting system using Au NCs for potential applications. Here, we have designed Au nanoclusters based dsDNA (double stranded deoxyribonucleic acid) nano assemblies where the Au nanocluster is covalently attached with Alexa Fluor 488 (A488) dye tagged dsDNA. Investigation reveals that the incorporation of Ag+ into dsDNA enhances the rate of energy transfer from A488 to Au NCs. In addition cadmium telluride quantum dot (CdTe QDs) based Au NCs hybrid material shows the significant enhancement of energy transfer 35% to 83% with changing the capping ligand of Au NCs from glutathione (GSH) to bovine serum albumin (BSA) protein. Another hybrid system is developed using carbon dots and dye encapsulated BSA-protein capped Au NCs for efficient light harvesting system with 83% energy transfer efficiency. Thus, Au NCs base nano bio assemblies may open up new possibilities for the construction of artificial light harvesting system.

Biodegradable FeMnSi Sputter-Coated Macroporous Polypropylene Membranes for the Sustained Release of Drugs

PubMed Central

Fornell, Jordina; Soriano, Jorge; Guerrero, Miguel; Sirvent, Juan de Dios; Ferran-Marqués, Marta; Ibáñez, Elena; Barrios, Leonardo; Baró, Maria Dolors; Suriñach, Santiago; Nogués, Carme; Sort, Jordi; Pellicer, Eva

2017-01-01

Pure Fe and FeMnSi thin films were sputtered on macroporous polypropylene (PP) membranes with the aim to obtain biocompatible, biodegradable and, eventually, magnetically-steerable platforms. Room-temperature ferromagnetic response was observed in both Fe- and FeMnSi-coated membranes. Good cell viability was observed in both cases by means of cytotoxicity studies, though the FeMnSi-coated membranes showed higher biodegradability than the Fe-coated ones. Various strategies to functionalize the porous platforms with transferrin-Alexa Fluor 488 (Tf-AF488) molecules were tested to determine an optimal balance between the functionalization yield and the cargo release. The distribution of Tf-AF488 within the FeMnSi-coated PP membranes, as well as its release and uptake by cells, was studied by confocal laser scanning microscopy. A homogeneous distribution of the drug within the membrane skeleton and its sustained release was achieved after three consecutive impregnations followed by the addition of a layer made of gelatin and maltodextrin, which prevented exceedingly fast release. The here-prepared organic-inorganic macroporous membranes could find applications as fixed or magnetically-steerable drug delivery platforms. PMID:28672792

Novel Micro/Nano Approaches for Glucose Measurement Using pH-Sensitive Hydrogels

DTIC Science & Technology

2005-06-01

hydrochloride ) (PAH) (polycation). A total of three bilayers of GOx/PAH were assembled, and enzyme activity was confirmed by colorimetric assay...D- glucosamine residues in chitosan can be conjugated with amine-reactive dyes, such as succinimidyl esters (Alexa Fluor 647TM, CY5®), isothiocyanates...were found to be: A 4 94 nm=0.153276 MW=161 [D- glucosamine ]=0.77g/L Sdye=68000cm-M-1 DOL=4.71 x 10-’ Therefore, the labeling ratio of FITC:chitosan

Near-infrared squaraine dyes for fluorescence enhanced surface assay

PubMed Central

Matveeva, Evgenia G.; Terpetschnig, Ewald A.; Stevens, Megan; Patsenker, Leonid; Kolosova, Olga S.; Gryczynski, Zygmunt; Gryczynski, Ignacy

2009-01-01

Commercially available, near-infrared fluorescent squaraine dyes (Seta-635 and Seta-670) were covalently bound to antibodies and employed insurface enhanced immunoassay. From fluorescence intensity and lifetime changes determined for a surface which had been coated with silver nanoparticles as well as a non-coated glass surface, both labelled compounds exhibited a 15 to 20-fold enhancement of fluorescence on the silver coated surface compared to that achieved on the non-coated surface. In addition, the fluorescence lifetime changes drastically for both labels in the case of silver-coated surfaces. The fluorescence signal enhancement obtained for the two dyes was greater than that previously recorded for Rhodamine Red-X and AlexaFluor-647 labels. PMID:20046935

Single Cell Fluorescence Imaging Using Metal Plasmon-Coupled Probe

PubMed Central

Zhang, Jian; Fu, Yi; Lakowicz, Joseph R.

2009-01-01

This work constitutes the first fluorescent imaging of cells using metal plasmon-coupled probes (PCPs) at single cell resolution. N-(2-Mercapto-propionyl)glycine-coated silver nanoparticles were synthesized by reduction of silver nitrate using sodium borohyride and then succinimidylated via ligand exchange. Alexa Fluor 647-labeled concanavalin A (con A) was chemically bound to the silver particles to make the fluorescent metal plasmon-coupled probes. The fluorescence images were collected using a scanning confocal microscopy. The fluorescence intensity was observed to enhance 7-fold when binding the labeled con A on a single silver particle. PCPs were conjugated on HEK 293 A cells. Imaging results demonstrate that cells labeled by PCPs were 20-fold brighter than those by free labeled con A. PMID:17375898

Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging

PubMed Central

Feng, Juan; Navratil, Marian; Thompson, LaDora V.

2011-01-01

The increase in the levels of protein carbonyls, biomarkers of oxidative stress, appears to play an important role in aging skeletal muscle. However, the exact distributions of carbonyls among various skeletal muscle microstructures still remain largely unknown, partly owing to the lack of adequate techniques to carry out these measurements. This report describes an immunohistochemical approach to determine the relative abundance of carbonyls in the intermyofibrillar mitochondria (IFM), the subsarcolemmal mitochondria (SSM), the cytoplasm, and the extracellular space of skeletal muscle. These morphological features were defined by labeling the nucleus, the Z-lines, and mitochondria. Carbonyls were detected by derivatization with dinitrophenylhydrazine followed by labeling with an Alexa 488-labeled anti-dinitrophenyl primary antibody. Alexa 488 fluorescence (green) in different fiber microstructures was used to estimate the relative abundance of carbonyls. On the basis of the samples examined, preliminary results suggest that the most dramatic age-related changes in carbonyl levels occur in the extracellular space, followed in a decreasing order by SSM, IFM, and the cytoplasm. These observations were confirmed in the soleus and semimembranosus muscles composed predominantly of type I and type II fibers, respectively. This approach could easily be extended to the investigation of carbonyl levels in other muscles (composed of mixed skeletal muscle fiber types) or other tissues in which protein carbonyls are present. PMID:18548236

Targeting of Cancer Stem Cells and Their Microenvironment in Early-Stage MutantK-ras Lung Cancer

DTIC Science & Technology

2015-10-01

a combination of SHH/ALDH/ NOTCH3 as potential stem cell markers. We will also test the hypothesis that SHH+ cell population may be a quiescent stem...and SHH are expressed within the same cell population. We will also test NOTCH3 in combination with 5E1-647 alone or in combination with 5E1-647 and...Aldefluor assays. Anti- NOTCH3 antibody will be labeled with Alexa Fluor 594 (red color spectrum) in an analogous manner to 5E1-594. As ALDH+ and

An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots

NASA Astrophysics Data System (ADS)

Samanta, Anirban; Walper, Scott A.; Susumu, Kimihiro; Dwyer, Chris L.; Medintz, Igor L.

2015-04-01

The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiple-sequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles from the resulting sequential BRET-FRET-FRET processes allow us to estimate the efficiency of each of the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of the energy transfer process(es) along with potential applications of such devices are finally discussed.The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiple-sequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles from the resulting sequential BRET-FRET-FRET processes allow us to estimate the efficiency of each of the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of the energy transfer process(es) along with potential applications of such devices are finally discussed. Electronic supplementary information (ESI) available: This material includes control experimental data and select deconvoluted spectra. See DOI: 10.1039/c5nr00828j

Fluorescent Labeling of Collagen Production by Cells for Noninvasive Imaging of Extracellular Matrix Deposition.

PubMed

Bardsley, Katie; Yang, Ying; El Haj, Alicia J

2017-04-01

Extracellular matrix (ECM) is an essential component of tissues and provides both integrity and biological cues for cells. Collagen is one of the major proteins found within the ECM and therefore is an essential component of all engineered tissues. Therefore, in this article, we present a method for the online real-time monitoring of collagen deposition in three-dimensional engineered constructs. This method revolves around modification of collagen through the addition of azide-L-proline to cell culture media. The incorporation of azide-L-proline into the neocollagen produced by cells can then be detected by reaction with 10 mM of a Click-IT Alexa Fluor 488 DIBO Alkyne. The reaction was shown as being specific to the collagen as little background staining was observed in cultures, which did not contain the modified proline, and the staining was also depleted after treatment with collagenase and colocalization of collagen type I staining by immunochemistry assay. Real-time online staining of collagen deposition was observed under different culture conditions without affecting proliferation. Collagen deposition was observed to be increased under mechanical stimulation; however, the localization varied across stimulation regimes. This is a new technique for real-time monitoring of cell-produced collagen and will be a valuable addition to the tissue engineering field.

Specific Adsorption of Osteopontin and Synthetic Polypeptides to Calcium Oxalate Monohydrate Crystals

PubMed Central

Taller, Adam; Grohe, Bernd; Rogers, Kem A.; Goldberg, Harvey A.; Hunter, Graeme K.

2007-01-01

Protein-crystal interactions are known to be important in biomineralization. To study the physicochemical basis of such interactions, we have developed a technique that combines confocal microscopy of crystals with fluorescence imaging of proteins. In this study, osteopontin (OPN), a protein abundant in urine, was labeled with the fluorescent dye AlexaFluor-488 and added to crystals of calcium oxalate monohydrate (COM), the major constituent of kidney stones. In five to seven optical sections along the z axis, scanning confocal microscopy was used to visualize COM crystals and fluorescence imaging to map OPN adsorbed to the crystals. To quantify the relative adsorption to different crystal faces, fluorescence intensity was measured around the perimeter of the crystal in several sections. Using this method, it was shown that OPN adsorbs with high specificity to the edges between {100} and {121} faces of COM and much less so to {100}, {121}, or {010} faces. By contrast, poly-L-aspartic acid adsorbs preferentially to {121} faces, whereas poly-L-glutamic acid adsorbs to all faces approximately equally. Growth of COM in the presence of rat bone OPN results in dumbbell-shaped crystals. We hypothesize that the edge-specific adsorption of OPN may be responsible for the dumbbell morphology of COM crystals found in human urine. PMID:17496021

Apoferritin fibers: a new template for 1D fluorescent hybrid nanostructures

NASA Astrophysics Data System (ADS)

Jurado, Rocío; Castello, Fabio; Bondia, Patricia; Casado, Santiago; Flors, Cristina; Cuesta, Rafael; Domínguez-Vera, José M.; Orte, Angel; Gálvez, Natividad

2016-05-01

Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials.Recently, research in the field of protein amyloid fibers has gained great attention due to the use of these materials as nanoscale templates for the construction of functional hybrid materials. The formation of apoferritin amyloid-like protein fibers is demonstrated herein for the first time. The morphology, size and stiffness of these one-dimensional structures are comparable to the fibers formed by β-lactoglobulin, a protein frequently used as a model in the study of amyloid-like fibrillar proteins. Nanometer-sized globular apoferritin is capable of self-assembling to form 1D micrometer-sized structures after being subjected to a heating process. Depending on the experimental conditions, fibers with different morphologies and sizes are obtained. The wire-like protein structure is rich in functional groups and allows chemical functionalization with diverse quantum dots (QD), as well as with different Alexa Fluor (AF) dyes, leading to hybrid fluorescent fibers with variable emission wavelengths, from green to near infrared, depending on the QD and AFs coupled. For fibers containing the pair AF488 and AF647, efficient fluorescence energy transfer from the covalently coupled donor (AF488) to acceptor tags (AF647) takes place. Apoferritin fibers are proposed here as a new promising template for obtaining hybrid functional materials. Electronic supplementary information (ESI) available: TEM images of ferritin protein fiber formation, and apoferritin after 18 days of heat treatment; FLIM-PIE technique details; fluorescence emission spectra of apoferritin and β-lactoglobulin fibers functionalized with different QDs. See DOI: 10.1039/c6nr01044j

The Interaction Affinity between Vascular Cell Adhesion Molecule-1 (VCAM-1) and Very Late Antigen-4 (VLA-4) Analyzed by Quantitative FRET

PubMed Central

Wu, Shu-Han; Karmenyan, Artashes; Chiou, Arthur

2015-01-01

Very late antigen-4 (VLA-4), a member of integrin superfamily, interacts with its major counter ligand vascular cell adhesion molecule-1 (VCAM-1) and plays an important role in leukocyte adhesion to vascular endothelium and immunological synapse formation. However, irregular expressions of these proteins may also lead to several autoimmune diseases and metastasis cancer. Thus, quantifying the interaction affinity of the VCAM-1/VLA-4 interaction is of fundamental importance in further understanding the nature of this interaction and drug discovery. In this study, we report an ‘in solution’ steady state organic fluorophore based quantitative fluorescence resonance energy transfer (FRET) assay to quantify this interaction in terms of the dissociation constant (Kd). We have used, in our FRET assay, the Alexa Fluor 488-VLA-4 conjugate as the donor, and Alexa Fluor 546-VCAM-1 as the acceptor. From the FRET signal analysis, Kd of this interaction was determined to be 41.82 ± 2.36 nM. To further confirm our estimation, we have employed surface plasmon resonance (SPR) technique to obtain Kd = 39.60 ± 1.78 nM, which is in good agreement with the result obtained by FRET. This is the first reported work which applies organic fluorophore based ‘in solution’ simple quantitative FRET assay to obtain the dissociation constant of the VCAM-1/VLA-4 interaction, and is also the first quantification of this interaction. Moreover, the value of Kd can serve as an indicator of abnormal protein-protein interactions; hence, this assay can potentially be further developed into a drug screening platform of VLA-4/VCAM-1 as well as other protein-ligand interactions. PMID:25793408

Enhancing Tumor Cell Response to Chemotherapy through the Targeted Delivery of Platinum Drugs Mediated by Highly Stable, Multifunctional Carboxymethylcellulose-Coated Magnetic Nanoparticles.

PubMed

Medříková, Zdenka; Novohradsky, Vojtech; Zajac, Juraj; Vrána, Oldřich; Kasparkova, Jana; Bakandritsos, Aristides; Petr, Martin; Zbořil, Radek; Brabec, Viktor

2016-07-04

The fabrication of nanoparticles using different formulations, and which can be used for the delivery of chemotherapeutics, has recently attracted considerable attention. We describe herein an innovative approach that may ultimately allow for the selective delivery of anticancer drugs to tumor cells by using an external magnet. A conventional antitumor drug, cisplatin, has been incorporated into new carboxymethylcellulose-stabilized magnetite nanoparticles conjugated with the fluorescent marker Alexa Fluor 488 or folic acid as targeting agent. The magnetic nanocarriers possess exceptionally high biocompatibility and colloidal stability. These cisplatin-loaded nanoparticles overcome the resistance mechanisms typical of free cisplatin. Moreover, experiments aimed at the localization of the nanoparticles driven by an external magnet in a medium that mimics physiological conditions confirmed that this localization can inhibit tumor cell growth site-specifically. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands.

PubMed

He, Wei; Kularatne, Sumith A; Kalli, Kimberly R; Prendergast, Franklyn G; Amato, Robert J; Klee, George G; Hartmann, Lynn C; Low, Philip S

2008-10-15

Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients. A CTC-enriched fraction was isolated from the peripheral blood of ovarian and prostate cancer patients by an optimized density gradient centrifugation protocol and labeled with the aforementioned fluorescent ligands. CTCs were then quantitated by flow cytometry. CTCs were detected in 18 of 20 ovarian cancer patients (mean 222 CTCs/ml; median 15 CTCs/ml; maximum 3,118 CTCs/ml), whereas CTC numbers in 16 gender-matched normal volunteers were negligible (mean 0.4 CTCs/ml; median 0.3 CTCs/ml; maximum 1.5 CTCs/ml; p < 0.001, chi(2)). CTCs were also detected in 10 of 13 prostate cancer patients (mean 26 CTCs/ml, median 14 CTCs/ml, maximum 94 CTCs/ml) but not in 18 gender-matched healthy donors (mean 0.8 CTCs/ml, median 1, maximum 3 CTC/ml; p < 0.0026, chi(2)). Tumor-specific fluorescent antibodies were much less efficient in quantitating CTCs because of their lower CTC labeling efficiency. Use of tumor-specific fluorescent ligands to label CTCs in peripheral blood can provide a simple, accurate and sensitive method for determining the number of cancer cells circulating in the bloodstream.

Use of Complementary Approaches to Imaging Biomolecules and Endogenous and Exogenous Trace Elements and Nanoparticles in Biological Samples

NASA Astrophysics Data System (ADS)

Brown, Koshonna Dinettia

X-ray Fluorescence Microscopy (XFM) is a useful technique for study of biological samples. XFM was used to map and quantify endogenous biological elements as well as exogenous materials in biological samples, such as the distribution of titanium dioxide (TiO2) nanoparticles. TiO 2 nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic particles for cancer detection and treatment, drug delivery, and induction of DNA breaks. Delivery of such nanoparticles can be targeted to specific cells and subcellular structures. In this work, we develop two novel approaches to stain TiO2 nanoparticles for optical microscopy and to confirm that staining by XFM. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called CLICK chemistry, for labeling of azide conjugated TiO2 nanoparticles with "clickable" dyes such as alkyne Alexa Fluor dyes with a high fluorescent yield. To confirm that the optical fluorescence signals of nanoparticles stained in situ match the distribution of the Ti element, we used high resolution synchrotron X-Ray Fluorescence Microscopy (XFM) using the Bionanoprobe instrument at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific X-ray fluorescence showed excellent overlap with the location of Alexa Fluor optical fluorescence detected by confocal microscopy. In this work XFM was also used to investigate native elemental differences between two different types of head and neck cancer, one associated with human papilloma virus infection, the other virus free. Future work may see a cross between these themes, for example, exploration of TiO2 nanoparticles as anticancer treatment for these two different types of head and neck cancer.

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

PubMed Central

Sakamoto, Soichiro; Kawabata, Hiroshi; Masuda, Taro; Uchiyama, Tatsuki; Mizumoto, Chisaki; Ohmori, Katsuyuki; Koeffler, H. Phillip; Kadowaki, Norimitsu; Takaori-Kondo, Akifumi

2015-01-01

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin. PMID:26441243

Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

PubMed

Gao, Chunyan; Ji, Shuting; Dong, Weijun; Qi, Yushan; Song, Wen; Cui, Debin; Shi, Jialan

2015-10-28

Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca(2+) ([Ca(2+)]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca(2+)]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

Quantification of retinal pigment epithelial phenotypic variation using laser scanning cytometry.

PubMed

Hjelmeland, L M; Fujikawa, A; Oltjen, S L; Smit-McBride, Z; Braunschweig, D

2010-06-16

Quantifying phenotypic variation at the level of protein expression (variegation) within populations of retinal pigment epithelium (RPE) cells may be important in the study of pathologies associated with this variation. The lack of quantitative methods for examining single cells, however, and the variable presence of pigment and/or lipofuscin complicate this experimental goal. We have applied the technique of laser scanning cytometry (LSC) to paraffin sections of mouse and human eyes to evaluate the utility of LSC for these measurements. Mouse eyes were perfusion fixed in 4% paraformaldehyde and embedded in paraffin. Postmortem human eyes were fixed and dissected to obtain a 9-mm punch, which was then embedded in paraffin. A laser scanning cytometer equipped with violet, argon, and helium-neon lasers and the detectors for blue, green, and long red were used to record the fluorescence of each individual cell at all three wavelengths. Raw data were recorded and processed using the WinCyte software. Individual nuclei were identified by the fluorescence of the 4',6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Next, RPE cells were uniquely identified in the green channel using an anti-retinal pigment epithelium-specific protein 65 kDa (anti-RPE65) monoclonal antibody with an Alexa Fluor 488-labeled secondary antibody. Mn-superoxide dismutase (MnSOD) was quantified in the long-red channel using an anti-MnSOD antibody and an Alexa Fluor 647-labeled secondary antibody. MnSOD(+) and RPE65(+) cells exhibited peaks in the plot of fluorescence intensity versus cell number, which could be characterized by the mean fluorescence intensity (MFI), the coefficient of variation (CV), and the percentage of total RPE cells that were also labeled for MnSOD. RPE cells can be uniquely identified in human and mouse paraffin sections by immunolabeling with anti-RPE65 antibody. A second antigen, such as MnSOD, can then be probed only within this set of RPE. Results are plotted primarily with the population frequency diagram, which can be subdivided into multiple regions. The data collected for each region include the MFI, the CV, and the number of cells that are immunolabeled in that region. Background interference from pigment or autofluorescent material can be successfully overcome by elevating the concentrations of fluorescent secondary antibodies. In the human and mouse eyes, age-related changes in MFI, CV, and percent RPE cells immunolabeled for MnSOD were observed. The extent of the variability of gene expression in RPE cells at the protein level can be quantified by LSC. Relative changes in the MFI, the CV, and/or percentage of RPE cells double labeled for a second antigen quantify the changes observed. The analysis of these data also suggest whether the effects observed are related to local changes in transcription (alterations of CV) or major changes of protein expression (MFI), which are likely to be due to changes in the chromatin structure. The changes of these variables with age suggest that the observed age-related variegation is primarily due to changes in the chromatin structure in individual cells.

Anisotropic diffusion of fluorescently labeled ATP in rat cardiomyocytes determined by raster image correlation spectroscopy

PubMed Central

Vendelin, Marko; Birkedal, Rikke

2008-01-01

A series of experimental data points to the existence of profound diffusion restrictions of ADP/ATP in rat cardiomyocytes. This assumption is required to explain the measurements of kinetics of respiration, sarcoplasmic reticulum loading with calcium, and kinetics of ATP-sensitive potassium channels. To be able to analyze and estimate the role of intracellular diffusion restrictions on bioenergetics, the intracellular diffusion coefficients of metabolites have to be determined. The aim of this work was to develop a practical method for determining diffusion coefficients in anisotropic medium and to estimate the overall diffusion coefficients of fluorescently labeled ATP in rat cardiomyocytes. For that, we have extended raster image correlation spectroscopy (RICS) protocols to be able to discriminate the anisotropy in the diffusion coefficient tensor. Using this extended protocol, we estimated diffusion coefficients of ATP labeled with the fluorescent conjugate Alexa Fluor 647 (Alexa-ATP). In the analysis, we assumed that the diffusion tensor can be described by two values: diffusion coefficient along the myofibril and that across it. The average diffusion coefficients found for Alexa-ATP were as follows: 83 ± 14 μm2/s in the longitudinal and 52 ± 16 μm2/s in the transverse directions (n = 8, mean ± SD). Those values are ∼2 (longitudinal) and ∼3.5 (transverse) times smaller than the diffusion coefficient value estimated for the surrounding solution. Such uneven reduction of average diffusion coefficient leads to anisotropic diffusion in rat cardiomyocytes. Although the source for such anisotropy is uncertain, we speculate that it may be induced by the ordered pattern of intracellular structures in rat cardiomyocytes. PMID:18815224

Central melanopsin projections in the diurnal rodent, Arvicanthis niloticus

PubMed Central

Langel, Jennifer L.; Smale, Laura; Esquiva, Gema; Hannibal, Jens

2015-01-01

The direct effects of photic stimuli on behavior are very different in diurnal and nocturnal species, as light stimulates an increase in activity in the former and a decrease in the latter. Studies of nocturnal mice have implicated a select population of retinal ganglion cells that are intrinsically photosensitive (ipRGCs) in mediation of these acute responses to light. ipRGCs are photosensitive due to the expression of the photopigment melanopsin; these cells use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitters. PACAP is useful for the study of central ipRGC projections because, in the retina, it is found exclusively within melanopsin cells. Little is known about the central projections of ipRGCs in diurnal species. Here, we first characterized these cells in the retina of the diurnal Nile grass rat using immunohistochemistry (IHC). The same basic subtypes of melanopsin cells that have been described in other mammals were present, but nearly 25% of them were displaced, primarily in its superior region. PACAP was present in 87.7% of all melanopsin cells, while 97.4% of PACAP cells contained melanopsin. We then investigated central projections of ipRGCs by examining the distribution of immunoreactive PACAP fibers in intact and enucleated animals. This revealed evidence that these cells project to the suprachiasmatic nucleus, lateral geniculate nucleus (LGN), pretectum, and superior colliculus. This distribution was confirmed with injections of cholera toxin subunit β coupled with Alexa Fluor 488 in one eye and Alexa Fluor 594 in the other, combined with IHC staining of PACAP. These studies also revealed that the ventral and dorsal LGN and the caudal olivary pretectal nucleus receive less innervation from ipRGCs than that reported in nocturnal rodents. Overall, these data suggest that although ipRGCs and their projections are very similar in diurnal and nocturnal rodents, they may not be identical. PMID:26236201

Central melanopsin projections in the diurnal rodent, Arvicanthis niloticus.

PubMed

Langel, Jennifer L; Smale, Laura; Esquiva, Gema; Hannibal, Jens

2015-01-01

The direct effects of photic stimuli on behavior are very different in diurnal and nocturnal species, as light stimulates an increase in activity in the former and a decrease in the latter. Studies of nocturnal mice have implicated a select population of retinal ganglion cells that are intrinsically photosensitive (ipRGCs) in mediation of these acute responses to light. ipRGCs are photosensitive due to the expression of the photopigment melanopsin; these cells use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitters. PACAP is useful for the study of central ipRGC projections because, in the retina, it is found exclusively within melanopsin cells. Little is known about the central projections of ipRGCs in diurnal species. Here, we first characterized these cells in the retina of the diurnal Nile grass rat using immunohistochemistry (IHC). The same basic subtypes of melanopsin cells that have been described in other mammals were present, but nearly 25% of them were displaced, primarily in its superior region. PACAP was present in 87.7% of all melanopsin cells, while 97.4% of PACAP cells contained melanopsin. We then investigated central projections of ipRGCs by examining the distribution of immunoreactive PACAP fibers in intact and enucleated animals. This revealed evidence that these cells project to the suprachiasmatic nucleus, lateral geniculate nucleus (LGN), pretectum, and superior colliculus. This distribution was confirmed with injections of cholera toxin subunit β coupled with Alexa Fluor 488 in one eye and Alexa Fluor 594 in the other, combined with IHC staining of PACAP. These studies also revealed that the ventral and dorsal LGN and the caudal olivary pretectal nucleus receive less innervation from ipRGCs than that reported in nocturnal rodents. Overall, these data suggest that although ipRGCs and their projections are very similar in diurnal and nocturnal rodents, they may not be identical.

An LED light source and novel fluorophore combinations improve fluorescence laparoscopic detection of metastatic pancreatic cancer in orthotopic mouse models.

PubMed

Metildi, Cristina A; Kaushal, Sharmeela; Lee, Claudia; Hardamon, Chanae R; Snyder, Cynthia S; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

2012-06-01

The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of a light-emitting diode (LED) light source and optimal fluorophore combinations. Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks postimplantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail-vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were also obtained with the OV-100 Olympus and Maestro CRI Small Animal Imaging Systems, serving as a positive control. Tumors were collected for histologic analysis. Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555), conjugated to antibodies, brightened the fluorescence signal, enhancing detection of submillimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence. Using an LED light source with adjustments to the red, blue, and green wavelengths, it is possible to simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this fluorescence laparoscopy technology for clinical use can improve staging and resection of pancreatic cancer. Copyright © 2012 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

Fluorescence-based sensing of 2,4,6-trinitrotoluene (TNT) using a multi-channeled poly(methyl methacrylate) (PMMA) microimmunosensor.

PubMed

Charles, Paul T; Adams, Andre A; Howell, Peter B; Trammell, Scott A; Deschamps, Jeffrey R; Kusterbeck, Anne W

2010-01-01

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1-10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT.

Fluorescence-based Sensing of 2,4,6-Trinitrotoluene (TNT) Using a Multi-channeled Poly(methyl methacrylate) (PMMA) Microimmunosensor

PubMed Central

Charles, Paul T.; Adams, Andre A.; Howell, Peter B.; Trammell, Scott A.; Deschamps, Jeffrey R.; Kusterbeck, Anne W.

2010-01-01

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1–10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT. PMID:22315573

Inhibition of beta-amyloid aggregation by fluorescent dye labels

NASA Astrophysics Data System (ADS)

Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.; Rolinski, Olaf J.

2014-02-01

The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelled Aβ peptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

Nanoparticles in porous microparticles prepared by supercritical infusion and pressure quench technology for sustained delivery of bevacizumab.

PubMed

Yandrapu, Sarath K; Upadhyay, Arun K; Petrash, J Mark; Kompella, Uday B

2013-12-02

Nanoparticles in porous microparticles (NPinPMP), a novel delivery system for sustained delivery of protein drugs, was developed using supercritical infusion and pressure quench technology, which does not expose proteins to organic solvents or sonication. The delivery system design is based on the ability of supercritical carbon dioxide (SC CO2) to expand poly(lactic-co-glycolic) acid (PLGA) matrix but not polylactic acid (PLA) matrix. The technology was applied to bevacizumab, a protein drug administered once a month intravitreally to treat wet age related macular degeneration. Bevacizumab coated PLA nanoparticles were encapsulated into porosifying PLGA microparticles by exposing the mixture to SC CO2. After SC CO2 exposure, the size of PLGA microparticles increased by 6.9-fold. Confocal and scanning electron microscopy studies demonstrated the expansion and porosification of PLGA microparticles and infusion of PLA nanoparticles inside PLGA microparticles. In vitro release of bevacizumab from NPinPMP was sustained for 4 months. Size exclusion chromatography, fluorescence spectroscopy, circular dichroism spectroscopy, SDS-PAGE, and ELISA studies indicated that the released bevacizumab maintained its monomeric form, conformation, and activity. Further, in vivo delivery of bevacizumab from NPinPMP was evaluated using noninvasive fluorophotometry after intravitreal administration of Alexa Fluor 488 conjugated bevacizumab in either solution or NPinPMP in a rat model. Unlike the vitreal signal from Alexa-bevacizumab solution, which reached baseline at 2 weeks, release of Alexa-bevacizumab from NPinPMP could be detected for 2 months. Thus, NPinPMP is a novel sustained release system for protein drugs to reduce frequency of protein injections in the therapy of back of the eye diseases.

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

PubMed Central

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-01-01

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting. PMID:26068810

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

NASA Astrophysics Data System (ADS)

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-06-01

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells.

PubMed

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-06-12

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

2013-01-01

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.

The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelled Aβ peptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

Aging-associated shifts in functional status of mast cells located by adult and aged mesenteric lymphatic vessels

PubMed Central

Chatterjee, Victor

2012-01-01

We had previously proposed the presence of permanent stimulatory influences in the tissue microenvironment surrounding the aged mesenteric lymphatic vessels (MLV), which influence aged lymphatic function. In this study, we performed immunohistochemical labeling of proteins known to be present in mast cells (mast cell tryptase, c-kit, prostaglandin D2 synthase, histidine decarboxylase, histamine, transmembrane protein 16A, and TNF-α) with double verification of mast cells in the same segment of rat mesentery containing MLV by labeling with Alexa Fluor 488-conjugated avidin followed by toluidine blue staining. Additionally, we evaluated the aging-associated changes in the number of mast cells located by MLV and in their functional status by inducing mast cell activation by various activators (substance P; anti-rat DNP Immunoglobulin E; peptidoglycan from Staphyloccus aureus and compound 48/80) in the presence of ruthenium red followed by subsequent staining by toluidine blue. We found that there was a 27% aging-associated increase in the total number of mast cells, with an ∼400% increase in the number of activated mast cells in aged mesenteric tissue in resting conditions with diminished ability of mast cells to be newly activated in the presence of inflammatory or chemical stimuli. We conclude that higher degree of preactivation of mast cells in aged mesenteric tissue is important for development of aging-associated impairment of function of mesenteric lymphatic vessels. The limited number of intact aged mast cells located close to the mesenteric lymphatic compartments to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response. PMID:22796537

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stratton, Dan; Lange, Sigrun; Kholia, Sharad

Highlights: • Microvesiculating cells record loss of mass on a Quartz Crystal Microbalance. • Using the Quartz Crystal Microbalance microvesicles are measured at 0.24 pg. • The QCM-D reveals loss in viscoelastic properties in microvesiculating cells. - Abstract: Using a Quartz Crystal Microbalance with dissipation monitoring, QCM-D (label-free system) measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, we showed the attachment, over a 60 min period, of a monolayer of PC3 cells to the gold electrodes of the quartz crystal sensor, which had been rendered hydrophilic. That MVs were released upon BzATP stimulation ofmore » cells was confirmed by NTA analysis (average 250 nm diameter), flow cytometry, showing high phosphatidylserine exposition and by fluorescent (Annexin V Alexa Fluor® 488-positive) and electron microscopy. Over a period of 1000s (16.7 min) during which early apoptosis increased from 4% plateauing at 10% and late apoptosis rose to 2%, the Δf increased 20 Hz, thereupon remaining constant for the last 1000s of the experiment. Using the Sauerbrey equation, the loss in mass, which corresponded to the release of 2.36 × 10{sup 6} MVs, was calculated to be 23 ng. We therefore estimated the mass of an MV to be 0.24 pg. With the deposition on the QCM-D of 3.5 × 10{sup 7} MVs over 200s, the decrease in Δf (Hz) gave an estimate of 0.235 pg per MV.« less

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong

2016-01-08

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively boundmore » the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.« less

pH shift assembly of adenoviral serotype 5 capsid protein nanosystems for enhanced delivery of nanoparticles, proteins and nucleic acids.

PubMed

Rao, Vidhya R; Upadhyay, Arun K; Kompella, Uday B

2013-11-28

Empty adenovirus serotype 5 (Ad5) capsids devoid of viral genome were developed as a novel delivery system for nanoparticles, proteins, and nucleic acids. Ad5 capsids of 110 nm diameter undergo an increase in particle size to 1637 nm in 1mM acetic acid at pH4.0 and then shrink to 60 nm, following pH reversal to 7.4. These pH shifts induced reversible changes in capsid zeta potential and secondary structure and irreversible changes in tertiary structure of capsid proteins. Using pH shift dependent changes in capsid size and structure, 20 nm fluorescent nanoparticles, FITC-BSA, and Alexa Fluor® 488 conjugated siRNA were encapsulated with high efficiency in Ad5 capsids, as confirmed by electron microscopy and/or flow cytometry. HEK cell uptake with capsid delivery system was 7.8-, 7.4-, and 2.9-fold greater for nanoparticles, FITC-BSA, and Alexa-siRNA, respectively, when compared to plain solutes. Physical mixtures of capsids and fluorescent solutes exhibited less capsid associated fluorescence intensity and cell uptake. Further, unlike physical mixture, pH shift assembled Ad5 capsids protected siRNA from RNase degradation. Ad5 capsids before and after pH shift exhibited endolysosomal escape. Thus, empty Ad5 capsids can encapsulate a variety of solutes based on pH shift assembly, resulting in enhanced cellular delivery. © 2013. Published by Elsevier B.V. All rights reserved.

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

PubMed

Hasan, Md Mehedi; Alam, Mohammad Wajih; Wahid, Khan A; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size.

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability

PubMed Central

Hasan, Md. Mehedi; Wahid, Khan A.; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size. PMID:27977709

A potential nanobiotechnology platform based on infectious bursal disease subviral particles.

PubMed

Taghavian, Omid; Mandal, Manoj K; Steinmetz, Nicole F; Rasche, Stefan; Spiegel, Holger; Fischer, Rainer; Schillberg, Stefan

2012-03-07

We describe a novel nanobiotechnology platform based on subviral particles derived from infectious bursal disease virus (IBD-SVPs). The major virus coat protein VP2 assembles into spherical, 23 nm SVPs when expressed as a heterologous protein in the yeast Pichia pastoris . We recovered up to 38 mg of IBD-SVPs at > 95% purity from 1 L of recombinant yeast culture. The purified particles were able to tolerate organic solvents up to 20% concentration (ethanol or dimethylsulfoxide), they resisted temperatures up to 65 °C and remained stable over a wide pH range (2.5-9.0). We achieved bioconjugation to the amine groups of lysine residues and to the carboxyl groups of aspartic and glutamic acid residues, allowing the functionalization of IBD-SVPs with biotin. The accessibility of surface amine groups was measured using Alexa Fluor 488 N -hydroxysuccinimide (NHS) ester, an amine-selective fluorescent dye, revealing that approximately 60 dye molecules were attached to the surface of each particle. IBD-SVPs can therefore be exploited as a robust and versatile nanoscaffold to display diverse functional ligands.

A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin.

PubMed

Popova, Dina; Jacobsson, Stig O P

2014-04-01

The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity. In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Probing minority population of antibiotic-resistant bacteria.

PubMed

Huang, Tianxun; Zheng, Yan; Yan, Ya; Yang, Lingling; Yao, Yihui; Zheng, Jiaxin; Wu, Lina; Wang, Xu; Chen, Yuqing; Xing, Jinchun; Yan, Xiaomei

2016-06-15

The evolution and spread of antibiotic-resistant pathogens has become a major threat to public health. Advanced tools are urgently needed to quickly diagnose antibiotic-resistant infections to initiate appropriate treatment. Here we report the development of a highly sensitive flow cytometric method to probe minority population of antibiotic-resistant bacteria via single cell detection. Monoclonal antibody against TEM-1 β-lactamase and Alexa Fluor 488-conjugated secondary antibody were used to selectively label resistant bacteria green, and nucleic acid dye SYTO 62 was used to stain all the bacteria red. A laboratory-built high sensitivity flow cytometer (HSFCM) was applied to simultaneously detect the side scatter and dual-color fluorescence signals of single bacteria. By using E. coli JM109/pUC19 and E. coli JM109 as the model systems for antibiotic-resistant and antibiotic-susceptible bacteria, respectively, as low as 0.1% of antibiotic-resistant bacteria were accurately quantified. By monitoring the dynamic population change of a bacterial culture with the administration of antibiotics, we confirmed that under the antimicrobial pressure, the original low population of antibiotic-resistant bacteria outcompeted susceptible strains and became the dominant population after 5hours of growth. Detection of antibiotic-resistant infection in clinical urine samples was achieved without cultivation, and the bacterial load of susceptible and resistant strains can be faithfully quantified. Overall, the HSFCM-based quantitative method provides a powerful tool for the fundamental studies of antibiotic resistance and holds the potential to provide rapid and precise guidance in clinical therapies. Copyright © 2016 Elsevier B.V. All rights reserved.

PSMA-11-Derived Dual-Labeled PSMA Inhibitors for Preoperative PET Imaging and Precise Fluorescence-Guided Surgery of Prostate Cancer.

PubMed

Baranski, Ann-Christin; Schäfer, Martin; Bauder-Wüst, Ulrike; Roscher, Mareike; Schmidt, Jana; Stenau, Esther; Simpfendörfer, Tobias; Teber, Dogu; Maier-Hein, Lena; Hadaschik, Boris; Haberkorn, Uwe; Eder, Matthias; Kopka, Klaus

2018-04-01

Resection of tumors using targeted dual-modality probes combining preoperative imaging with intraoperative guidance is of high clinical relevance and might considerably affect the outcome of prostate cancer therapy. This work aimed at the development of dual-labeled prostate-specific membrane antigen (PSMA) inhibitors derived from the established N,N' -bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine- N,N' -diacetic acid (HBED-CC)-based PET tracer 68 Ga-Glu-urea-Lys(Ahx)-HBED-CC ( 68 Ga-PSMA-11) to allow accurate intraoperative detection of PSMA-positive tumors. Methods: A series of novel PSMA-targeting fluorescent dye conjugates of Glu-urea-Lys-HBED-CC was synthesized, and their biologic properties were determined in cell-based assays and confocal microscopy. As a preclinical proof of concept, specific tumor uptake, pharmacokinetics, and feasibility for intraoperative fluorescence guidance were investigated in tumor-bearing mice and healthy pigs. Results: The designed dual-labeled PSMA inhibitors exhibited high binding affinity and PSMA-specific effective internalization. Conjugation of fluorescein isothiocyanate (10.86 ± 0.94 percentage injected dose [%ID]/g), IRDye800CW (13.66 ± 3.73 %ID/g), and DyLight800 (15.62 ± 5.52 %ID/g) resulted in a significantly increased specific tumor uptake, whereas 68 Ga-Glu-urea-Lys-HBED-CC-AlexaFluor488 (9.12 ± 5.47 %ID/g) revealed a tumor uptake similar to that of 68 Ga-PSMA-11 (4.89 ± 1.34 %ID/g). The first proof-of-concept studies with the clinically relevant candidate 68 Ga-Glu-urea-Lys-HBED-CC-IRDye800CW reinforced a fast, specific enrichment in PSMA-positive tumors, with rapid background clearance. With regard to intraoperative navigation, a specific fluorescence signal was detected in PSMA-expressing tissue. Conclusion: This study demonstrated that PSMA-11-derived dual-labeled dye conjugates are feasible for providing PSMA-specific pre-, intra-, and postoperative detection of prostate cancer lesions and have high potential for future clinical translation. © 2018 by the Society of Nuclear Medicine and Molecular Imaging.

Enzyme-directed assembly of nanoparticles in tumors monitored by in vivo whole animal imaging and ex vivo super-resolution fluorescence imaging.

PubMed

Chien, Miao-Ping; Carlini, Andrea S; Hu, Dehong; Barback, Christopher V; Rush, Anthony M; Hall, David J; Orr, Galya; Gianneschi, Nathan C

2013-12-18

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Simultaneous dual modality optical and MR imaging of mouse dorsal skin-fold window chamber

NASA Astrophysics Data System (ADS)

Salek, Mir Farrokh; Pagel, Mark D.; Gmitro, Arthur F.

2011-02-01

Optical imaging and MRI have both been used extensively to study tumor microenvironment. The two imaging modalities are complementary and can be used to cross-validate one another for specific measurements. We have developed a modular platform that is capable of doing optical microscopy inside an MRI instrument. To do this, an optical relay system transfers the image to outside of the MR bore to a commercial grade CCD camera. This enables simultaneous optical and MR imaging of the same tissue and thus creates the ideal situation for comparative or complementary studies using both modalities. Initial experiments have been done using GFP labeled prostate cancer cells implanted in mouse dorsal skin fold window chamber. Vascular hemodynamics and vascular permeability were studied using our imaging system. Towards this goal, we developed a dual MR-Optical contrast agent by labeling BSA with both Gd-DTPA and Alexa Fluor. Overall system design and results of these preliminary vascular studies are presented.

Development of test models to quantify encapsulated bioburden in spacecraft polymer materials by cultivation-dependent and molecular methods

NASA Astrophysics Data System (ADS)

Bauermeister, Anja; Moissl-Eichinger, Christine; Mahnert, Alexander; Probst, Alexander; Flier, Niwin; Auerbach, Anna; Weber, Christina; Haberer, Klaus; Boeker, Alexander

Bioburden encapsulated in spacecraft polymers (such as adhesives and coatings) poses a potential risk to scientific exploration of other celestial bodies, but it is not easily detectable. In this study, we developed novel testing strategies to estimate the quantity of intrinsic encapsulated bioburden in polymers used frequently on spaceflight hardware. In particular Scotch-Weld (TM) 2216 B/A (Epoxy adhesive); MAP SG121FD (Silicone coating), Solithane (®) 113 (Urethane resin); ESP 495 (Silicone adhesive); and Dow Corning (®) 93-500 (Silicone encapsulant) were investigated. As extraction of bioburden from polymerized (solid) materials did not prove feasible, a method was devised to extract contaminants from uncured polymer precursors by dilution in organic solvents. Cultivation-dependent analyses showed less than 0.1-2.5 colony forming units (cfu) per cm³ polymer, whereas quantitative PCR with extracted DNA indicated considerably higher values, despite low DNA extraction efficiency. Results obtained by this method reflected the most conservative proxy for encapsulated bioburden. To observe the effect of physical and chemical stress occurring during polymerization on the viability of encapsulated contaminants, Bacillus safensis spores were embedded close to the surface in cured polymer, which facilitated access for different analytical techniques. Staining by AlexaFluor succinimidyl ester 488 (AF488), propidium monoazide (PMA), CTC (5-cyano-2,3-diotolyl tetrazolium chloride) and subsequent confocal laser scanning microscopy (CLSM) demonstrated that embedded spores retained integrity, germination and cultivation ability even after polymerization of the adhesive Scotch-Weld™ 2216 B/A.

Development of Antidepressants as Novel Agents To Treat Small Cell Lung Cancer

DTIC Science & Technology

2014-08-01

least in part by the capacity of these drugs to disrupt autocrine survival loops between neuro - transmitters and their receptors at the surface of SCLC...Ser10 (p-H3; Millipore), cleaved caspase-3 (CC3; Cell Signal- ing Technology), insulin (DAKO), and synaptophysin (SYN; Neuro - mics). Alexa Fluor

Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate.

PubMed

Berguig, Geoffrey Y; Convertine, Anthony J; Shi, Julie; Palanca-Wessels, Maria Corinna; Duvall, Craig L; Pun, Suzie H; Press, Oliver W; Stayton, Patrick S

2012-12-03

Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody-poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells, where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-release dynamics of the polymer-mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alexa Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH distribution of the HD39/SA-polymer conjugates was quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH value experienced by the conjugates was also characterized as a function of time by flow cytometry. PPAA was shown to alter the intracellular trafficking kinetics strongly relative to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 h, only 11% of the HD39/SA-PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast, the average intracellular pH of HD39/SA alone dropped from 6.7 ± 0.2 at 1 h to 5.6 ± 0.5 after 3 h and 4.7 ± 0.6 after 6 h. Conjugation of the control polymer PMAA to HD39/SA showed an average pH drop similar to that of HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 h, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA-PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody-polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time.

Detection of substrate binding of a collagen-specific molecular chaperone HSP47 in solution using fluorescence correlation spectroscopy.

PubMed

Kitamura, Akira; Ishida, Yoshihito; Kubota, Hiroshi; Pack, Chan-Gi; Homma, Takayuki; Ito, Shinya; Araki, Kazutaka; Kinjo, Masataka; Nagata, Kazuhiro

2018-02-26

Heat shock protein 47 kDa (HSP47), an ER-resident and collagen-specific molecular chaperone, recognizes collagenous hydrophobic amino acid sequences (Gly-Pro-Hyp) and assists in secretion of correctly folded collagen. Elevated collagen production is correlated with HSP47 expression in various diseases, including fibrosis and keloid. HSP47 knockdown ameliorates liver fibrosis by inhibiting collagen secretion, and inhibition of the interaction of HSP47 with procollagen also prevents collagen secretion. Therefore, a high-throughput system for screening of drugs capable of inhibiting the interaction between HSP47 and collagen would aid the development of novel therapies for fibrotic diseases. In this study, we established a straightforward method for rapidly and quantitatively measuring the interaction between HSP47 and collagen in solution using fluorescence correlation spectroscopy (FCS). The diffusion rate of HSP47 labeled with Alexa Fluor 488 (HSP47-AF), a green fluorescent dye, decreased upon addition of type I or III collagen, whereas that of dye-labeled protein disulfide isomerase (PDI) or bovine serum albumin (BSA) did not, indicating that specific binding of HSP47 to collagen could be detected using FCS. Using this method, we calculated the dissociation constant of the interaction between HSP47 and collagen. The binding ratio between HSP47-AF and collagen did not change in the presence of sodium chloride, confirming that the interaction was hydrophobic in nature. In addition, we observed dissociation of collagen from HSP47 at low pH and re-association after recovery to neutral pH. These observations indicate that this system is appropriate for detecting the interaction between HSP47 and collagen, and could be applied to high-throughput screening for drugs capable of suppressing and/or curing fibrosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Screening Nylon-3 Polymers, a New Class of Cationic Amphiphiles, for siRNA Delivery

PubMed Central

2015-01-01

Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20–65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent. PMID:25437915

Reconstruction of Toll-like receptor 9-mediated responses in HEK-Blue hTLR9 cells by transfection of human macrophage scavenger receptor 1 gene.

PubMed

Ohtsuki, Shozo; Takahashi, Yuki; Inoue, Takao; Takakura, Yoshinobu; Nishikawa, Makiya

2017-10-20

We used human Toll-like receptor 9 (hTLR9)-expressing HEK-Blue hTLR9 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon response to CpG DNA, to evaluate the immunological properties of nucleic acid drug candidates. Our preliminary studies showed that phosphodiester CpG DNA hardly induced any SEAP secretion in HEK-Blue hTLR9 cells. In the current study, therefore, we developed HEK-Blue hTLR9 cells transduced with human macrophage scavenger receptor-1 (hMSR1), a cell-surface DNA receptor, and determined whether HEK-Blue hTLR9/hMSR1 cells respond to phosphorothioate (PS) CpG DNA and phosphodiester (PO) CpG DNA. We selected PS CpG2006, a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA (tetrapodna) containing ssCpG (tetraCpG) as model TLR9 ligands. Alexa Fluor 488-labeled ligands were used for flow cytometry. Unlike the mock-transfected HEK-Blue hTLR9 cells, the HEK-Blue hTLR9/hMSR1 cells efficiently took up all three CpG DNAs. SEAP release was almost proportional to the uptake. Treatment of HEK-Blue hTLR9/hMSR1 cells with an anti-hMSR1 antibody significantly reduced the uptake of ssCpG and tetraCpG. Collectively, reconstruction of TLR9-mediated responses to CpG DNA in HEK-Blue hTLR9 cells can be used to evaluate the toxicity of nucleic acid drug candidates with diverse physicochemical properties.

Screening nylon-3 polymers, a new class of cationic amphiphiles, for siRNA delivery.

PubMed

Nadithe, Venkatareddy; Liu, Runhui; Killinger, Bryan A; Movassaghian, Sara; Kim, Na Hyung; Moszczynska, Anna B; Masters, Kristyn S; Gellman, Samuel H; Merkel, Olivia M

2015-02-02

Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20-65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent.

Nanoencapsulation of pomegranate bioactive compounds for breast cancer chemoprevention.

PubMed

Shirode, Amit B; Bharali, Dhruba J; Nallanthighal, Sameera; Coon, Justin K; Mousa, Shaker A; Reliene, Ramune

2015-01-01

Pomegranate polyphenols are potent antioxidants and chemopreventive agents but have low bioavailability and a short half-life. For example, punicalagin (PU), the major polyphenol in pomegranates, is not absorbed in its intact form but is hydrolyzed to ellagic acid (EA) moieties and rapidly metabolized into short-lived metabolites of EA. We hypothesized that encapsulation of pomegranate polyphenols into biodegradable sustained release nanoparticles (NPs) may circumvent these limitations. We describe here the development, characterization, and bioactivity assessment of novel formulations of poly(D,L-lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) NPs loaded with pomegranate extract (PE) or individual polyphenols such as PU or EA. Monodispersed, spherical 150-200 nm average diameter NPs were prepared by the double emulsion-solvent evaporation method. Uptake of Alexa Fluor-488-labeled NPs was evaluated in MCF-7 breast cancer cells over a 24-hour time course. Confocal fluorescent microscopy revealed that PLGA-PEG NPs were efficiently taken up, and the uptake reached the maximum at 24 hours. In addition, we examined the antiproliferative effects of PE-, PU-, and/or EA-loaded NPs in MCF-7 and Hs578T breast cancer cells. We found that PE, PU, and EA nanoprototypes had a 2- to 12-fold enhanced effect on cell growth inhibition compared to their free counterparts, while void NPs did not affect cell growth. PU-NPs were the most potent nanoprototype of pomegranates. Thus, PU may be the polyphenol of choice for further chemoprevention studies with pomegranate nanoprototypes. These data demonstrate that nanotechnology-enabled delivery of pomegranate polyphenols enhances their anticancer effects in breast cancer cells. Thus, pomegranate polyphenols are promising agents for nanochemoprevention of breast cancer.

Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolff, Natascha A.; Lee, Wing-Kee; Abouhamed, Marouan

2008-07-01

Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membranemore » (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 {mu}M for 24 h) toxicity was significantly attenuated from 27.3 {+-} 3.9% in ARF6-WT to 11.1 {+-} 4.0% in ARF6-DN cells (n = 6, P < 0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0 {+-} 4.6% versus 3.9 {+-} 3.9% of ARF6-WT cells, n = 3, P < 0.01) and/or remained near the PM (89.3 {+-} 5. 6% versus 45.2 {+-} 14.3% of ARF6-WT cells, n = 3, P < 0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells.« less

Analysis of UV-excited fluorochromes by flow cytometry using near-ultraviolet laser diodes.

PubMed

Telford, William G

2004-09-01

Violet laser diodes have become common and reliable laser sources for benchtop flow cytometers. While these lasers are very useful for a variety of violet and some ultraviolet-excited fluorochromes (e.g., DAPI), they do not efficiently excite most UV-stimulated probes. In this study, the next generation of InGaN near-UV laser diodes (NUVLDs) emitting in the 370-375-nm range have been evaluated as laser sources for cuvette-based flow cytometers. Several NUVLDs, ranging in wavelength from 370 to 374 nm and in power level from 1.5 to 10 mW, were mounted on a BD Biosciences LSR II and evaluated for their ability to excite cells labeled with the UV DNA binding dye DAPI, several UV phenotyping fluorochromes (including Alexa Fluor 350, Marina Blue, and quantum dots), and the fluorescent calcium chelator indo-1. NUVLDs at the 8-10-mW power range gave detection sensitivity levels comparable to more powerful solid-state and ion laser sources, using low-fluorescence microsphere beads as measurement standards. NUVLDs at all tested power levels allowed extremely high-resolution DAPI cell cycle analysis, and sources in the 8-10-mW power range excited Alexa Fluor 350, Marina Blue, and a variety of quantum dots at virtually the same signal-to-noise ratios as more powerful UV sources. These evaluations indicate that near-UV laser diodes installed on a cuvette-based flow cytometer performed nearly as well as more powerful solid-state UV lasers on the same instrumentation, and comparably to more powerful ion lasers on a jet-in-air system, and. Despite their limited power, integration of these small and inexpensive lasers into benchtop flow cytometers should allow the use of flow cytometric applications requiring UV excitation on a wide variety of instruments. Copyright 2004 Wiley-Liss, Inc.

Utilizing hyaluronic acid as a versatile platform for fluorescence resonance energy transfer-based glucose sensing.

PubMed

Ge, Minghao; Bai, Pengli; Chen, Mingli; Tian, Jingjing; Hu, Jun; Zhi, Xu; Yin, Huancai; Yin, Jian

2018-03-01

Here, we utilized the ultrasonic emulsification technique to generate hyaluronic acid microspheres incorporating a fluorescence-based glucose biosensor. We synthesized a novel lanthanide ion luminophore based on Eu 3+ . Eu sulfosuccinimidyl dextran (Eu-dextran) and Alexa Fluor 647 sulfosuccinimidyl-ConA (Alexa Fluor 647-ConA) were encapsulated in hyaluronic acid hydrogel to generate microspheres. Glucose sensing was carried out using a fluorescence resonance energy transfer (FRET)-based assay principle. A proportional fluorescence intensity increase was found within a 0.5-10-mM glucose concentration range. The glucose-sensing strategy showed an excellent tolerance for potential interferents. Meanwhile, the fluorescent signal of hyaluronic acid microspheres was very stable after testing for 72 h in glucose solution. Overall, hyaluronic acid microspheres encapsulating sensing biomolecules offer a stable and biocompatible biosensor for a variety of applications including cell culture systems, tissue engineering, detection of blood glucose, etc. Graphical abstract We report an ingenious biosensor encapsulated in hyaluronic acid microspheres for monitoring of glucose. Glucose sensing is carried out using a fluorescence resonance energy transfer-based assay principle with a novel lanthanide ions luminophore. The glucose detection system has excellent biocompatibility and stability for monitoring of glucose.

Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by In Vivo Whole Animal and Ex Vivo Super-Resolution Fluorescence Imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chien, Miao-Ping; Carlini, Andrea S.; Hu, Dehong

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block, and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo, and in ex vivo organ analysis following intratumoral injectionmore » into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micron-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.« less

Light emitting diode, photodiode-based fluorescence detection system for DNA analysis with microchip electrophoresis.

PubMed

Hall, Gordon H; Glerum, D Moira; Backhouse, Christopher J

2016-02-01

Electrophoretic separation of fluorescently end-labeled DNA after a PCR serves as a gold standard in genetic diagnostics. Because of their size and cost, instruments for this type of analysis have had limited market uptake, particularly for point-of-care applications. This might be changed through a higher level of system integration and lower instrument costs that can be realized through the use of LEDs for excitation and photodiodes for detection--if they provide sufficient sensitivity. Here, we demonstrate an optimized microchip electrophoresis instrument using polymeric fluidic chips with fluorescence detection of end-labeled DNA with a LOD of 0.15 nM of Alexa Fluor 532. This represents orders of magnitude improvement over previously reported instruments of this type. We demonstrate the system with an electrophoretic separation of two PCR products and their respective primers. We believe that this is the first LED-induced fluorescence microchip electrophoresis system with photodiode-based detection that could be used for standard applications of PCR and electrophoresis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biocatalytic response of multi-layer assembled collagen/hyaluronic acid nanoengineered capsules.

PubMed

Sousa, Fernanda; Kreft, Oliver; Sukhorukov, Gleb B; Möhwald, Helmuth; Kokol, Vanja

2014-01-01

Biodegradable hollow capsules filled with fluorescently labelled bovine serum albumin (BSA) as a model drug were prepared via layer-by-layer (LbL) self-assembly of type-I collagen (COL) and hyaluronic acid (HA) using calcium carbonate micro-particles and co-precipitation method. Capsules loaded with fluorescein isothiocyanate (FITC)-BSA, tetramethylrhodamin isothiocyanate (TRITC)-BSA or Alex-Fluor-488-BSA, respectively, were characterised before and after core removal using Confocal Laser Scanning Microscopy (CLSM), whilst the morphologies of individual hollow capsules were assessed using Atomic Force Microscopy (AFM). The sustained release of the encapsulated FITC-BSA protein was attained using enzymatic degradation of the capsule shells by collagenase. The released profile of the fluorescently-labelled BSA indicated that it could be successfully controlled by modulating the number of layers and/or by collagen crosslinking either before or after the capsule's assembly.

A Novel PET Imaging Using 64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

PubMed Central

Kobayashi, Kazuko; Sasaki, Takanori; Takenaka, Fumiaki; Yakushiji, Hiromasa; Fujii, Yoshihiro; Kishi, Yoshiro; Kita, Shoichi; Shen, Lianhua; Kumon, Hiromi; Matsuura, Eiji

2015-01-01

Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions. PMID:25883990

Improving Joint Function Using Photochemical Hydrogels for Articular Surface Repair

DTIC Science & Technology

2013-10-01

riboflavin and blue light in hypoxic conditions. Control gels were not photochemically crosslinked . New cartilage matrix was formed in vivo in mice after 4...Sections were probed with AlexaFluor 568- conjugated secondary antibodies and counterstained with DAPI for cell nuclei. All samples were processed at...calcium deposits demonstrated with von Kossa stains; 2) A degradable form of photochemically crosslinked PEG norbomene gel was formulated and growth

Activatable Optical Imaging with a Silica-Rhodamine Based Near Infrared (SiR700) Fluorophore: A comparison with cyanine based dyes

PubMed Central

McCann, Thomas E.; Kosaka, Nobuyuki; Koide, Yuichiro; Mitsunaga, Makoto; Choyke, Peter L.; Nagano, Tetsuo; Urano, Yasuteru; Kobayashi, Hisataka

2011-01-01

Optical imaging is emerging as an important tool to visualize tumors. However, there are many potential choices among the available fluorophores. Optical imaging probes that emit in the visible range can image superficial tumors with high quantum yields, however, if deeper imaging is needed then near infrared (NIR) fluorophores are necessary. Most commercially available NIR fluorophores are cyanine based and are prone to non-specific binding and relatively limited photostability. Silica-containing rhodamine (SiR) fluorophores represent a new class of NIR fluorophores, which permit photoactivation via H-dimer formation as well as demonstrate improved photostability. This permits higher tumor-to-background ratios (TBRs) to be achieved over longer periods of time. Here, we compared an avidin conjugated with SiR700 (Av-SiR700) to similar compounds based on cyanine dyes (Av-Cy5.5 and Av-Alexa Fluor 680) in a mouse tumor model of ovarian cancer metastasis. We found that the Av-SiR700 probe demonstrated superior quenching enabling activation after binding-internalization to the target cell. As a result, Av-SiR700 had higher TBRs compared to Av-Cy5.5, and better biostability compared to Av-Alexa Fluor 680. PMID:22034863

A molecular-sized optical logic circuit for digital modulation of a fluorescence signal

NASA Astrophysics Data System (ADS)

Nishimura, Takahiro; Tsuchida, Karin; Ogura, Yusuke; Tanida, Jun

2018-03-01

Fluorescence measurement allows simultaneous detection of multiple molecular species by using spectrally distinct fluorescence probes. However, due to the broad spectra of fluorescence emission, the multiplicity of fluorescence measurement is generally limited. To overcome this limitation, we propose a method to digitally modulate fluorescence output signals with a molecular-sized optical logic circuit by using optical control of fluorescence resonance energy transfer (FRET). The circuit receives a set of optical inputs represented with different light wavelengths, and then it switches high and low fluorescence intensity from a reporting molecule according to the result of the logic operation. By using combinational optical inputs in readout of fluorescence signals, the number of biomolecular species that can be identified is increased. To implement the FRET-based circuits, we designed two types of basic elements, YES and NOT switches. An YES switch produces a high-level output intensity when receiving a designated light wavelength input and a low-level intensity without the light irradiation. A NOT switch operates inversely to the YES switch. In experiments, we investigated the operation of the YES and NOT switches that receive a 532-nm light input and modulate the fluorescence intensity of Alexa Fluor 488. The experimental result demonstrates that the switches can modulate fluorescence signals according to the optical input.

Direct Quantification of Solute Diffusivity in Agarose and Articular Cartilage Using Correlation Spectroscopy.

PubMed

Shoga, Janty S; Graham, Brian T; Wang, Liyun; Price, Christopher

2017-10-01

Articular cartilage is an avascular tissue; diffusive transport is critical for its homeostasis. While numerous techniques have been used to quantify diffusivity within porous, hydrated tissues and tissue engineered constructs, these techniques have suffered from issues regarding invasiveness and spatial resolution. In the present study, we implemented and compared two separate correlation spectroscopy techniques, fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), for the direct, and minimally-invasive quantification of fluorescent solute diffusion in agarose and articular cartilage. Specifically, we quantified the diffusional properties of fluorescein and Alexa Fluor 488-conjugated dextrans (3k and 10k) in aqueous solutions, agarose gels of varying concentration (i.e. 1, 3, 5%), and in different zones of juvenile bovine articular cartilage explants (i.e. superficial, middle, and deep). In agarose, properties of solute diffusion obtained via FCS and RICS were inversely related to molecule size, gel concentration, and applied strain. In cartilage, the diffusional properties of solutes were similarly dependent upon solute size, cartilage zone, and compressive strain; findings that agree with work utilizing other quantification techniques. In conclusion, this study established the utility of FCS and RICS as simple and minimally invasive techniques for quantifying microscale solute diffusivity within agarose constructs and articular cartilage explants.

Ocular Drug Delivery through pHEMA-Hydrogel Contact Lenses Co-Loaded with Lipophilic Vitamins

NASA Astrophysics Data System (ADS)

Lee, Dasom; Cho, Seungkwon; Park, Hwa Sung; Kwon, Inchan

2016-09-01

Ocular drug delivery through hydrogel contact lenses has great potential for the treatment of ocular diseases. Previous studies showed that the loading of lipophilic vitamin E to silicone-hydrogel contact lenses was beneficial in ocular drug delivery. We hypothesized that vitamin E loading to another type of popular hydrogel contact lenses, pHEMA-hydrogel contact lenses, improves ocular drug delivery by increasing the drug loading or the duration of drug release. Loading of vitamin E to pHEMA-hydrogel contact lenses significantly increased the loading of a hydrophilic drug surrogate (Alexa Fluor 488 dye) and two hydrophilic glaucoma drugs (timolol and brimonidine) to the lenses by 37.5%, 19.1%, and 18.7%, respectively. However, the release duration time was not significantly altered. Next, we hypothesized that the lipophilic nature of vitamin E attributes to the enhanced drug loading. Therefore, we investigated the effects of co-loading of another lipophilic vitamin, vitamin A, on drug surrogate delivery. We found out that vitamin A loading also increased the loading of the drug surrogate to pHEMA-hydrogel contact lenses by 30.3%. Similar to vitamin E loading, vitamin A loading did not significantly alter the release duration time of the drug or drug surrogate.

Altered osteoblast structure and function in parabolic flight

NASA Astrophysics Data System (ADS)

Zhong-Quan, Dai; Ying-Hui, Li; Fen, Yang; Bai, Ding; Ying-Jun, Tan

Introduction Bone loss has a significant impact on astronauts during spaceflight being one of the main obstacles preventing interplanetary missions However the exact mechanism is not well understood In the present study we investigated the effects of acute gravitational changes generated by parabolic flight on the structure and function of osteoblasts ROS17 2 8 carried by airbus A300 Methods The alteration of microfilament cytoskeleton was observed by the Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I immunofluorescence stain ALP activity and expression COL1A1 expression osteocalcin secrete which presenting the osteoblast function were detected by modified calcium and cobalt method RT-PCR and radioimmunity methods respectively Results The changed gravity induced the reorganization of microfilament cytoskeleton of osteoblast After 3 hours parabolic flight F-actin of osteoblast cytoskeleton became more thickness and directivity whereas G-actin reduced and relatively concentrated at the edge of nucleus observed by confocal fluorescence microscopy This phenomenon is identical with structure alternation observed in hypergravity but the osteoblast function decrease The excretion of osteocalcin the activity and mRNA expression of ALP decrease but the COL1A1 expression has no changes These results were similar to the changes in simulated or real microgravity Conclusion Above results suggest that short time gravity alternative change induce osteoblast structure and function

Diffusion properties of molecules at the blood-brain interface: potential contributions of astrocyte endfeet to diffusion barrier functions.

PubMed

Nuriya, Mutsuo; Shinotsuka, Takanori; Yasui, Masato

2013-09-01

Molecular diffusion in the extracellular space (ECS) plays a key role in determining tissue physiology and pharmacology. The blood-brain barrier regulates the exchange of substances between the brain and the blood, but the diffusion properties of molecules at this blood-brain interface, particularly around the astrocyte endfeet, are poorly characterized. In this study, we used 2-photon microscopy and acute brain slices of mouse neocortex and directly assessed the diffusion patterns of fluorescent molecules. By observing the diffusion of unconjugated and 10-kDa dextran-conjugated Alexa Fluor 488 from the ECS of the brain parenchyma to the blood vessels, we find various degrees of diffusion barriers at the endfeet: Some allow the invasion of dye inside the endfoot network while others completely block it. Detailed analyses of the time course for dye clearance support the existence of a tight endfoot network capable of acting as a diffusion barrier. Finally, we show that this diffusion pattern collapses under pathological conditions. These data demonstrate the heterogeneous nature of molecular diffusion dynamics around the endfeet and suggest that these structures can serve as the diffusion barrier. Therefore, astrocyte endfeet may add another layer of regulation to the exchange of molecules between blood vessels and brain parenchyma.

Quantification of encapsulated bioburden in spacecraft polymer materials by cultivation-dependent and molecular methods.

PubMed

Bauermeister, Anja; Mahnert, Alexander; Auerbach, Anna; Böker, Alexander; Flier, Niwin; Weber, Christina; Probst, Alexander J; Moissl-Eichinger, Christine; Haberer, Klaus

2014-01-01

Bioburden encapsulated in spacecraft polymers (such as adhesives and coatings) poses a potential risk to jeopardize scientific exploration of other celestial bodies. This is particularly critical for spacecraft components intended for hard landing. So far, it remained unclear if polymers are indeed a source of microbial contamination. In addition, data with respect to survival of microbes during the embedding/polymerization process are sparse. In this study we developed testing strategies to quantitatively examine encapsulated bioburden in five different polymers used frequently and in large quantities on spaceflight hardware. As quantitative extraction of the bioburden from polymerized (solid) materials did not prove feasible, contaminants were extracted from uncured precursors. Cultivation-based analyses revealed <0.1-2.5 colony forming units (cfu) per cm3 polymer, whereas quantitative PCR-based detection of contaminants indicated considerably higher values, despite low DNA extraction efficiency. Results obtained from this approach reflect the most conservative proxy for encapsulated bioburden, as they give the maximum bioburden of the polymers irrespective of any additional physical and chemical stress occurring during polymerization. To address the latter issue, we deployed an embedding model to elucidate and monitor the physiological status of embedded Bacillus safensis spores in a cured polymer. Staining approaches using AlexaFluor succinimidyl ester 488 (AF488), propidium monoazide (PMA), CTC (5-cyano-2,3-diotolyl tetrazolium chloride) demonstrated that embedded spores retained integrity, germination and cultivation ability even after polymerization of the adhesive Scotch-Weld 2216 B/A. Using the methods presented here, we were able to estimate the worst case contribution of encapsulated bioburden in different polymers to the bioburden of spacecraft. We demonstrated that spores were not affected by polymerization processes. Besides Planetary Protection considerations, our results could prove useful for the manufacturing of food packaging, pharmacy industry and implant technology.

Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays: a new Rapid Catch and Signal (RCS) technology.

PubMed

Dragan, Anatoliy I; Golberg, Karina; Elbaz, Amit; Marks, Robert; Zhang, Yongxia; Geddes, Chris D

2011-03-07

For analyses of DNA fragment sequences in solution we introduce a 2-color DNA assay, utilizing a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization. The assay is based on a new "Catch and Signal" technology, i.e. the simultaneous specific recognition of two target DNA sequences in one well by complementary anchor-ssDNAs, attached to silver island films (SiFs). It is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver NPs, which is ideal for MEF. Subsequently the emission dramatically increases, while the excited state lifetime decreases. It is also shown that 30s microwave irradiation of wells, containing DNA molecules, considerably (~1000-fold) speeds up the highly selective hybridization of DNA fragments at ambient temperature. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis. Copyright © 2010 Elsevier B.V. All rights reserved.

Competitive FRET-aptamer-based detection of methylphosphonic acid, a common nerve agent metabolite.

PubMed

Bruno, John G; Carrillo, Maria P; Phillips, Taylor; Vail, Neal K; Hanson, Douglas

2008-09-01

Competitive fluorescence resonance energy transfer (FRET)-aptamer-based assay formats are described for one-step detection of methylphosphonic acid (MPA; a metabolite of several organophosphorus (OP) nerve agents). AminoMPA was attached to tosyl-magnetic beads and used for DNA aptamer selection from which one dominant aptamer sequence emerged. Two different FRET approaches were attempted. In one approach, the complementary DNA sequence was used as a template for labeling the aptamer with Alexa Fluor 546 (AF 546)-14-dUTP by asymmetric PCR. Following 3-dimensional (3-D), molecular modeling of the aptamer-MPA complex, a series of three fluoresceinated aptamers labeled at positions 50, 51, and 52 in the putative optimal binding pocket were synthesized. In both FRET formats, aminoMPA was linked to Black Hole Quencher (BHQ-1 or BHQ-2)-succinimides and allowed to bind the fluorescein or AF 546-labeled MPA aptamer. Following gel filtration to purify the labeled MPA aptamer-BHQ-aminoMPA FRET complexes, the complexes were competed against various concentrations of unlabeled MPA, MPA derivatives, and unrelated compounds in titration and cross-reactivity studies. Both approaches yielded low microgram per milliliter detection limits for MPA with generally low levels of cross-reactivity for unrelated compounds. However, the data suggest a pattern of traits that may effect the direction (lights on or off) and intensity of the FRET.

Novel Micro/Nano Approaches for Glucose Measurement Using pH-Sensitive

DTIC Science & Technology

2006-06-01

step, the anionic enzyme was assembled in multilayers in alternation with poly(allylamine hydrochloride ) (PAH) (polycation). A total of three...group of D- glucosamine residues in chitosan can be conjugated with amine-reactive dyes, such as succinimidyl esters (Alexa Fluor 647™, CY5...measured values for FITC-chitosan were found to be: A494nm=0.153276 MW=161 [D- glucosamine ]=0.77g/L εdye=68000cm-1M-1

How Giardia Swim and Divide

PubMed Central

Ghosh, Sudip; Frisardi, Marta; Rogers, Rick; Samuelson, John

2001-01-01

To determine how binuclear giardia swim, we used video microscopy to observe trophozoites of Giardia intestinalis, which were labeled with an amino-specific Alexa Fluor dye that highlighted the flagella and adherence disc. Giardia swam forward by means of the synchronous beating of anterior, posterolateral, and ventral flagella in the plane of the ventral disc, while caudal flagella swam in a plane perpendicular to the disc. Giardia turned in the plane of the disc by means of a rudder-like motion of its tail, which was constant rather than beating. To determine how giardia divide, we used three-dimensional confocal microscopy, the same surface label, nuclear stains, and antitubulin antibodies. Giardia divided with mirror-image symmetry in the plane of the adherence disc, so that the right nucleus of the mother became the left nucleus of the daughter. Pairs of nuclei were tethered together by microtubules which surrounded nuclei and prevented mother or daughter giardia from receiving two copies of the same nucleus. New adherence discs formed upon a spiral backbone of microtubules, which had a clockwise rotation when viewed from the ventral surface. These dynamic observations of the parasite begin to reveal how giardia swim and divide. PMID:11705969

Continuously manufactured magnetic polymersomes - a versatile tool (not only) for targeted cancer therapy

NASA Astrophysics Data System (ADS)

Bleul, Regina; Thiermann, Raphael; Marten, Gernot U.; House, Michael J.; Pierre, Timothy G. St.; Häfeli, Urs O.; Maskos, Michael

2013-11-01

Micromixer technology was used to prepare polymeric vesicles (Pluronic® L-121) dual loaded with the anti-cancer drug camptothecin and magnetic nanoparticles. Successful incorporation of the magnetic nanoparticles was confirmed by transmission electron microscopy. Dynamic light scattering measurements showed a relatively narrow size distribution of the hybrid polymersomes. Camptothecin polymersomes reduced the cell viability of prostate cancer cells (PC-3) measured after 72 h significantly, while drug-free polymersomes showed no cytotoxic effects. Covalent attachment of a cancer targeting peptide (bombesin) as well as a fluorescent label (Alexa Fluor® 647) to the hybrid polymersomes was performed and specific cell binding and internalization were shown by flow cytometry and confocal microscopy. Relaxometry measurements clearly demonstrated the capacity of magnetic polymersomes to generate significant T2-weighted MRI contrast and potentially allow for direct monitoring of the biodistribution of the polymersomes. Micromixer technology as an easy, fast and efficient way to manufacture hybrid polymersomes as theranostic drug delivery devices is a further step from basic research to personalized medicine.

Multifunctional nanocarriers for biomedical applications

NASA Astrophysics Data System (ADS)

Bleul, R.; Thiermann, R.; Saatchi, K.; Häfeli, U. O.; Maskos, M.

2013-02-01

Polymeric vesicles (Pluronic® L-121) loaded with magnetic nanoparticles (MNP) and an anti-cancer drug (camptothecin) were prepared continuously in a micro mixing device. Characterization by TEM confirmed the successful incorporation of the MNP and DLS measurements showed a relatively narrow size distribution of the hybrid polymersomes. A very high drug loading of camptothecin (100 μg/ml in the polymersome formulation) was reached and a drug release study of loaded magnetic polymersomes has shown a sustained camptothecin release over several days. Carboxylation of Pluronic® L-121 was performed and enabled a further surface functionalization with bombesin, a 14 amino acid peptide, which binds specifically to the GRPR (gastrin releasing peptide receptor). This receptor is often overexpressed in tumor cells (e.g., human prostate cancer cells) and therefore a suitable target for cancer treatment. An additional fluorescence label with Alexa Fluor® 647 allow tracking of the polymersomes e.g., in cell experiments. Relaxivity measurements to evaluate the potential of magnetic polymersomes as MR contrast agent for in vivo imaging are in progress.

Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells

NASA Astrophysics Data System (ADS)

Rehman, Shagufta; Brown-Steinke, Kathleen; Palmer, Lisa; Periasamy, Ammasi

2015-03-01

Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.

Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans.

PubMed

Barkó, Szilvia; Szatmári, Dávid; Bódis, Emőke; Türmer, Katalin; Ujfalusi, Zoltán; Popp, David; Robinson, Robert C; Nyitrai, Miklós

2016-09-01

Weil's syndrome is caused by Leptospira interrogans infections, a Gram negative bacterium with a distinct thin corkscrew cell shape. The molecular basis for this unusual morphology is unknown. In many bacteria, cell wall synthesis is orchestrated by the actin homolog, MreB. Here we have identified the MreB within the L. interrogans genome and expressed the His-tagged protein product of the synthesized gene (Li-MreB) in Escherichia coli. Li-MreB did not purify under standard nucleotide-free conditions used for MreBs from other species, requiring the continual presence of ATP to remain soluble. Covalent modification of Li-MreB free thiols with Alexa488 produced a fluorescent version of Li-MreB. We developed native and denaturing/refolding purification schemes for Li-MreB. The purified product was shown to assemble and disassemble in MgCl2 and KCl dependent manners, as monitored by light scattering and sedimentation studies. The fluorescence spectrum of labeled Li-MreB-Alexa488 showed cation-induced changes in line with an activation process followed by a polymerization phase. The resulting filaments appeared as bundles and sheets under the fluorescence microscope. Finally, since the Li-MreB polymerization was cation dependent, we developed a simple method to measure monovalent cation concentrations within a test case prokaryote, E. coli. We have identified and initially characterized the cation-dependent polymerization properties of a novel MreB from a non-rod shaped bacterium and developed a method to measure cation concentrations within prokaryotes. This initial characterization of Li-MreB will enable future structural determination of the MreB filament from this corkscrew-shaped bacterium. Copyright © 2016 Elsevier B.V. All rights reserved.

Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding, Endocytosis, and Degradation*

PubMed Central

Date, Kimie; Satoh, Ayano; Iida, Kaoruko; Ogawa, Haruko

2015-01-01

α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na+/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104–23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption stage in the intestine. PMID:26023238

Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA.

PubMed

Zelenka, Jaroslav; Alán, Lukáš; Jabůrek, Martin; Ježek, Petr

2014-04-01

Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

Real-time association rate constant measurement using combination tapered fiber-optic biosensor (CTFOB) dip-probes

NASA Astrophysics Data System (ADS)

Simmonds, Boris; Wang, Chun-Wei; Kapoor, Rakesh

2010-02-01

This document reports a novel method of measuring association rate constant (ka) for antibody-antigen interaction using evanescent wave-based combination tapered fiber-optic biosensor (CTFOB) dip-probes. The method was demonstrated by measuring association rate constant for bovine serum albumin (BSA) and anti-BSA antibody interaction. "Direct method" was used for detection; goat anti-BSA "capture" antibodies were immobilized on the probe surfaces while the antigen (BSA) was directly labeled with Alexa 488 dye. The probes were subsequently submerged in 3nM Labeled BSA in egg albumin (1 mg/ml). The fluorescence signal recorded was proportional to BSA anti-BSA conjugates and continuous signal was acquired suing a fiber optic spectrometer (Ocean Optics, Inc.). A 476 nm diode laser was use as an excitation source. Association constant was estimated from a plot of signal as a function of time. Measured association rate constant ka for the binding of BSA with anti-BSA at room temperature is (8.33 +/- 0.01) x 104 M-1s-1.

Improved Dye Stability in Single-Molecule Fluorescence Experiments

NASA Astrophysics Data System (ADS)

EcheverrÍa Aitken, Colin; Marshall, R. Andrew; Pugi, Joseph D.

Complex biological systems challenge existing single-molecule methods. In particular, dye stability limits observation time in singlemolecule fluorescence applications. Current approaches to improving dye performance involve the addition of enzymatic oxygen scavenging systems and small molecule additives. We present an enzymatic oxygen scavenging system that improves dye stability in single-molecule experiments. Compared to the currently-employed glucose-oxidase/catalase system, the protocatechuate-3,4-dioxygenase system achieves lower dissolved oxygen concentration and stabilizes single Cy3, Cy5, and Alexa488 fluorophores. Moreover, this system possesses none of the limitations associated with the glucose oxidase/catalase system. We also tested the effects of small molecule additives in this system. Biological reducing agents significantly destabilize the Cy5 fluorophore as a function of reducing potential. In contrast, anti-oxidants stabilize the Cy3 and Alexa488 fluorophores. We recommend use of the protocatechuate-3,4,-dioxygenase system with antioxidant additives, and in the absence of biological reducing agents. This system should have wide application to single-molecule fluorescence experiments.

Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5 Monoclonal Antibody

DTIC Science & Technology

2013-08-01

Janelia Farm , VA, travel award 2010 -­‐ Translational Medicine Symposium, HHMI Peer Cluster Meeting, travel award 2010 -­‐ AACR Annual Conference...lab. Science Education Alliance, Janelia Farm , VA, 2001 3. Londoño-Joshi AI, Forero-Torres A, Fineberg NS, Oliver PG, Zhou T, LoBuglio AF, Buchsbaum...rabbit mAb and cleaved caspase 3 rabbit mAb were purchased from Cell Signaling (Billerica, MA). Secondary antibodies, Alexa fluor 405 goat anti-rabbit

X-ray microbeam stand-alone facility for cultured cells irradiation

NASA Astrophysics Data System (ADS)

Bożek, Sebastian; Bielecki, Jakub; Wiecheć, Anna; Lekki, Janusz; Stachura, Zbigniew; Pogoda, Katarzyna; Lipiec, Ewelina; Tkocz, Konrad; Kwiatek, Wojciech M.

2017-03-01

The article describes an X-ray microbeam standalone facility dedicated for irradiation of living cultured cells. The article can serve as an advice for such facilities construction, as it begins from engineering details, through mathematical modeling and experimental procedures, ending up with preliminary experimental results and conclusions. The presented system consists of an open type X-ray tube with microfocusing down to about 2 μm, an X-ray focusing system with optical elements arranged in the nested Kirckpatrick-Baez (or Montel) geometry, a sample stand and an optical microscope with a scientific digital CCD camera. For the beam visualisation an X-ray sensitive CCD camera and a spectral detector are used, as well as a scintillator screen combined with the microscope. A method of precise one by one irradiation of previously chosen cells is presented, as well as a fast method of uniform irradiation of a chosen sample area. Mathematical models of beam and cell with calculations of kerma and dose are presented. The experiments on dose-effect relationship, kinetics of DNA double strand breaks repair, as well as micronuclei observation were performed on PC-3 (Prostate Cancer) cultured cells. The cells were seeded and irradiated on Mylar foil, which covered a hole drilled in the Petri dish. DNA lesions were visualised with γ-H2AX marker combined with Alexa Fluor 488 fluorescent dye.

Preliminary investigation of proton and helium ion radiation effects on fluorescent dyes for use in astrobiology applications.

PubMed

Thompson, Daniel P; Wilson, Paul K; Sims, Mark R; Cullen, David C; Holt, John M C; Parker, David J; Smith, Mike D

2006-04-15

The Specific Molecular Identification of Life Experiment (SMILE) instrument (Sims et al. Planet. Space Science 2005, 53, 781-791) proposes to use specific molecular receptors for the detection of organic biomarkers on future astrobiology missions (e.g., to Mars). Such receptors will be used in assays with fluorescently labeled assay reagents. A key uncertainty of this approach is whether the fluorescent labels used in the system will survive exposure to levels of solar and galactic particle radiation encountered during a flight to Mars. Therefore, two fluorescent dyes (fluorescein and Alexa Fluor 633) have been exposed to low-energy proton and alpha radiation with total fluences comparable or exceeding that expected during an unshielded cruise to Mars. The results of these initial experiments are presented, which show that both dyes retain their fluorescent properties after irradiation. No significant alteration in the absorption and emission wavelengths or the quantum yields of the dyes with either radiation exposure was found. These results suggest other structurally similar fluorophores will likely retain their fluorescent properties after exposure to similar levels of proton and alpha radiation. However, more extensive radiation fluorophore testing is needed before their suitability for astrobiology missions to Mars can be fully confirmed.

Fractone-heparan sulphates mediate FGF-2 stimulation of cell proliferation in the adult subventricular zone.

PubMed

Douet, V; Kerever, A; Arikawa-Hirasawa, E; Mercier, F

2013-04-01

Fractones are extracellular matrix structures that form a niche for neural stem cells and their immediate progeny in the subventricular zone of the lateral ventricle (SVZa), the primary neurogenic zone in the adult brain. We have previously shown that heparan sulphates (HS) associated with fractones bind fibroblast growth factor-2 (FGF-2), a powerful mitotic growth factor in the SVZa. Here, our objective was to determine whether the binding of FGF-2 to fractone-HS is implicated in the mechanism leading to cell proliferation in the SVZa. Heparitinase-1 was intracerebroventricularly injected with FGF-2 to N-desulfate HS proteoglycans and determine whether the loss of HS and of FGF-2 binding to fractones modifies FGF-2 effect on cell proliferation. We also examined in vivo the binding of Alexa-Fluor-FGF-2 in relationship with the location of HS immunoreactivity in the SVZa. Heparatinase-1 drastically reduced the stimulatory effect of FGF-2 on cell proliferation in the SVZa. Alexa-Fluor-FGF-2 binding was strictly co-localized with HS immunoreactivity in fractones and adjacent vascular basement membranes in the SVZa. Our results demonstrate that FGF-2 requires HS to stimulate cell proliferation in the SVZa and suggest that HS associated with fractones and vascular basement membranes are responsible for activating FGF-2. Therefore, fractones and vascular basement membranes may function as a HS niche to drive cell proliferation in the adult neurogenic zone. © 2013 Blackwell Publishing Ltd.

Sustained subconjunctival protein delivery using a thermosetting gel delivery system.

PubMed

Rieke, Erin R; Amaral, Juan; Becerra, S Patricia; Lutz, Robert J

2010-02-01

An effective treatment modality for posterior eye diseases would provide prolonged delivery of therapeutic agents, including macromolecules, to eye tissues using a safe and minimally invasive method. The goal of this study was to assess the ability of a thermosetting gel to deliver a fluorescently labeled protein, Alexa 647 ovalbumin, to the choroid and retina of rats following a single subconjunctival injection of the gel. Additional experiments were performed to compare in vitro to in vivo ovalbumin release rates from the gel. The ovalbumin content of the eye tissues was monitored by spectrophotometric assays of tissue extracts of Alexa 647 ovalbumin from dissected sclera, choroid, and retina at time points ranging from 2 h to 14 days. At the same time points, fluorescence microscopy images of tissue samples were also obtained. Measurement of intact ovalbumin was verified by LDS-PAGE analysis of the tissue extract solutions. In vitro release of Alexa 488 ovalbumin into 37 degrees C PBS solutions from ovalbumin-loaded gel pellets was also monitored over time by spectrophotometric assay. In vivo ovalbumin release rates were determined by measurement of residual ovalbumin extracted from gel pellets removed from rat eyes at various time intervals. Our results indicate that ovalbumin concentrations can be maintained at measurable levels in the sclera, choroid, and retina of rats for up to 14 days using the thermosetting gel delivery system. The concentration of ovalbumin exhibited a gradient that decreased from sclera to choroid and to retina. The in vitro release rate profiles were similar to the in vivo release profiles. Our findings suggest that the thermosetting gel system may be a feasible method for safe and convenient sustained delivery of proteins to choroidal and retinal tissue in the posterior segments of the eye.

Sustained Subconjunctival Protein Delivery Using a Thermosetting Gel Delivery System

PubMed Central

2010-01-01

Purpose: An effective treatment modality for posterior eye diseases would provide prolonged delivery of therapeutic agents, including macromolecules, to eye tissues using a safe and minimally invasive method. The goal of this study was to assess the ability of a thermosetting gel to deliver a fluorescently labeled protein, Alexa 647 ovalbumin, to the choroid and retina of rats following a single subconjunctival injection of the gel. Additional experiments were performed to compare in vitro to in vivo ovalbumin release rates from the gel. Methods: The ovalbumin content of the eye tissues was monitored by spectrophotometric assays of tissue extracts of Alexa 647 ovalbumin from dissected sclera, choroid, and retina at time points ranging from 2 h to 14 days. At the same time points, fluorescence microscopy images of tissue samples were also obtained. Measurement of intact ovalbumin was verified by LDS-PAGE analysis of the tissue extract solutions. In vitro release of Alexa 488 ovalbumin into 37°C PBS solutions from ovalbumin-loaded gel pellets was also monitored over time by spectrophotometric assay. In vivo ovalbumin release rates were determined by measurement of residual ovalbumin extracted from gel pellets removed from rat eyes at various time intervals. Results: Our results indicate that ovalbumin concentrations can be maintained at measurable levels in the sclera, choroid, and retina of rats for up to 14 days using the thermosetting gel delivery system. The concentration of ovalbumin exhibited a gradient that decreased from sclera to choroid and to retina. The in vitro release rate profiles were similar to the in vivo release profiles. Conclusions: Our findings suggest that the thermosetting gel system may be a feasible method for safe and convenient sustained delivery of proteins to choroidal and retinal tissue in the posterior segments of the eye. PMID:20148655

Tracking Antibody Distribution with Near-Infrared Fluorescent Dyes: Impact of Dye Structure and Degree of Labeling on Plasma Clearance.

PubMed

Cilliers, Cornelius; Nessler, Ian; Christodolu, Nikolas; Thurber, Greg M

2017-05-01

Monoclonal antibodies labeled with near-infrared (NIR) fluorophores have potential use in disease detection, intraoperative imaging, and pharmacokinetic characterization of therapeutic antibodies in both the preclinical and clinical setting. Recent work has shown conjugation of NIR fluorophores to antibodies can potentially alter antibody disposition at a sufficiently high degree of labeling (DoL); however, other reports show minimal impact after labeling with NIR fluorophores. In this work, we label two clinically approved antibodies, Herceptin (trastuzumab) and Avastin (bevacizumab), with NIR dyes IRDye 800CW (800CW) or Alexa Fluor 680 (AF680), at 1.2 and 0.3 dyes/antibody and examine the impact of fluorophore conjugation on antibody plasma clearance and tissue distribution. At 0.3 DoL, AF680 conjugates exhibited similar clearance to unlabeled antibody over 17 days while 800CW conjugates diverged after 4 days, suggesting AF680 is a more suitable choice for long-term pharmacokinetic studies. At the 1.2 DoL, 800CW conjugates cleared faster than unlabeled antibodies after several hours, in agreement with other published reports. The tissue biodistribution for bevacizumab-800CW and -AF680 conjugates agreed well with literature reported biodistributions using radiolabels. However, the greater tissue autofluorescence at 680 nm resulted in limited detection above background at low (∼2 mg/kg) doses and 0.3 DoL for AF680, indicating that 800CW is more appropriate for short-term biodistribution measurements and intraoperative imaging. Overall, our work shows a DoL of 0.3 or less for non-site-specifically labeled antibodies (with a Poisson distribution) is ideal for limiting the impact of NIR fluorophores on antibody pharmacokinetics.

Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

PubMed

Hagihara, Kenta; Tsukagoshi, Kazuhiko; Nakajima, Chinami; Esaki, Shinsuke; Hashimoto, Masahiko

2016-01-01

We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent.

Highly sensitive optical detection of specific protein in breast cancer cells using microstructured fiber in extremely low sample volume

NASA Astrophysics Data System (ADS)

Padmanabhan, Saraswathi; Shinoj, Vengalathunadakal K.; Murukeshan, Vadakke M.; Padmanabhan, Parasuraman

2010-01-01

A simple optical method using hollow-core photonic crystal fiber for protein detection has been described. In this study, estrogen receptor (ER) from a MCF-7 breast carcinoma cell lysates immobilized inside a hollow-core photonic crystal fiber was detected using anti-ER primary antibody with either Alexa™ Fluor 488 (green fluorescent dye) or 555 (red Fluorescent dye) labeled Goat anti-rabbit IgG as the secondary antibody. The fluorescence fingerprints of the ERα protein were observed under fluorescence microscope, and its optical characteristics were analyzed. The ERα protein detection by this proposed method is based on immuno binding from sample volume as low as 50 nL. This method is expected to offer great potential as a biosensor for medical diagnostics and therapeutics applications.

ConA-based glucose sensing using the long-lifetime azadioxatriangulenium fluorophore

NASA Astrophysics Data System (ADS)

Cummins, Brian; Simpson, Jonathan; Gryczynski, Zygmunt; Sørensen, Thomas Just; Laursen, Bo W.; Graham, Duncan; Birch, David; Coté, Gerard

2014-02-01

Fluorescent glucose sensing technologies have been identified as possible alternatives to current continuous glucose monitoring approaches. We have recently introduced a new, smart fluorescent ligand to overcome the traditional problems of ConA-based glucose sensors. For this assay to be translated into a continuous glucose monitoring device where both components are free in solution, the molecular weight of the smart fluorescent ligand must be increased. We have identified ovalbumin as a naturally-occurring glycoprotein that could serve as the core-component of a 2nd generation smart fluorescent ligand. It has a single asparagine residue that is capable of displaying an N-linked glycan and a similar isoelectric point to ConA. Thus, binding between ConA and ovalbumin can potentially be monovalent and sugar specific. This work is the preliminary implementation of fluorescently-labeled ovalbumin in the ConA-based assay. We conjugate the red-emitting, long-lifetime azadioxatriangulenium (ADOTA+) dye to ovalbumin, as ADOTA have many advantageous properties to track the equilibrium binding of the assay. The ADOTA-labeled ovalbumin is paired with Alexa Fluor 647-labeled ConA to create a Förster Resonance Energy Transfer (FRET) assay that is glucose dependent. The assay responds across the physiologically relevant glucose range (0-500 mg/dL) with increasing intensity from the ADOTA-ovalbumin, showing that the strategy may allow for the translation of the smart fluorescent ligand concept into a continuous glucose monitoring device.

Semi-automated image analysis: detecting carbonylation in subcellular regions of skeletal muscle

PubMed Central

Kostal, Vratislav; Levar, Kiara; Swift, Mark; Skillrud, Erik; Chapman, Mark; Thompson, LaDora V.

2011-01-01

The level of carbonylation in skeletal muscle is a marker of oxidative damage associated with disease and aging. While immunofluorescence microscopy is an elegant method to identify carbonylation sites in muscle cross-sections, imaging analysis is manual, tedious, and time consuming, especially when the goal is to characterize carbonyl contents in subcellular regions. In this paper, we present a semi-automated method for the analysis of carbonylation in subcellular regions of skeletal muscle cross-sections visualized with dual fluorescent immunohistochemistry. Carbonyls were visualized by their reaction with 2,4-dinitrophenylhydrazine (DNPH) followed by immunolabeling with an Alexa488-tagged anti-DNP antibody. Mitochondria were probed with an anti-COXI primary antibody followed by the labeling with an Alexa568-tagged secondary antibody. After imaging, muscle fibers were individually analyzed using a custom-designed, lab-written, computer-aided procedure to measure carbonylation levels in subsarcolemmal and interfibrillar mitochondrial regions, and in the cytoplasmic and extracellular regions. Using this procedure, we were able to decrease the time necessary for the analysis of a single muscle fiber from 45 min to about 1 min. The procedure was tested by four independent analysts and found to be independent on inter-person and intra-person variations. This procedure will help increase highly needed throughput in muscle studies related to ageing, disease, physical performance, and inactivity that use carbonyl levels as markers of oxidative damage. PMID:21327623

Expression and putative function of fibronectin and its receptor (integrin alpha(5)beta(1)) in male and female gametes during bovine fertilization in vitro.

PubMed

Thys, Mirjan; Nauwynck, Hans; Maes, Dominiek; Hoogewijs, Maarten; Vercauteren, Dries; Rijsselaere, Tom; Favoreel, Herman; Van Soom, Ann

2009-09-01

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn--when present during bovine IVF--strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (alpha(5)beta(1)) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin alpha(5) on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin alpha(5) at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin alpha(5)beta(1) receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin alpha(5) expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.

[Changes of monocyte and monocyte-platelet aggregates in different subgroups of thrombotic events in patients with acute myocardial infarction during PCI].

PubMed

Wang, Sheng; Sun, Cuifang; Liao, Wang; Wu, Zhongwei; Wang, Yudai; Huang, Xiuxian; Lu, Sijia; Dong, Xiaoli; Shuai, Fujie; Li, Bin

2017-07-01

Objective To investigate the impact of thrombotic events on the alterations of monocyte and monocyte-platelet aggregates (MPAs) in patients with acute myocardial infarction (AMI) during percutaneous coronary intervention (PCI). Methods Blood was collected before PCI for flow cytometry. Monocyte subsets and MPAs were detected by four-color platform (CDl4-APC, CDl6-PE-Cy7, CD86-PE and CD41-Alexa Fluor R 488). According to the expression of the platelet surface marker CD41, the number of monocyte subsets and MPAs was analyzed using the fluorescent microspheres of absolute counting tube. The Wilcoxon rank sum test and receiver operating characteristic (ROC) curve analysis were performed. Results CD14 + CD16 ++ monocytes in intraprocedural thrombotic events (IPTE) group were significantly fewer than those in non-IPTE group, and the percentage in total mononuclear cells decreased. Compared with non-IPTE group, MPA binding ratio and monocyte subset MPA binding ratio were significantly higher in IPTE group. ROC analysis showed that MPA binding ratio and subgroup MPA binding ratio had a better predictive value for IPTE in patients with AMI. Conclusion The CD14 + CD16 ++ monocytes in IPTE group were significantly fewer than those in the non-IPTE group. MPA binding ratio and MPA binding ratio of monocyte subsets were significantly higher in the IPTE group than in the non-IPTE group, so they have a good predictive value for IPTE in patients with AMI.

Enhanced Transdermal Delivery by Combined Application of Dissolving Microneedle Patch on Serum-Treated Skin.

PubMed

Kim, Suyong; Dangol, Manita; Kang, Geonwoo; Lahiji, Shayan F; Yang, Huisuk; Jang, Mingyu; Ma, Yonghao; Li, Chengguo; Lee, Sang Gon; Kim, Chang Hyun; Choi, Young Wook; Kim, So Jeong; Ryu, Ja Hyun; Baek, Ji Hwoon; Koh, Jaesuk; Jung, Hyungil

2017-06-05

Dissolving microneedle (DMN), a transdermal drug delivery system in which drugs are encapsulated in a biodegradable polymeric microstructure, is designed to dissolve after skin penetration and release the encapsulated drugs into the body. However, because of limited loading capacity of drugs within microsized structures, only a small dosage can be delivered, which is often insufficient for patients. We propose a novel DMN application that combines topical and DMN application simultaneously to improve skin permeation efficiency. Drugs in pretreated topical formulation and encapsulated drugs in DMN patch are delivered into the skin through microchannels created by DMN application, thus greatly increasing the delivered dose. We used 4-n-butylresorcinol to treat human hyperpigmentation and found that sequential application of serum formulation and DMNs was successful. In skin distribution experiments using Alexa Fluor 488 and 568 dyes as model drugs, we confirmed that the pretreated serum formulation was delivered into the skin through microchannels created by the DMNs. In vitro skin permeation and retention experiments confirmed that this novel combined application delivered more 4-n-butylresorcinol into the skin than traditional DMN-only and serum-only applications. Moreover, this combined application showed a higher efficacy in reducing patients' melanin index and hyperpigmented regions compared with the serum-only application. As combined application of DMNs on serum-treated skin can overcome both dose limitations and safety concerns, this novel approach can advance developments in transdermal drug delivery.

Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

PubMed Central

Chou, Cheng-Chung; Huang, Yi-Han

2012-01-01

This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection. PMID:23211753

Development of new PTK7-targeting aptamer-fluorescent and -radiolabelled probes for evaluation as molecular imaging agents: Lymphoma and melanoma in vivo proof of concept.

PubMed

Calzada, Victoria; Moreno, María; Newton, Jessica; González, Joel; Fernández, Marcelo; Gambini, Juan Pablo; Ibarra, Manuel; Chabalgoity, Alejandro; Deutscher, Susan; Quinn, Thomas; Cabral, Pablo; Cerecetto, Hugo

2017-02-01

Aptamers are single-stranded oligonucleotides that recognize molecular targets with high affinity and specificity. Aptamer that selectively bind to the protein tyrosine kinase-7 (PTK7) receptor, overexpressed on many cancers, has been labelled as probes for molecular imaging of cancer. Two new PTK7-targeting aptamer probes were developed by coupling frameworks from the fluorescent dye AlexaFluor647 or the 6-hydrazinonicotinamide (HYNIC) chelator-labelled to 99m Tc. The derivatizations via a 5'-aminohexyl terminal linker were done at room temperature and under mild buffer conditions. Physicochemical and biological controls for both imaging agents were performed verifying the integrity of the aptamer-conjugates by HPLC. Recognition of melanoma (B16F1) and lymphoma (A20) mouse cell lines by the aptamer was studied using cell binding, flow cytometry and confocal microscopy. Finally, in vivo imaging studies in tumour-bearing mice were performed. The new probes were able to bind to melanoma and lymphoma cell lines in vitro, the in vivo imaging in tumour-bearing mice showed different uptake behaviours showing for the fluorescent conjugate good uptake by B cell lymphoma while the radiolabelled conjugate did not display tumour uptake due to its high extravascular distribution, and both showed rapid clearance properties in tumour-bearing mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

PubMed

Benítez, Francisco Moreno; Camacho, Antonio Letrán; del Cuvillo Bernal, Alfonso; de Medina, Pedro Lobatón Sánchez; García Cózar, Francisco J; Romeu, Marisa Espinazo

2014-01-01

There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count, and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labeled with AlexaFluor(®) 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter(®) ). Optical microscopy study was realized with a Leica optical microscope. Bland and Altman was used to determine agreement between both techniques measured. We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a P-value: 0.0008 E(-2) and 0.0002 with regard to smaller particles, so the Bland and Altman measurement showed a good correlation between them, P-value: 0.0003. Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. Copyright © 2013 Clinical Cytometry Society.

Optical tomograph optimized for tumor detection inside highly absorbent organs

NASA Astrophysics Data System (ADS)

Boutet, Jérôme; Koenig, Anne; Hervé, Lionel; Berger, Michel; Dinten, Jean-Marc; Josserand, Véronique; Coll, Jean-Luc

2011-05-01

This paper presents a tomograph for small animal fluorescence imaging. The compact and cost-effective system described in this article was designed to address the problem of tumor detection inside highly absorbent heterogeneous organs, such as lungs. To validate the tomograph's ability to detect cancerous nodules inside lungs, in vivo tumor growth was studied on seven cancerous mice bearing murine mammary tumors marked with Alexa Fluor 700. They were successively imaged 10, 12, and 14 days after the primary tumor implantation. The fluorescence maps were compared over this time period. As expected, the reconstructed fluorescence increases with the tumor growth stage.

Quantification and visualization of glutathione S-transferase omega 1 in cells using inductively coupled plasma mass spectrometry (ICP-MS) and fluorescence microscopy.

PubMed

Liang, Yong; Jiang, Xin; Tang, Nannan; Yang, Limin; Chen, Haifeng; Wang, Qiuquan

2015-03-01

We report a novel activity-based and Cu-free click chemistry (CC) mediated methodology for glutathione S-transferase omega 1 (GSTO1) quantification using species-unspecific isotope dilution inductively coupled plasma mass spectrometry (SUID ICP-MS), in which dibenzylcyclooctyne-modified 2-chloroacetamide (DBCO-ChAcA) was designed and synthesized, meanwhile, as a navigator towards GSTO1 for subsequent N3-DOTA-Eu-tagging via Cu-free CC. Using (153)Eu-SUID ICP-MS coupled with size exclusion chromatography (SEC), the LOD (3σ) of GSTO1 reached 6.9 fmol with an RSD of 2.4% at the 0.1 μM level (n = 5) considering the recovery of GSTO1 on the SEC was 96.5 ± 2.4%. The GSTO1 contents in the cells of human hepatocellular carcinoma C7721 and breast carcinoma MCF-7 as well as normal hepatic C7701 without or with cis-platin administration were quantified to be from 1.2 μg/10,000 cells (n = 3, RSD = 4.5%) corresponding to 1.2 × 10(-2) ng per cell to 4.76 μg/10,000 cells (n = 3, RSD = 2.9%) corresponding to 4.76 × 10(-2) ng per cell. For a comparative study, DBCO-ChAcA-fluor 488-based fluorescence microscopy could not alone visualize GSTO1 in the cells but could together with those from the small SH-containing molecules such as GSH and that from extra N3-fluor 488 in the cells. This activity-based CC-mediated tagging/labeling strategy provided an opportunity for ICP-MS-based targeted protein quantification, and is very much expected to find its applications in biological mechanism study and the subsequent drug design.

An Ultra-High Fluorescence Enhancement and High Throughput Assay for Revealing Expression and Internalization of Chemokine Receptor CXCR4.

PubMed

He, Hua; Wang, Xiaojuan; Cheng, Tiantian; Xia, Yongqing; Lao, Jun; Ge, Baosheng; Ren, Hao; Khan, Naseer Ullah; Huang, Fang

2016-04-18

Revealing chemokine receptor CXCR4 expression, distribution, and internalization levels in different cancers helps to evaluate cancer progression or prognosis and to set personalized treatment strategy. We here describe a sensitive and high-throughput immunoassay for determining CXCR4 expression and distribution in cancer cells. The assay is accessible to a wide range of users in an ordinary lab only by dip-coating poly(styrene-co-N-isopropylacrylamide) spheres on the glass substrate. The self- assembled spheres form three-dimensional photonic colloidal crystals which enhance the fluorescence of CF647 and Alexa Fluor 647 by a factor of up to 1000. CXCR4 in cells is detected by using the sandwich immunoassay, where the primary antibody recognizes CXCR4 and the secondary antibody is labeled with CF647. With the newly established assay, we quantified the total expression of CXCR4, its distribution on the cell membrane and cytoplasm, and revealed their internalization level upon SDF-1α activation in various cancer cells, even for those with extremely low expression level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wide-field lifetime-based FRET imaging for the assessment of early functional distribution of transferrin-based delivery in breast tumor-bearing small animals

NASA Astrophysics Data System (ADS)

Sinsuebphon, Nattawut; Rudkouskaya, Alena; Barroso, Margarida; Intes, Xavier

2016-02-01

Targeted drug delivery is a critical aspect of successful cancer therapy. Assessment of dynamic distribution of the drug provides relative concentration and bioavailability at the target tissue. The most common approach of the assessment is intensity-based imaging, which only provides information about anatomical distribution. Observation of biomolecular interactions can be performed using Förster resonance energy transfer (FRET). Thus, FRET-based imaging can assess functional distribution and provide potential therapeutic outcomes. In this study, we used wide-field lifetime-based FRET imaging for the study of early functional distribution of transferrin delivery in breast cancer tumor models in small animals. Transferrin is a carrier for cancer drug delivery. Its interaction with its receptor is within a few nanometers, which is suitable for FRET. Alexa Fluor® 700 and Alexa Fluor® 750 were conjugated to holo-transferrin which were then administered via tail vein injection to the mice implanted with T47D breast cancer xenografts. Images were continuously acquired for 60 minutes post-injection. The results showed that transferrin was primarily distributed to the liver, the urinary bladder, and the tumor. The cellular uptake of transferrin, which was indicated by the level of FRET, was high in the liver but very low in the urinary bladder. The results also suggested that the fluorescence intensity and FRET signals were independent. The liver showed increasing intensity and increasing FRET during the observation period, while the urinary bladder showed increasing intensity but minimal FRET. Tumors gave varied results corresponding to their FRET progression. These results were relevant to the biomolecular events that occurred in the animals.

Demonstration of prominent actin filaments in the root columella

NASA Technical Reports Server (NTRS)

Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

2001-01-01

The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.

PubMed

Woda, Marcia; Friberg, Heather; Currier, Jeffrey R; Srikiatkhachorn, Anon; Macareo, Louis R; Green, Sharone; Jarman, Richard G; Rothman, Alan L; Mathew, Anuja

2016-10-01

The development of reagents to identify and characterize antigen-specific B cells has been challenging. We recently developed Alexa Fluor-labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV(+) class-switched memory B cells (IgD(-)CD27(+) CD19(+) cells) reached up to 8% during acute infection and early convalescence. AF DENV-labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38(-)CD27(+)) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. AF DENVs reveal changes in the phenotype of DENV serotype-specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Detection of endolithic spatial distribution in marble stone.

PubMed

Casanova Municchia, A; Percario, Z; Caneva, G

2014-10-01

The penetration of endolithic microorganisms, which develop to depths of several millimetres or even centimetres into the stone, and the diffusion of their extracellular substances speeds up the stone deterioration process. The aim of this study was to investigate, using a confocal laser scanning microscopy with a double-staining, a marble rock sample by observing the endolithic spatial distribution and quantifying the volume they occupied within the stone, in order to understand the real impact of these microorganisms on the conservation of stone monuments. Often the only factors taken into account by biodeterioration studies regarding endolithic microorganisms, are spread and depth of penetration. Despite the knowledge of three-dimensional spatial distribution and quantification of volume, it is indispensable to understand the real damage caused by endolithic microorganisms to stone monuments. In this work, we analyze a marble rock sample using a confocal laser scanning microscopy stained with propidium iodide and Concavalin-A conjugate with the fluorophore Alexa Fluor 488, comparing these results with other techniques (SEM microscope, microphotographs of polished cross-sections and thin-section, PAS staining methods), An image analysis approach has also been applied. The use of confocal laser scanning microscopy with double staining shows clear evidence of the presence of endolithic microorganisms (cyanobacteria and fungi) as well as the extracellular polymeric substance matrix in a three-dimensional architecture as part of the rock sample, this technique, therefore, seems very useful when applied to restoration interventions on stone monuments when endolithic growth is suspected. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms.

PubMed

Skogman, Malena Elise; Vuorela, Pia Maarit; Fallarero, Adyary

2012-09-01

Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.

Localisation of the high-affinity choline transporter-1 in the rat skeletal motor unit.

PubMed

Lips, Katrin S; Pfeil, Uwe; Haberberger, Rainer V; Kummer, Wolfgang

2002-03-01

The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled alpha-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.

Multiphoton imaging with a nanosecond supercontinuum source

NASA Astrophysics Data System (ADS)

Lefort, Claire; O'Connor, Rodney P.; Blanquet, Véronique; Baraige, Fabienne; Tombelaine, Vincent; Lévêque, Philippe; Couderc, Vincent; Leproux, Philippe

2016-03-01

Multiphoton microscopy is a well-established technique for biological imaging of several kinds of targets. It is classically based on multiphoton processes allowing two means of contrast simultaneously: two-photon fluorescence (TPF) and second harmonic generation (SHG). Today, the quasi exclusive laser technology used in that aim is femtosecond titanium sapphire (Ti: Sa) laser. We experimentally demonstrate that a nanosecond supercontinuum laser source (STM-250-VIS-IR-custom, Leukos, France; 1 ns, 600-2400 nm, 250 kHz, 1 W) allows to obtain the same kind of image quality in the case of both TPF and SHG, since it is properly filtered. The first set of images concerns the muscle of a mouse. It highlights the simultaneous detection of TPF and SHG. TPF is obtained thanks to the labelling of alpha-actinin with Alexa Fluor® 546 by immunochemistry. SHG is created from the non-centrosymmetric organization of myosin. As expected, discs of actin and myosin are superimposed alternatively. The resulting images are compared with those obtained from a standard femtosecond Ti: Sa source. The physical parameters of the supercontinuum are discussed. Finally, all the interest of using an ultra-broadband source is presented with images obtained in vivo on the brain of a mouse where tumor cells labeled with eGFP are grafted. Texas Red® conjugating Dextran is injected into the blood vessels network. Thus, two fluorophores having absorption wavelengths separated by 80 nm are imaged simultaneously with a single laser source.

High-sensitivity detection of breast tumors in vivo by use of a pH-sensitive near-infrared fluorescence probe

NASA Astrophysics Data System (ADS)

Mathejczyk, Julia Eva; Pauli, Jutta; Dullin, Christian; Resch-Genger, Ute; Alves, Frauke; Napp, Joanna

2012-07-01

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

Csf1r-mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System.

PubMed

Hawley, Catherine A; Rojo, Rocio; Raper, Anna; Sauter, Kristin A; Lisowski, Zofia M; Grabert, Kathleen; Bain, Calum C; Davis, Gemma M; Louwe, Pieter A; Ostrowski, Michael C; Hume, David A; Pridans, Clare; Jenkins, Stephen J

2018-03-15

CSF1 is the primary growth factor controlling macrophage numbers, but whether expression of the CSF1 receptor differs between discrete populations of mononuclear phagocytes remains unclear. We have generated a Csf1r -mApple transgenic fluorescent reporter mouse that, in combination with lineage tracing, Alexa Fluor 647-labeled CSF1-Fc and CSF1, and a modified Δ Csf1- enhanced cyan fluorescent protein (ECFP) transgene that lacks a 150 bp segment of the distal promoter, we have used to dissect the differentiation and CSF1 responsiveness of mononuclear phagocyte populations in situ. Consistent with previous Csf1r- driven reporter lines, Csf1r -mApple was expressed in blood monocytes and at higher levels in tissue macrophages, and was readily detectable in whole mounts or with multiphoton microscopy. In the liver and peritoneal cavity, uptake of labeled CSF1 largely reflected transgene expression, with greater receptor activity in mature macrophages than monocytes and tissue-specific expression in conventional dendritic cells. However, CSF1 uptake also differed between subsets of monocytes and discrete populations of tissue macrophages, which in macrophages correlated with their level of dependence on CSF1 receptor signaling for survival rather than degree of transgene expression. A double Δ Csf1r -ECFP- Csf1r -mApple transgenic mouse distinguished subpopulations of microglia in the brain, and permitted imaging of interstitial macrophages distinct from alveolar macrophages, and pulmonary monocytes and conventional dendritic cells. The Csf1r- mApple mice and fluorescently labeled CSF1 will be valuable resources for the study of macrophage and CSF1 biology, which are compatible with existing EGFP-based reporter lines. Copyright © 2018 The Authors.

Complex logic functions implemented with quantum dot bionanophotonic circuits.

PubMed

Claussen, Jonathan C; Hildebrandt, Niko; Susumu, Kimihiro; Ancona, Mario G; Medintz, Igor L

2014-03-26

We combine quantum dots (QDs) with long-lifetime terbium complexes (Tb), a near-IR Alexa Fluor dye (A647), and self-assembling peptides to demonstrate combinatorial and sequential bionanophotonic logic devices that function by time-gated Förster resonance energy transfer (FRET). Upon excitation, the Tb-QD-A647 FRET-complex produces time-dependent photoluminescent signatures from multi-FRET pathways enabled by the capacitor-like behavior of the Tb. The unique photoluminescent signatures are manipulated by ratiometrically varying dye/Tb inputs and collection time. Fluorescent output is converted into Boolean logic states to create complex arithmetic circuits including the half-adder/half-subtractor, 2:1 multiplexer/1:2 demultiplexer, and a 3-digit, 16-combination keypad lock.

Use of a mixture of n-dodecyl-beta-D-maltoside and sodium dodecyl sulfate in poly(dimethylsiloxane) microchips to suppress adhesion and promote separation of proteins.

PubMed

Huang, Bo; Kim, Samuel; Wu, Hongkai; Zare, Richard N

2007-12-01

Dynamic modification of poly(dimethylsiloxane) channels using a mixture of n-dodecyl-beta-D-maltoside (DDM) and sodium dodecyl sulfate (SDS) is able to suppress analyte adsorption and control electroosmotic flow (EOF). In this mixed surfactant system, the nonionic surfactant DDM functions as a surface blocking reagent, whereas the anionic surfactant SDS introduces negative charges to the channel walls. Changing the DDM/SDS mixing ratio tunes the surface charge density and the strength of EOF. Using 0.1% (w/v) DDM and 0.03% (w/v) SDS, Alexa Fluor 647 labeled streptavidin can be analyzed according to the charges added by the fluorophores. Protein molecules with different numbers of fluorophores are well resolved. DDM and SDS also form negatively charged mixed micelles, which act as a separation medium. The low critical micellar concentration of DDM/SDS mixed micelles also allows the use of SDS at a nondenaturing concentration, which enables the analysis of proteins in their native state. The immunocomplex between a membrane protein, beta2 adrenergic receptor, and anti-FLAG antibody has been fully separated using 0.1% (w/v) DDM and 0.03% (w/v) SDS. We have also analyzed the composition of light-harvesting protein-chromophore complexes in cyanobacteria.

Oligonucleotide-arrayed TFT photosensor applicable for DNA chip technology.

PubMed

Tanaka, Tsuyoshi; Hatakeyama, Keiichi; Sawaguchi, Masahiro; Iwadate, Akihito; Mizutani, Yasushi; Sasaki, Kazuhiro; Tateishi, Naofumi; Takeyama, Haruko; Matsunaga, Tadashi

2006-09-05

A thin film transistor (TFT) photosensor fabricated by semiconductor integrated circuit (IC) technology was applied to DNA chip technology. The surface of the TFT photosensor was coated with TiO2 using a vapor deposition technique for the fabrication of optical filters. The immobilization of thiolated oligonucleotide probes onto a TiO2-coated TFT photosensor using gamma-aminopropyltriethoxysilane (APTES) and N-(gamma-maleimidobutyloxy) sulfosuccinimide ester (GMBS) was optimized. The coverage value of immobilized oligonucleotides reached a plateau at 33.7 pmol/cm2, which was similar to a previous analysis using radioisotope-labeled oligonucleotides. The lowest detection limits were 0.05 pmol/cm2 for quantum dot and 2.1 pmol/cm2 for Alexa Fluor 350. Furthermore, single nucleotide polymorphism (SNP) detection was examined using the oligonucleotide-arrayed TFT photosensor. A SNP present in the aldehyde dehydrogenase 2 (ALDH2) gene was used as a target. The SNPs in ALDH2*1 and ALDH2*2 target DNA were detected successfully using the TFT photosensor. DNA hybridization in the presence of both ALDH2*1 and ALDH2*2 target DNA was observed using both ALDH2*1 and ALDH2*2 detection oligonucleotides-arrayed TFT photosensor. Use of the TFT photosensor will allow the development of a disposable photodetecting device for DNA chip systems. (c) 2006 Wiley Periodicals, Inc.

Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

PubMed

Benítez, Francisco Moreno; Camacho, Antonio Letrán; Del Cuvillo Bernal, Alfonso; de Medina, Pedro Lobatón Sánchez; Cózar, Francisco J García; Romeu, Ma Luisa Espinazo

2013-07-10

Background: There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Methods: Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labelled with AlexaFluor ® 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter ® ). Optical microscopy study was realized with a Leica optical microscope. Bland & Altman was used to determine agreement between both techniques measured. Results: We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a p-value: 0.0008E -2 and 0.0002 with regard to smaller particles, so the Bland & Altman measurement showed a good correlation between them, p-value: 0,0003. Conclusion: Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

PubMed

Brito-Argáez, Ligia; Tamayo-Sansores, José A; Madera-Piña, Dianeli; García-Villalobos, Francisco J; Moo-Puc, Rosa E; Kú-González, Ángela; Villanueva, Marco A; Islas-Flores, Ignacio

2016-12-01

The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an ∼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A Direct Synergistic Effect of Immunotherapy and Chemotherapy as a New Paradigm in Treatment of Breast Cancer

DTIC Science & Technology

2009-04-01

Untreated and TAX treated EL4 targets were labeled with DDAO-SE and loaded with control (C.P.) or specific (S.P.) peptide. The tumor cells were...C ell Vehicle treated EL4 cells 100 101 102 103 104 FL4-H: anti mouse granzyme B alexa647 0 100 200 300 400 500 # Cells 2.5297.5 100 101 102 103...mouse granzyme B alexa647 0 100 200 300 400 500 # Cells 7.292.8 Taxol treated EL4 cells 100 101 102 103 104 FL4-H: anti mouse granzyme B alexa647 0

A Murine Model of Inflammatory Bladder Disease: Cathelicidin Peptide Induced Bladder Inflammation and Treatment With Sulfated Polysaccharides

PubMed Central

Oottamasathien, Siam; Jia, Wanjian; McCoard, Lindsi; Slack, Sean; Zhang, Jianxing; Skardal, Aleksander; Job, Kathleen; Kennedy, Thomas P.; Dull, Randal O.; Prestwich, Glenn D.

2013-01-01

Purpose Studies show that LL-37 is a naturally occurring urinary defensin peptide that is up-regulated during urinary tract infections. Although normal urinary LL-37 levels are antimicrobial, we propose that increased LL-37 may trigger bladder inflammation. We further suggest that anti-inflammatory sulfated polysaccharides known as semi-synthetic glycosaminoglycan ether compounds can treat/prevent LL-37 mediated bladder inflammation. Materials and Methods C57BL/6 mice were catheterized/instilled with LL-37 (320 μM at 150 μl) for 45 minutes. Animals were sacrificed at 12 and 24 hours, and tissues were examined using hematoxylin and eosin. Separate experiments were performed for myeloperoxidase to quantify inflammation. GM-1111 semi-synthetic glycosaminoglycan ether treatments involved instillation of 10 mg/ml for 45 minutes directly before or after LL-37. Tissues were harvested at 24 hours. To compare semi-synthetic glycosaminoglycan ether efficacy experiments were performed using 10 mg/ml heparin. Finally, tissue localization of semi-synthetic glycosaminoglycan ether was examined using a fluorescent GM-1111-Alexa Fluor® 633 conjugate. Results Profound bladder inflammation developed after LL-37. Greater tissue inflammation occurred after 24 hours compared to that at 12 hours. Myeloperoxidase assays revealed a 21 and 61-fold increase at 12 and 24 hours, respectively. Semi-synthetic glycosaminoglycan ether treatment after LL-37 showed mild attenuation of inflammation with myeloperoxidase 2.5-fold below that of untreated bladders. Semi-synthetic glycosaminoglycan ether treatment before LL-37 demonstrated almost complete attenuation of inflammation. Myeloperoxidase results mirrored those in controls. In heparin treated bladders minimal attenuation of inflammation occurred. Finally, instillation of GM-1111-Alexa Fluor 633 revealed urothelial coating, significant tissue penetration and binding to endovasculature. Conclusions We developed what is to our knowledge a new model of inflammatory bladder disease by challenge with the naturally occurring urinary peptide LL-37. We also noted that a new class of anti-inflammatory sulfated polysaccharides prevents and mitigates bladder inflammation. PMID:21855919

A fluorescence resonance energy transfer quantum dot explosive nanosensor (Invited Paper)

NASA Astrophysics Data System (ADS)

Medintz, Igor L.; Goldman, Ellen R.; Clapp, Aaron R.; Uyeda, H. T.; Lassman, Michael E.; Hayhurst, Andrew; Mattoussi, Hedi

2005-04-01

Quantum dots (QDs) are a versatile synthetic photoluminescent nanomaterial whose chemical and photo-physical properties suggest that they may be superior to conventional organic fluorophores for a variety of biosensing applications. We have previously investigated QD-fluorescence resonance energy transfer (FRET) interactions by using the E. coli bacterial periplasmic binding protein - maltose binding protein (MBP) which was site-specifically dye-labeled and self assembled onto the QD surface and allowed us to monitor FRET between the QD donor and the acceptor dye. FRET efficiency increased as a function of the number of dye-acceptor moieties arrayed around the QD donor. We used this system to further demonstrate a prototype FRET based biosensor that functioned in the chemical/nutrient sensing of maltose. There are a number of potential benefits to using this type of QD-FRET based biosensing strategy. The protein attached to the QDs surface functions as a biosensing and biorecognition element in this configuration while the QD acts as both nanoscaffold and FRET energy donor. In this report, we show that the sensor design can be extended to target a completely unrelated analyte, namely the explosive TNT. The sensor consists of anti-TNT antibody fragments self-assembled onto the QD surface with a dye-labeled analog of TNT (TNB coupled to AlexaFluor 555 dye) prebound in the fragment binding site. The close proximity of dye to QD establishes a baseline level of FRET and addition of TNT displaces the TNB-dye analog, recovering QD photoluminescence in a concentration dependent manner. Potential benefits of this QD sensing strategy are discussed.

In vivo fluorescence imaging of hepatocellular carcinoma using a novel GPC3-specific aptamer probe

PubMed Central

Zhao, Menglong; Dong, Lili; Liu, Zhuang; Yang, Shuohui

2018-01-01

Background Glypican-3 (GPC3) is highly expressed in most of the hepatocellular carcinomas (HCCs), even in small HCCs. It may be used as a potential biomarker for early detection of HCC. The aptamer is a promising targeting agent with unique advantages over antibody. This study was to introduce a novel GPC3 specific aptamer (AP613-1), to verify its specific binding property in vitro, and to evaluate its targeting efficiency in vivo by performing near-infrared (NIR) fluorescence imaging on an HCC xenograft model. Methods AP613-1 was generated from the systematic evolution of ligands by exponential enrichment. Flow cytometry and aptamer-based immunofluorescence imaging were performed to verify the binding affinity of AP613-1 to GPC3 in vitro. NIR Fluorescence images of nude mice with unilateral (n=12) and bilateral (n=4) subcutaneous xenograft tumors were obtained. Correlation between the tumor fluorescence intensities in vivo and ex vivo was analyzed. Results AP613-1 could specifically bind to GPC3 in vitro. In vivo and ex vivo tumors, fluorescence intensities were in excellent correlation (P<0.001, r=0.968). The fluorescence intensity is significantly higher in tumors given Alexa Fluor 750 (AF750) labeled AP613-1 than in those given AF750 labeled initial ssDNA library both in vivo (P<0.001) and ex vivo (P=0.022). In the mice with bilateral subcutaneous tumors injected with AF750 labeled AP613-1, Huh-7 tumors showed significantly higher fluorescence intensities than A549 tumors both in vivo (P=0.016) and ex vivo (P=0.004). Conclusions AP613-1 displays a specific binding affinity to GPC3 positive HCC. Fluorescently labeled AP613-1 could be used as an imaging probe to subcutaneous HCC in xenograft models. PMID:29675356

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

PubMed

Gehring, Andrew G; Brewster, Jeffrey D; He, Yiping; Irwin, Peter L; Paoli, George C; Simons, Tawana; Tu, Shu-I; Uknalis, Joseph

2015-12-04

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 10⁵ cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

Activatable clinical fluorophore-quencher antibody pairs as dual molecular probes for the enhanced specificity of image-guided surgery

NASA Astrophysics Data System (ADS)

Obaid, Girgis; Spring, Bryan Q.; Bano, Shazia; Hasan, Tayyaba

2017-12-01

The emergence of fluorescently labeled therapeutic antibodies has given rise to molecular probes for image-guided surgery. However, the extraneous interstitial presence of an unbound and nonspecifically accumulated probe gives rise to false-positive detection of tumor tissue and margins. Thus, the concept of tumor-cell activation of smart probes provides a potentially superior mechanism of delineating tumor margins as well as small tumor deposits. The combination of molecular targeting with intracellular activation circumvents the presence of extracellular, nonspecific signals of targeted probe accumulation. Here, we present a demonstration of the clinical antibodies cetuximab (cet, anti-EGFR mAb) and trastuzumab (trast, anti-HER-2 mAb) conjugated to Alexa Fluor molecules and IRDye QC-1 quencher optimized at the ratio of 1∶2∶6 to provide the greatest degree of proteolytic fluorescence activation, synonymous with intracellular lysosomal degradation. The cet-AF-Q-C1 conjugate (1∶2∶6) provides up to 9.8-fold proteolytic fluorescence activation. By preparing a spectrally distinct, irrelevant sham IgG-AF-QC-1 conjugate, a dual-activatable probe approach is shown to enhance the specificity of imaging within an orthotopic AsPC-1 pancreatic cancer xenograft model. The dual-activatable approach warrants expedited clinical translation to improve the specificity of image-guided surgery by spectrally decomposing specific from nonspecific probe accumulation, binding, and internalization.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site.

PubMed

Kadamur, Ganesh; Ross, Elliott M

2016-05-20

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Intrinsic Pleckstrin Homology (PH) Domain Motion in Phospholipase C-β Exposes a Gβγ Protein Binding Site*

PubMed Central

Kadamur, Ganesh

2016-01-01

Mammalian phospholipase C-β (PLC-β) isoforms are stimulated by heterotrimeric G protein subunits and members of the Rho GTPase family of small G proteins. Although recent structural studies showed how Gαq and Rac1 bind PLC-β, there is a lack of consensus regarding the Gβγ binding site in PLC-β. Using FRET between cerulean fluorescent protein-labeled Gβγ and the Alexa Fluor 594-labeled PLC-β pleckstrin homology (PH) domain, we demonstrate that the PH domain is the minimal Gβγ binding region in PLC-β3. We show that the isolated PH domain can compete with full-length PLC-β3 for binding Gβγ but not Gαq, Using sequence conservation, structural analyses, and mutagenesis, we identify a hydrophobic face of the PLC-β PH domain as the Gβγ binding interface. This PH domain surface is not solvent-exposed in crystal structures of PLC-β, necessitating conformational rearrangement to allow Gβγ binding. Blocking PH domain motion in PLC-β by cross-linking it to the EF hand domain inhibits stimulation by Gβγ without altering basal activity or Gαq response. The fraction of PLC-β cross-linked is proportional to the fractional loss of Gβγ response. Cross-linked PLC-β does not bind Gβγ in a FRET-based Gβγ-PLC-β binding assay. We propose that unliganded PLC-β exists in equilibrium between a closed conformation observed in crystal structures and an open conformation where the PH domain moves away from the EF hands. Therefore, intrinsic movement of the PH domain in PLC-β modulates Gβγ access to its binding site. PMID:27002154

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

2013-07-25

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1. Copyright © 2013 Elsevier B.V. All rights reserved.

Fluorescent nanodiamond and lanthanide labelled in situ hybridization for the identification of RNA transcripts in fixed and CLARITY-cleared central nervous system tissues (Conference Presentation)

NASA Astrophysics Data System (ADS)

Parker, Lindsay M.; Staikopoulos, Vicky; Cordina, Nicole M.; Sayyadi, Nima; Hutchinson, Mark R.; Packer, Nicolle H.

2016-03-01

Despite significant advancement in the methodology used to conjugate, incorporate and visualize fluorescent molecules at the cellular and tissue levels, biomedical imaging predominantly relies on the limitations of established fluorescent molecules such as fluorescein, cyanine and AlexaFluor dyes or genetic incorporation of fluorescent proteins by viral or other means. These fluorescent dyes and conjugates are highly susceptible to photobleaching and compete with cellular autofluorescence, making biomedical imaging unreliable, difficult and time consuming in many cases. In addition, some proteins have low copy numbers and/or poor antibody recognition, further making detection and imaging difficult. We are developing better methods for imaging central nervous system neuroinflammatory markers using targeted mRNA transcripts labelled with fluorescent nanodiamonds or lanthanide chelates. These tags have increased signal and photostability and can also discriminate against tissue/cell autofluorescence. Brains and spinal cords from BALB/c mice with a chronic constriction model of neuropathic pain (neuroinflammation group) or that have undergone sham surgeries (control group) were collected. A subset of brains and spinal cords were perfused and fixed with paraformaldehyde (n=3 sham and n=3 pain groups) prior to sectioning and in situ hybridization using nanodiamond or lanthanide chelate conjugated complementary RNA probes. Another subset of brains and spinal cords from the same cohort of animals were perfused and processed for CLARITY hydrogel based clearing prior to in situ hybridization with the same probes. We will present our findings on the photostability, sensitivity and discrimination from background tissue autofluorescence of our novel RNA probes, compared to traditional fluorophore tags.

Direct comparison of single- and multi-walled carbon nanotubes in fluorescence quenching phenomenon

NASA Astrophysics Data System (ADS)

Oura, Shusuke; Umemura, Kazuo

2018-03-01

Here, we report the fluorescence quenching ability of single-stranded DNA (ssDNA)-wrapped single- and multi-walled carbon nanotubes (ssDNA-SWNTs and ssDNA-MWNTs, respectively) using fluorescein dye-labeled ssDNA (Fluor-ssDNA). To compare the quenching abilities of SWNTs and MWNTs, we measured the quenching ratios of fluorescence emission from fluorescein when Fluor-ssDNA reacted with the hybrids of 30-mers of thymine (T30) and SWNTs or MWNTs (T30-SWNTs and T30-MWNTs, respectively). The fluorescence quenching ratios of Fluor-T30 in SWNT and MWNT samples were 28 ± 3.1 and 36 ± 2.0% relative to free fluorescein at the same concentration, respectively. On the other hand, those of Fluor-A30 with SWNT and MWNT hybrids were 11 ± 1.9 and 32 ± 1.9%, respectively. Our results suggest that although the fluorescence quenching ability of MWNT was greater than that of SWNT, SWNT quenching ratios were more sensitive to the base sequences of Fluor-ssDNA.

Synthesis, characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels.

PubMed

Pragl, Bernt; Koschak, Alexandra; Trieb, Maria; Obermair, Gerald; Kaufmann, Walter A; Gerster, Uli; Blanc, Eric; Hahn, Christoph; Prinz, Heino; Schütz, Gerhard; Darbon, Herve; Gruber, Hermann J; Knaus, Hans-Günther

2002-01-01

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.

Capillary electrophoretic analysis reveals subcellular binding between individual mitochondria and cytoskeleton

PubMed Central

Kostal, Vratislav; Arriaga, Edgar A.

2011-01-01

Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for non-bound ‘free’ subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count and quantify properties of individual binding events of mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zeptomole F-actin, which theoretically represents a ~ 2.5-μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections. PMID:21309532

Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2

NASA Astrophysics Data System (ADS)

Dai, Zhongquan; Tan, Yingjun; Yang, Fen; Qu, Lina; Zhang, Hongyu; Wan, Yumin; Li, Yinghui

2011-01-01

Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure alteration inhibits the function performing.

Assessing the efficacy of co-inoculation of wheat seedlings with the associative bacteria Paenibacillus polymyxa 1465 and Azospirillum brasilense Sp245.

PubMed

Yegorenkova, Irina V; Tregubova, Kristina V; Burygin, Gennady L; Matora, Larisa Y; Ignatov, Vladimir V

2016-03-01

Co-inoculation of associative bacteria, which have high nitrogen-fixing activity, tolerance for environmental conditions, and the ability to compete with the natural microflora, is used widely to enhance the growth and yields of agricultural plants. We evaluated the ability of 2 co-inoculated plant-growth-promoting rhizobacteria, Paenibacillus polymyxa 1465 and Azospirillum brasilense Sp245, to colonize roots of wheat (Triticum aestivum L. 'Saratovskaya 29') seedlings, and we assessed the morphometric parameters of wheat early in its development. Analysis by ELISA with polyclonal antibodies raised against the exopolysaccharide of P. polymyxa 1465 and the lipopolysaccharide of A. brasilense Sp245 demonstrated that the root-colonizing activity of A. brasilense was higher when the bacterium was co-inoculated with P. polymyxa than when it was inoculated singly. Immunofluorescence microscopy with Alexa Fluor 532-labeled antibodies revealed sites of attachment of co-inoculated P. polymyxa and A. brasilense and showed that the 2 bacteria colonized similar regions of the roots. Co-inoculation exerted a negative effect on wheat seedling development, inhibiting root length by 17.6%, total root weight by 11%, and total shoot weight by 12%. Under certain conditions, dual inoculation of wheat may prove ineffective, apparently owing to the competition between the rhizobacteria for colonization sites on the plant roots. The findings from this study may aid in developing techniques for mixed bacterial inoculation of cultivated plants.

Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

PubMed Central

Lidke, Diane S.; Lidke, Keith A.

2015-01-01

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique. PMID:25860558

Targeting hepatocellular carcinoma with aptamer-functionalized PLGA/PLA-PEG nanoparticles

NASA Astrophysics Data System (ADS)

Weigum, Shannon E.; Sutton, Melissa; Barnes, Eugenia; Miller, Sarah; Betancourt, Tania

2014-08-01

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, particularly in regions where chronic Hepatitis B and C infections are common. Nanoparticle assemblies that incorporate high-affinity aptamers which specifically bind malignant hepatocellular carcinoma cells could be useful for targeted drug delivery or enhancing contrast with existing ablation therapies. The in vitro interactions of a tumor-specific aptamer, TLS11a, were characterized in a hepatoma cell line via live-cell fluorescence imaging, SDS-PAGE and Western Blotting techniques. Cell surface binding of the aptamer-AlexaFluor®546 conjugate was found to occur within 20 minutes of initial exposure, followed by internalization and localization to late endosomes or lysosomes using a pH-sensitive LysoSensor™ Green dye and confocal microscopy. Aptamer-functionalized polymer nanoparticles containing poly(lactic-co-glycolic acid) (PLGA) and poly(lactide)-b-poly(ethylene glycol) (PLA-PEG) were then prepared by nanoprecipitation and passively loaded with the chemotherapeutic agent, doxorubicin, yielding spherical nanoparticles approximately 50 nm in diameter. Targeted drug delivery and cytotoxicity was assessed using live/dead fluorescent dyes and a MTT colorimetric viability assay with elevated levels of cell death found in cultures treated with either the aptamer-coated and uncoated polymer nanoparticles. Identification and characterization of the cell surface protein epitope(s) recognized by the TLS11a aptamer are ongoing along with nanoparticle optimization, but these preliminary studies support continued investigation of this aptamer and functionalized nanoparticle conjugates for targeted labeling and drug delivery within malignant hepatocellular carcinomas.

WGA-Alexa conjugates for axonal tracing

PubMed Central

Levy, Sabrina L.; White, Joshua J.; Lackey, Elizabeth P.; Schwartz, Lindsey; Sillitoe, Roy V.

2017-01-01

Anatomical labeling approaches are essential for understanding brain organization. Among these approaches are various methods of performing tract tracing. However, a major hurdle to overcome when marking neurons in vivo is visibility. Poor visibility makes it challenging to image a desired neuronal pathway so that it can be easily differentiated from a closely neighboring pathway. As a result, it becomes impossible to analyze individual projections or their connections. The tracer that is chosen for a given purpose has a major influence on the quality of the tracing. Here, we describe the wheat germ agglutinin (WGA) tracer conjugated to Alexa fluorophores for reliable high-resolution tracing of central nervous system projections. Using the mouse cerebellum as a model system, we implement WGA-Alexa tracing for marking and mapping neural circuits that control motor function. We also show its utility for marking localized regions of the cerebellum after performing single-unit extracellular recordings in vivo. PMID:28398642

Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

PubMed Central

Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

2016-01-01

Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity. PMID:26843283

Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

NASA Astrophysics Data System (ADS)

Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

2016-02-01

Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.

Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

PubMed

Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

2012-01-01

Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.

Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy.

PubMed

Belle, Valérie; Rouger, Sabrina; Costanzo, Stéphanie; Liquière, Elodie; Strancar, Janez; Guigliarelli, Bruno; Fournel, André; Longhi, Sonia

2008-12-01

Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.

Frequency Domain Fluorescent Molecular Tomography and Molecular Probes for Small Animal Imaging

NASA Astrophysics Data System (ADS)

Kujala, Naresh Gandhi

Fluorescent molecular tomography (FMT) is a noninvasive biomedical optical imaging that enables 3-dimensional quantitative determination of fluorochromes distributed in biological tissues. There are three methods for imaging large volume tissues based on different light sources: (a) using a light source of constant intensity, through a continuous or constant wave, (b) using a light source that is intensity modulated with a radio frequency (RF), and (c) using ultrafast pulses in the femtosecond range. In this study, we have developed a frequency domain fluorescent molecular tomographic system based on the heterodyne technique, using a single source and detector pair that can be used for small animal imaging. In our system, the intensity of the laser source is modulated with a RF frequency to produce a diffuse photon density wave in the tissue. The phase of the diffuse photon density wave is measured by comparing the reference signal with the signal from the tissue using a phasemeter. The data acquisition was performed by using a Labview program. The results suggest that we can measure the phase change from the heterogeneous inside tissue. Combined with fiber optics and filter sets, the system can be used to sensitively image the targeted fluorescent molecular probes, allowing the detection of cancer at an early stage. We used the system to detect the tumor-targeting molecular probe Alexa Fluor 680 and Alexa Fluor 750 bombesin peptide conjugates in phantoms as well as mouse tissues. We also developed and evaluated fluorescent Bombesin (BBN) probes to target gastrin-releasing peptide (GRP) receptors for optical molecular imaging. GRP receptors are over-expressed in several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. BBN is a 14 amino acid peptide that is an analogue to human gastrin-releasing peptide that binds specifically to GRPr receptors. BBN conjugates are significant in cancer detection and therapy. The optical molecular probe AF750 BBN peptide exhibits optimal pharmacokinetic properties for targeting GRPr in mice. Fluorescent microscopic imaging of the molecular probe in PC-3 prostate and T-47D breast cancer cell lines indicated specific uptake, internalization, and receptor blocking of these probes. In vivo investigations in severely compromised immunodeficient (SCID) mice bearing xenografted PC-3 prostate and T47-D breast cancer lesions demonstrated the ability of this new molecular probe to specifically target tumor tissue with high selectively and affinity.

Non-invasive Optical Molecular Imaging for Cancer Detection

NASA Astrophysics Data System (ADS)

Luo, Zhen

Cancer is a leading cause of death worldwide. It remains the second most common cause of death in the US, accounting for nearly 1 out of every 4 deaths. Improved fundamental understanding of molecular processes and pathways resulting in cancer development has catalyzed a shift towards molecular analysis of cancer using imaging technologies. It is expected that the non-invasive or minimally invasive molecular imaging analysis of cancer can significantly aid in improving the early detection of cancer and will result in reduced mortality and morbidity associated with the disease. The central hypothesis of the proposed research is that non-invasive imaging of changes in metabolic activity of individual cells, and extracellular pH within a tissue will improve early stage detection of cancer. The specific goals of this research project were to: (a) develop novel optical imaging probes to image changes in choline metabolism and tissue pH as a function of progression of cancer using clinically isolated tissue biopsies; (b) correlate changes in tissue extracellular pH and metabolic activity of tissues as a function of disease state using clinically isolated tissue biopsies; (c) provide fundamental understanding of relationship between tumor hypoxia, acidification of the extracellular space and altered cellular metabolism with progression of cancer. Three novel molecular imaging probes were developed to detect changes in choline and glucose metabolism and extracellular pH in model systems and clinically isolated cells and biopsies. Glucose uptake and metabolism was measured using a fluorescence analog of glucose, 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose), while choline metabolism was measured using a click chemistry analog of choline, propargyl choline, which can be in-situ labeled with a fluorophore Alexa-488 azide via a click chemistry reaction. Extracellular pH in tissue were measured by Alexa-647 labeled pHLIP (pH low insertion peptide), which can selectively target plasma membrane of cells based on lower extracellular pH. 20 pairs of clinically normal and abnormal biopsies were obtained from consenting patients at UCDMC. Fluorescence intensity of tissue biopsies before and after topical delivery of 2-NBDG and Alexa-647 labeled pHLIP was measured non-invasively by widefield imaging and confocal microscope. Uptake of propargyl choline was measured after topical delivery using confocal microscope. The results of all three molecular imagine probes were further correlated with pathological diagnosis. The imaging results of clinical biopsies demonstrated that 2-NBDG, propargyl choline and pHLIP peptide can accurately distinguish the pathologically normal and abnormal biopsies. Topical application of the contrast agents generated significantly higher fluorescence signal intensity in all neoplastic tissues as compared to clinically normal biopsies irrespective of the anatomic location or patient. This unpaired comparison across all the cancer patients in this study highlights the specificity of the imaging approach. Furthermore, the results indicated that changes in intracellular glucose, choline metabolism and cancer acidosis are initiated in the early stages of cancer and these changes are correlated with the progression of the disease. In conclusion, these novel optical molecular imaging approaches to measure multiple biomarkers in cancer have significant potential to be a useful tool for improving early detection and prognostic evaluation of oral neoplasia.

Brainstem structures are primarily affected in an experimental model of severe scorpion envenomation.

PubMed

Guidine, Patrícia Alves Maia; Cash, Diana; Drumond, Luciana Estefani; de Souza E Rezende, Gustavo Henrique; Massensini, André Ricardo; Williams, Steve Charles Rees; Moraes-Santos, Tasso; Moraes, Márcio Flávio Dutra; Mesquita, Michel Bernanos Soares

2014-01-01

Severe scorpion envenoming (SSE) is more frequent in children and is characterized by systemic dysfunctions with a mortality rate of up to 9%. Recent evidence shows that the central nervous system (CNS) plays a key role in triggering the cascade of symptoms present in SSE. The age-dependent role of the CNS in SSE lethality may be summarized in 3 hypotheses: (1) the shown increased blood brain barrier permeability of infants to the toxins would especially and primarily compromise neurovegetative control areas, (2) the neurons within these areas have high affinity to the toxins, and (3) the neurovascular interaction is such that SSE metabolically compromises proper function of toxin-targeted areas. A pharmacological magnetic resonance imaging paradigm was used to evaluate localized hemodynamic changes in relative cerebral blood volume (rCBV) for 30 min after the injection of TsTX, the most lethal toxin from the venom of the Tityus serrulatus scorpion. The brainstem showed significant rCBV reduction 1 min after TsTX administration, whereas rostral brain areas had delayed increase in rCBV (confirmed by laser Doppler measurements of cortical cerebral blood flow). Moreover, metabolic activity by 14C-2-deoxyglucose autoradiography showed the highest relative increase at the brainstem. To test whether TsTX has high affinity to brainstem neurons, the lateral ventricle was injected with Alexa Fluor 568 TsTX. Although some neurons showed intense fluorescence, the labeling pattern suggests that specific neurons were targeted. Altogether, these results suggest that brainstem areas involved in neurovegetative control are most likely within the primary structures triggering the cascade of symptoms present in SSE.

Super-resolution Microscopical Localization of Dopamine Receptors 1 and 2 in Rat Hippocampal Synaptosomes.

PubMed

Miklosi, Andras G; Del Favero, Giorgia; Bulat, Tanja; Höger, Harald; Shigemoto, Ryuichi; Marko, Doris; Lubec, Gert

2018-06-01

Although dopamine receptors D1 and D2 play key roles in hippocampal function, their synaptic localization within the hippocampus has not been fully elucidated. In order to understand precise functions of pre- or postsynaptic dopamine receptors (DRs), the development of protocols to differentiate pre- and postsynaptic DRs is essential. So far, most studies on determination and quantification of DRs did not discriminate between subsynaptic localization. Therefore, the aim of the study was to generate a robust workflow for the localization of DRs. This work provides the basis for future work on hippocampal DRs, in light that DRs may have different functions at pre- or postsynaptic sites. Synaptosomes from rat hippocampi isolated by a sucrose gradient protocol were prepared for super-resolution direct stochastic optical reconstruction microscopy (dSTORM) using Bassoon as a presynaptic zone and Homer1 as postsynaptic density marker. Direct labeling of primary validated antibodies against dopamine receptors D1 (D1R) and D2 (D2R) with Alexa Fluor 594 enabled unequivocal assignment of D1R and D2R to both, pre- and postsynaptic sites. D1R immunoreactivity clusters were observed within the presynaptic active zone as well as at perisynaptic sites at the edge of the presynaptic active zone. The results may be useful for the interpretation of previous studies and the design of future work on DRs in the hippocampus. Moreover, the reduction of the complexity of brain tissue by the use of synaptosomal preparations and dSTORM technology may represent a useful tool for synaptic localization of brain proteins.

Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

PubMed

Berndt-Paetz, Mandy; Herbst, Luise; Weimann, Annett; Gonsior, Andreas; Stolzenburg, Jens-Uwe; Neuhaus, Jochen

2018-05-01

Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (G αq/11 , G αs , G αi ) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor ® 488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP-subtype coupling patterns. Detection of M3 interactions with endocytic trafficking proteins by PLA resulted in object sizes correlating with well-documented vesicle sizes of the endocytosis pathway. PLA enabled highly specific detection of M3 receptor expression, demonstration of M3/GP differential coupling and intracellular M3 trafficking in human detrusor smooth muscle cells. This new approach minimized background fluorescence and antibody cross-reactions resulting from single antibody application, and enhanced specificity due to the use of two primary antibodies. Use of subcellular markers allowed visualization of subcellular receptor location. PLA/CLSM allows analyses of muscarinic "receptor - G protein - promiscuity" and intracellular trafficking even in bladder paraffin sections and may give new insights into the etiology and pathology of BPS/IC. Copyright © 2018 Elsevier GmbH. All rights reserved.

Nanoparticles in Porous Microparticles Prepared by Supercritical Infusion and Pressure Quench Technology for Sustained Delivery of Bevacizumab

PubMed Central

K.Yandrapu, Sarath; Upadhyay, Arun K.; Petrash, J. Mark; Kompella, Uday B.

2014-01-01

Nanoparticles in porous microparticles (NPinPMP), a novel delivery system for sustained delivery of protein drugs, was developed using supercritical infusion and pressure quench technology, which does not expose proteins to organic solvents or sonication. The delivery system design is based on the ability of supercritical carbon dioxide (SC CO2) to expand poly(lactic-co-glycolic) acid (PLGA) matrix but not polylactic acid (PLA) matrix. The technology was applied to bevacizumab, a protein drug administered once a month intravitreally to treat wet age related macular degeneration. Bevacizumab coated PLA nanoparticles were encapsulated into porosifying PLGA microparticles by exposing the mixture to SC CO2. After SC CO2 exposure, the size of PLGA microparticles increased by 6.9 fold. Confocal and scanning electron microscopy studies demonstrated the expansion and porosification of PLGA microparticles and infusion of PLA nanoparticles inside PLGA microparticles. In vitro release of bevacizumab from NPinPMP was sustained for 4 months. Size exclusion chromatography, fluorescence spectroscopy, circular dichroism spectroscopy, SDS-PAGE, and ELISA studies indicated that the released bevacizumab maintained its monomeric form, conformation, and activity. Further, in vivo delivery of bevacizumab from NPinPMP was evaluated using noninvasive fluorophotometry after intravitreal administration of Alexa Flour 488 conjugated bevacizumab in either solution or NPinPMP in a rat model. Unlike the vitreal signal from Alexa-bevacizumab solution, which reached baseline at 2 weeks, release of Alexa-bevacizumab from NPinPMP could be detected for 2 months. Thus, NPinPMP is a novel sustained release system for protein drugs to reduce frequency of protein injections in the therapy of back of the eye diseases. PMID:24131101

Gadolinium-loaded gel scintillators for neutron and antineutrino detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Riddle, Catherine Lynn; Akers, Douglas William; Demmer, Ricky Lynn

A gadolinium (Gd) loaded scintillation gel (Gd-ScintGel) compound allows for neutron and gamma-ray detection. The unique gel scintillator encompasses some of the best features of both liquid and solid scintillators, yet without many of the disadvantages associated therewith. Preferably, the gel scintillator is a water soluble Gd-DTPA compound and water soluble fluorophores such as: CdSe/ZnS (or ZnS) quantum dot (Q-dot) nanoparticles, coumarin derivatives 7-hydroxy-4-methylcoumarin, 7-hydroxy-4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-carboxylic acid, and Alexa Fluor 350 as well as a carbostyril compound, carbostyril 124 in a stable water-based gel, such as methylcellulose or polyacrylamide polymers. The Gd-loaded ScintGel allows for a homogenious distribution ofmore » the Gd-DTPA and the fluorophores, and yields clean fluorescent emission peaks. A moderator, such as deuterium or a water-based clear polymer, can be incorporated in the Gd-ScintGel. The gel scintillators can be used in compact detectors, including neutron and antineutrino detectors.« less

Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

2010-09-01

The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

New functionalized mercaptoundecahydrododecaborate derivatives for potential application in boron neutron capture therapy: synthesis, characterization and dynamic visualization in cells.

PubMed

Genady, Afaf R; Ioppolo, Joseph A; Azaam, Mohamed M; El-Zaria, Mohamed E

2015-03-26

A series of mercaptoundecahydrododecaborate (B12H11SH(2-), BSH) bearing mono- and dicarboxyalkyl derivatives was prepared, characterized, and their reactivity towards amidation and esterification in DMF was evaluated. Symmetrical alkylation of BSH was achieved by treatment with primary haloalkyl carboxylic acids in aqueous acetonitrile to produce S,S-bis(carboxyalkyl)sulfonium-undecahydro-closo-dodecaborate tetramethylammonium salts. Unsymmetrically substituted sulfonium salts were obtained through a similar treatment of cyanoethylthioether-undecahydro-closo-dodecaborate tetramethylammonium salt with haloalkyl carboxylic acid. Selective removal of the remaining cyanoethyl group upon treatment with tetramethylammonium hydroxide yielded S-carboxyalkyl-thioether-undecahydro-closo-dodecaborate ditetramethylammonium salts. N,N'-dicyclohexylcarbodiimide (DCC) activated amidation of S,S-bis(carboxyalkyl)sulfonium-undecahydro-closo-dodecaborate or S-carboxyalkyl-thioether-undecahydro-closo-dodecaborate tetramethylammonium salts with propargylamine provided the opportunity to install terminal acetylene groups for further conjugation. These compounds acted as powerful building blocks for the synthesis of a broad range of 1,4-disubstituted 1,2,3-triazole products in high yields, utilizing the Cu(I)-mediated click cycloaddition reaction. The synthesis of BSH-lipid with a two-tailed moiety was also achieved, by esterification of S,S-bis(carboxyethyl)sulfoniumundecahydro-closo-dodecaborate(1-) tetramethylammonium salt with 1,2-O-distearoyl-sn-3-glycerol, which may prove useful in the liposomal boron delivery system. The bio-compatibility of the azide-alkyne click reaction was then utilized by performing this reaction in cell culture. The distribution of BSH in HeLa cells could be visualized by treating the cells first with a BSH-alkyne compound and then with Alexa Fluor 488(®) azide dye. The BSH-dye conjugate, which did not wash out, revealed the distribution of boron in the HeLa cells. Cytotoxicity assays of these BSH derivatives revealed that the synthesized BSH-conjugated triazoles possessed low cytotoxicity in HeLa cancer cells. Of these compounds, BSH conjugated triazole 15 induced a significant increase in the level of boron accumulation in HeLa cells. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Evaluation of retinal nerve fiber layer thickness and axonal transport 1 and 2 weeks after 8 hours of acute intraocular pressure elevation in rats.

PubMed

Abbott, Carla J; Choe, Tiffany E; Lusardi, Theresa A; Burgoyne, Claude F; Wang, Lin; Fortune, Brad

2014-02-04

To compare in vivo retinal nerve fiber layer thickness (RNFLT) and axonal transport at 1 and 2 weeks after an 8-hour acute IOP elevation in rats. Forty-seven adult male Brown Norway rats were used. Procedures were performed under anesthesia. The IOP was manometrically elevated to 50 mm Hg or held at 15 mm Hg (sham) for 8 hours unilaterally. The RNFLT was measured by spectral-domain optical coherence tomography. Anterograde and retrograde axonal transport was assessed from confocal scanning laser ophthalmoscopy imaging 24 hours after bilateral injections of 2 μL 1% cholera toxin B-subunit conjugated to AlexaFluor 488 into the vitreous or superior colliculi, respectively. Retinal ganglion cell (RGC) and microglial densities were determined using antibodies against Brn3a and Iba-1. The RNFLT in experimental eyes increased from baseline by 11% at 1 day (P < 0.001), peaked at 19% at 1 week (P < 0.0001), remained 11% thicker at 2 weeks (P < 0.001), recovered at 3 weeks (P > 0.05), and showed no sign of thinning at 6 weeks (P > 0.05). There was no disruption of anterograde transport at 1 week (superior colliculi fluorescence intensity, 75.3 ± 7.9 arbitrary units [AU] for the experimental eyes and 77.1 ± 6.7 AU for the control eyes) (P = 0.438) or 2 weeks (P = 0.188). There was no obstruction of retrograde transport at 1 week (RCG density, 1651 ± 153 per mm(2) for the experimental eyes and 1615 ± 135 per mm(2) for the control eyes) (P = 0.63) or 2 weeks (P = 0.25). There was no loss of Brn3a-positive RGC density at 6 weeks (P = 0.74) and no increase in microglial density (P = 0.92). Acute IOP elevation to 50 mm Hg for 8 hours does not cause a persisting axonal transport deficit at 1 or 2 weeks or a detectable RNFLT or RGC loss by 6 weeks but does lead to transient RNFL thickening that resolves by 3 weeks.

Evaluation of Retinal Nerve Fiber Layer Thickness and Axonal Transport 1 and 2 Weeks After 8 Hours of Acute Intraocular Pressure Elevation in Rats

PubMed Central

Abbott, Carla J.; Choe, Tiffany E.; Lusardi, Theresa A.; Burgoyne, Claude F.; Wang, Lin; Fortune, Brad

2014-01-01

Purpose. To compare in vivo retinal nerve fiber layer thickness (RNFLT) and axonal transport at 1 and 2 weeks after an 8-hour acute IOP elevation in rats. Methods. Forty-seven adult male Brown Norway rats were used. Procedures were performed under anesthesia. The IOP was manometrically elevated to 50 mm Hg or held at 15 mm Hg (sham) for 8 hours unilaterally. The RNFLT was measured by spectral-domain optical coherence tomography. Anterograde and retrograde axonal transport was assessed from confocal scanning laser ophthalmoscopy imaging 24 hours after bilateral injections of 2 μL 1% cholera toxin B-subunit conjugated to AlexaFluor 488 into the vitreous or superior colliculi, respectively. Retinal ganglion cell (RGC) and microglial densities were determined using antibodies against Brn3a and Iba-1. Results. The RNFLT in experimental eyes increased from baseline by 11% at 1 day (P < 0.001), peaked at 19% at 1 week (P < 0.0001), remained 11% thicker at 2 weeks (P < 0.001), recovered at 3 weeks (P > 0.05), and showed no sign of thinning at 6 weeks (P > 0.05). There was no disruption of anterograde transport at 1 week (superior colliculi fluorescence intensity, 75.3 ± 7.9 arbitrary units [AU] for the experimental eyes and 77.1 ± 6.7 AU for the control eyes) (P = 0.438) or 2 weeks (P = 0.188). There was no obstruction of retrograde transport at 1 week (RCG density, 1651 ± 153 per mm2 for the experimental eyes and 1615 ± 135 per mm2 for the control eyes) (P = 0.63) or 2 weeks (P = 0.25). There was no loss of Brn3a-positive RGC density at 6 weeks (P = 0.74) and no increase in microglial density (P = 0.92). Conclusions. Acute IOP elevation to 50 mm Hg for 8 hours does not cause a persisting axonal transport deficit at 1 or 2 weeks or a detectable RNFLT or RGC loss by 6 weeks but does lead to transient RNFL thickening that resolves by 3 weeks. PMID:24398096

Delivery of macromolecules into the endothelium of whole ex vivo human cornea by femtosecond laser-activated carbon nanoparticles.

PubMed

Jumelle, Clotilde; Mauclair, Cyril; Houzet, Julien; Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Perrache, Chantal; Gain, Philippe; Thuret, Gilles

2016-08-01

The targeted delivery of drugs or genes into corneal endothelial cells (ECs) during eye banking could help improve graft quality and quantity. Physical methods raising less safety concerns than viral ones, we previously adapted, for in vitro ECs, a recent innovative technique of drug delivery based on the activation of carbon nanoparticles (CNPs) by a femtosecond laser (fsL). The aim of the present pilot study was to adapt this method to enable molecule delivery into the intact endothelium of ex vivo human corneas. ECs from 40 organ-cultured corneas were perforated by photoacoustic reaction induced by irradiation of CNPs by a fsL. This enabled intracellular delivery of Alexa Fluor 488 dextran, a 4000 Da fluorescent macromolecule. The influence of increasing laser fluences (15, 20, 30 and 40 mJ/cm(2)) and of protective additives (ROCK inhibitor and poloxamer 407) on delivery and mortality rates was quantified using ImageJ. No dextran was delivered with a fluence lower than 20 mJ/cm(2). Dextran was delivered into 3% (range 0%-7%) of cells at 20 mJ/cm(2), 7% (range 2%-12%) at 30 mJ/cm(2) and reaching a median 13% (range 3%-24%) for 40 mJ/cm(2), showing that dextran uptake by ECs increased significantly with fluence. Induced mortality varied from 0% to 53% irrespective of fluence, but likely to be related with the endothelial status (EC density and morphometry, donor age, storage duration and presence of Descemet's folds). ROCK inhibitor slightly increased uptake efficiency, unlike poloxamer. However, none of them decreased the mortality induced by laser. This study shows that a macromolecule can be delivered specifically into ECs of a whole organ-cultured human cornea, using fsL-activated CNPs. The delivery rate was relatively high for a non-viral method. Further optimisation is required to understand and reduce variability in cell mortality. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum.

PubMed

Tame, Akihiro; Yoshida, Takao; Ohishi, Kazue; Maruyama, Tadashi

2015-07-01

Deep-sea mytilid mussels harbor symbiotic bacteria in their gill epithelial cells that are horizontally or environmentally transmitted to the next generation of hosts. To understand the immune defense system in deep-sea symbiotic mussels, we examined the hemocyte populations of the symbiotic Bathymodiolus mussel species Bathymodiolus japonicus, Bathymodiolus platifrons, and Bathymodiolus septemdierum, and characterized three types of hemocytes: agranulocytes (AGs), basophilic granulocytes (BGs), and eosinophilic granulocytes (EGs). Of these, the EG cells were the largest (diameter, 8.4-10.0 μm) and had eosinophilic cytoplasm with numerous eosinophilic granules (diameter, 0.8-1.2 μm). Meanwhile, the BGs were of medium size (diameter, 6.7-8.0 μm) and contained small basophilic granules (diameter, 0.3-0.4 μm) in basophilic cytoplasm, and the AGs, the smallest of the hemocytes (diameter, 4.8-6.0 μm), had basophilic cytoplasm lacking granules. A lectin binding assay revealed that concanavalin A bound to all three hemocyte types, while wheat germ agglutinin bound exclusively to EGs and BGs. The total hemocyte population densities within the hemolymph of all three Bathymodiolus mussel species were similar (8.4-13.3 × 10(5) cells/mL), and the percentages of circulating AGs, BGs, and EGs in the hemolymph of these organisms were 44.7-48.5%, 14.3-17.6%, and 34.3-41.0%, respectively. To analyze the functional differences between these hemocytes, the phagocytic activity and post-phagocytic phagosome-lysosome fusion events were analyzed in each cell type using a fluorescent Alexa Fluor(®) 488-conjugated Escherichia coli bioparticle and a LysoTracker(®) lysosomal marker, respectively. While the AGs exhibited no phagocytic activity, both types of granulocytes were phagocytic. Of the three hemocyte types, the EGs exhibited the highest level of phagocytic activity as well as rapid phagosome-lysosome fusion, which occurred within 2 h of incubation. Meanwhile, the BGs showed lower phagocytic activity and lower rates of phagosome-lysosome fusion than the EGs. These findings indicate that the two types of granulocyte play distinct roles in the defense system. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Endogenously Generated Plasmin at the Vascular Wall Injury Site Amplifies Lysine Binding Site-Dependent Plasminogen Accumulation in Microthrombi

PubMed Central

Brzoska, Tomasz; Tanaka-Murakami, Aki; Suzuki, Yuko; Sano, Hideto; Kanayama, Naohiro; Urano, Tetsumei

2015-01-01

The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation. PMID:25806939

Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

PubMed

Petryayeva, Eleonora; Algar, W Russ

2014-03-18

Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.

Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Krull, Ulrich J

2013-08-06

A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

Plasmonically amplified bioassay - Total internal reflection fluorescence vs. epifluorescence geometry.

PubMed

Hageneder, Simone; Bauch, Martin; Dostalek, Jakub

2016-08-15

This paper investigates plasmonic amplification in two commonly used optical configurations for fluorescence readout of bioassays - epifluorescence (EPF) and total internal reflection fluorescence (TIRF). The plasmonic amplification in the EPF configuration was implemented by using crossed gold diffraction grating and Kretschmann geometry of attenuated total reflection method (ATR) was employed in the TIRF configuration. Identical assay, surface architecture for analyte capture, and optics for the excitation, collection and detection of emitted fluorescence light intensity were used in both TIRF and EPF configurations. Simulations predict that the crossed gold diffraction grating (EPF) can amplify the fluorescence signal by a factor of 10(2) by the combination of surface plasmon-enhanced excitation and directional surface plasmon-coupled emission in the red part of spectrum. This factor is about order of magnitude higher than that predicted for the Kretschmann geometry (TIRF) which only took advantage of the surface plasmon-enhanced excitation. When applied for the readout of sandwich interleukin 6 (IL-6) immunoassay, the plasmonically amplified EPF geometry designed for Alexa Fluor 647 labels offered 4-times higher fluorescence signal intensity compared to TIRF. Interestingly, both geometries allowed reaching the same detection limit of 0.4pM despite of the difference in the fluorescence signal enhancement. This is attributed to inherently lower background of fluorescence signal for TIRF geometry compared to that for EPF which compensates for the weaker fluorescence signal enhancement. The analysis of the inflammation biomarker IL-6 in serum at medically relevant concentrations and the utilization of plasmonic amplification for the fluorescence measurement of kinetics of surface affinity reactions are demonstrated for both EPF and TIRF readout. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Quantum Dot-Based Hybrid Nanostructures and Energy Transfer on the Nanoscale for Single- and Multi-Photon Imaging and Cancer Diagnostics

NASA Astrophysics Data System (ADS)

Nabiev, Igor

2017-01-01

An ideal single-photon (1P) or multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible 1P or two-photon (2P) absorption cross section and the smallest possible highly specific recognition molecules conjugated with the nanoparticle in an oriented manner. However, the conditions used for conjugation of typical recognition molecules (conventional antibodies, Abs) with nanoparticles often provoke their unfolding and/or yield nanoprobes with irregular orientation of Abs on the nanoparticle surface. Conjugation of smaller Ab fragments, such as single-domain antibodies (sdAbs), with quantum dots (QDs) in an oriented manner can be considered as an attractive approach to engineering of ultrasmall diagnostic nanoprobes. QDs conjugated to 13-kDa sdAbs derived from camelid IgG or streptavidin have been used as efficient 1P or 2P excitation probes for imaging of cancer markers. The 2P absorption cross sections (TPACSs) for some conjugates are higher than 49,000 GM (Goeppert-Mayer units), which is close to the theoretical value calculated for CdSe QDs and considerably exceeds that of organic dyes. A further step in advanced QD-based cancer diagnostics has been made through implementation of efficient FRET-based imaging with 2P excitation, which has been demonstrated for double immunostaining complexes formed on the surface of cancer cells from sdAb-QD conjugates (donor) and a combination of monoclonal Abs and secondary antibodies labeled with the AlexaFluor dye (acceptor). The proposed approach permits obtaining an exceptional contrast of 2P imaging of cancer biomarkers without any contribution of cell and tissue autofluorescence in the recorded images.

Magnetic luminescent nanoparticles as internal calibration for an immunoassay for ricin

NASA Astrophysics Data System (ADS)

Dosev, Dosi; Nichkova, Mikaela; Ma, Zhi-Ya; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

2008-02-01

Fluorescence techniques rely on measurement of relative fluorescence units and require calibration to obtain reliable and comparable quantitative data. Fluorescent immunoassays are a very sensitive and convenient method of choice for rapid detection of biotoxins, such as ricin. Here we present the application of magnetic luminescent nanoparticles (MLNPs) with a magnetic core of Fe 3O 4 and a fluorescent shell of Eu:Gd IIO 3 as carriers for a nanobead-immunoassay for the detection of ricin with internal calibration. A sandwich immunoassay for ricin was performed on the surface of the MLNPs. The particles were functionalized with capture polyclonal antibodies. Anti-ricin antibodies labeled with Alexa Fluor dye were used as the detecting antibodies. After magnetic extraction, the amount of ricin bound to the particle surface was quantified and related to the fluorescence signal of the nanoparticles. In this new platform, the MLNPs have three main functions: (1) a probe for the specific extraction of the target analyte from the sample; (2) a carrier in the quantitative immunoassay with magnetic separation; and (3) an internal standard in the fluorescence measurement of the dye reporter. The MLNPs serve as an internal control for the total analysis including extraction and assay performance. This approach eliminates the experimental error inherent in particle extraction and measurement of absolute organic dye fluorescence intensities. All fluorescent measurements were performed in a microplate reader. The standard curve for ricin had a dynamic range from 20 ng/ml to 100 μg/ml with a detection limit of 5 ng/ml. The configuration that has been developed can be easily adapted to a high throughput miniaturized system.

Optically-Induced Neuronal Activity Is Sufficient to Promote Functional Motor Axon Regeneration In Vivo.

PubMed

Ward, Patricia J; Jones, Laura N; Mulligan, Amanda; Goolsby, William; Wilhelm, Jennifer C; English, Arthur W

2016-01-01

Peripheral nerve injuries are common, and functional recovery is very poor. Beyond surgical repair of the nerve, there are currently no treatment options for these patients. In experimental models of nerve injury, interventions (such as exercise and electrical stimulation) that increase neuronal activity of the injured neurons effectively enhance axon regeneration. Here, we utilized optogenetics to determine whether increased activity alone is sufficient to promote motor axon regeneration. In thy-1-ChR2/YFP transgenic mice in which a subset of motoneurons express the light-sensitive cation channel, channelrhodopsin (ChR2), we activated axons in the sciatic nerve using blue light immediately prior to transection and surgical repair of the sciatic nerve. At four weeks post-injury, direct muscle EMG responses evoked with both optical and electrical stimuli as well as the ratio of these optical/electrical evoked EMG responses were significantly greater in mice that received optical treatment. Thus, significantly more ChR2+ axons successfully re-innervated the gastrocnemius muscle in mice that received optical treatment. Sections of the gastrocnemius muscles were reacted with antibodies to Synaptic Vesicle Protein 2 (SV2) to quantify the number of re-occupied motor endplates. The number of SV2+ endplates was greater in mice that received optical treatment. The number of retrogradely-labeled motoneurons following intramuscular injection of cholera toxin subunit B (conjugated to Alexa Fluor 555) was greater in mice that received optical treatment. Thus, the acute (1 hour), one-time optical treatment resulted in robust, long-lasting effects compared to untreated animals as well as untreated axons (ChR2-). We conclude that neuronal activation is sufficient to promote motor axon regeneration, and this regenerative effect is specific to the activated neurons.

Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes.

PubMed

Moniruzzaman, Md; Islam, Md Zahidul; Sharmin, Sabrina; Dohra, Hideo; Yamazaki, Masahito

2017-08-22

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.

Imaging heterostructured quantum dots in cultured cells with epifluorescence and transmission electron microscopy

PubMed Central

Rivera, Erin M.; Provencio, Casilda Trujillo; Steinbruck, Andrea; Rastogi, Pawan; Dennis, Allison; Hollingsworth, Jennifer; Serrano, Elba

2011-01-01

Quantum dots (QDs) are semiconductor nanocrystals with extensive imaging and diagnostic capabilities, including the potential for single molecule tracking. Commercially available QDs offer distinct advantages over organic fluorophores, such as increased photostability and tunable emission spectra, but their cadmium selenide (CdSe) core raises toxicity concerns. For this reason, replacements for CdSe-based QDs have been sought that can offer equivalent optical properties. The spectral range, brightness and stability of InP QDs may comprise such a solution. To this end, LANL/CINT personnel fabricated moderately thick-shell novel InP QDs that retain brightness and emission over time in an aqueous environment. We are interested in evaluating how the composition and surface properties of these novel QDs affect their entry and sequestration within the cell. Here we use epifluorescence and transmission electron microscopy (TEM) to evaluate the structural properties of cultured Xenopus kidney cells (A6; ATCC) that were exposed either to commercially available CdSe QDs (Qtracker® 565, Invitrogen) or to heterostructured InP QDs (LANL). Epifluorescence imaging permitted assessment of the general morphology of cells labeled with fluorescent molecular probes (Alexa Fluor® ® phalloidin; Hoechst 33342), and the prevalence of QD association with cells. In contrast, TEM offered unique advantages for viewing electron dense QDs at higher resolution with regard to subcellular sequestration and compartmentalization. Preliminary results show that in the absence of targeting moieties, InP QDs (200 nM) can passively enter cells and sequester nonspecifically in cytosolic regions whereas commercially available targeted QDs principally associate with membranous structures within the cell. Supported by: NIH 5R01GM084702. PMID:21808662

Human eyelid meibomian glands and tarsal muscle are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in El-Bagre, Colombia, South America.

PubMed

Abreu-Velez, Ana Maria; Howard, Michael S; Hashimoto, Takashi; Grossniklaus, Hans E

2010-03-01

Previously, we described a new variant of endemic pemphigus foliaceus (EPF) in Colombia, South America (El Bagre-EPF). Continuing our characterization of this variant of EPF, we now focus on one of our previously reported clinical findings: the presence of ocular lesions. These ocular lesions are seen in patients having extensive skin involvement, as measured by the Lund and Browder scale, which is generally used for patients with skin burns. We specifically searched for evidence of autoreactivity to various eyelid structures in these patients and correlated our immunologic data with the clinical findings. We performed indirect immunofluorescence studies using normal-appearing human eyelid skin from routine blepharoplasties as substrate tissue. We tested sera from 12 patients with El Bagre-EPF and ocular lesions, 5 patients with sporadic (nonendemic) pemphigus foliaceus, and 20 healthy control subjects (10 from the El Bagre-EPF endemic area and 10 from nonendemic areas). We used fluorescein isothiocyanate conjugated goat antiserum to human total IgG/IgA/IgM as a secondary antibody. In addition, we used fluorescein isothiocyanate conjugated antibodies to human fibrinogen, albumin, IgG, IgE, C1q, and C3, Texas Red (Rockland Immunochemicals, Inc, Gilbertsville, PA), Alexa Fluor 555, or Alexa Fluor 594 (Invitrogen, Carlsbad, CA). Ki-67 (a cell proliferation marker) was used to determine the cell proliferation rate, and nuclear counterstaining was performed with either 4', 6-diamidino-2-phenylindole or Topro III (Invitrogen, Carlsbad, CA). We observed autoreactivity to multiple eyelid structures, including meibomian glands and tarsal muscle bundles at different levels, and some areas of the epidermis and the dermis close to the isthmus of the eyelids. Tarsal plate autoreactivity was seen in 10 of 12 of the El Bagre-EPF sera and in one control with pemphigus erythematosus. Furthermore, immunoprecipitation using an eyelid sample as a substrate with 1 mmol/L of sodium orthovanodate showed autoreactivity to several antigens, including some of possible lipid origin. The main limitation of this study is the fact that the antigen or antigens remain unknown. We identified for the first time to our knowledge autoantibodies to meibomian glands and tarsal muscle in El Bagre-EPF. Our findings suggest that the autoantibodies to the ocular structures cause the clinical and histopathological findings in the ocular lesions in El Bagre-EPF. Copyright 2009 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

Human eyelid meibomian glands and tarsal muscle are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in El-Bagre, Colombia, South America

PubMed Central

Abreu-Velez, Ana Maria; Howard, Michael S.; Hashimoto, Takashi; Grossniklaus, Hans E.

2010-01-01

Background Previously, we described a new variant of endemic pemphigus foliaceus (EPF) in Colombia, South America (El Bagre-EPF). Objective Continuing our characterization of this variant of EPF, we now focus on one of our previously reported clinical findings: the presence of ocular lesions. These ocular lesions are seen in patients having extensive skin involvement, as measured by the Lund and Browder scale, which is generally used for patients with skin burns. Methods We specifically searched for evidence of autoreactivity to various eyelid structures in these patients and correlated our immunologic data with the clinical findings. We performed indirect immunofluorescence studies using normal-appearing human eyelid skin from routine blepharoplasties as substrate tissue. We tested sera from 12 patients with El Bagre-EPF and ocular lesions, 5 patients with sporadic (nonendemic) pemphigus foliaceus, and 20 healthy control subjects (10 from the El Bagre-EPF endemic area and 10 from nonendemic areas). We used fluorescein isothiocyanate conjugated goat antiserum to human total IgG/IgA/IgM as a secondary antibody. In addition, we used fluorescein isothiocyanate conjugated antibodies to human fibrinogen, albumin, IgG, IgE, C1q, and C3, Texas Red (Rockland Immunochemicals, Inc, Gilbertsville, PA), Alexa Fluor 555, or Alexa Fluor 594 (Invitrogen, Carlsbad, CA). Ki-67 (a cell proliferation marker) was used to determine the cell proliferation rate, and nuclear counterstaining was performed with either 4′, 6-diamidino-2-phenylindole or Topro III (Invitrogen, Carlsbad, CA). Results We observed autoreactivity to multiple eyelid structures, including meibomian glands and tarsal muscle bundles at different levels, and some areas of the epidermis and the dermis close to the isthmus of the eyelids. Tarsal plate autoreactivity was seen in 10 of 12 of the El Bagre-EPF sera and in one control with pemphigus erythematosus. Furthermore, immunoprecipitation using an eyelid sample as a substrate with 1 mmol/L of sodium orthovanodate showed autoreactivity to several antigens, including some of possible lipid origin. Limitations The main limitation of this study is the fact that the antigen or antigens remain unknown. Conclusion We identified for the first time to our knowledge autoantibodies to meibomian glands and tarsal muscle in El Bagre-EPF. Our findings suggest that the autoantibodies to the ocular structures cause the clinical and histopathological findings in the ocular lesions in El Bagre-EPF. PMID:20061054

Adapter reagents for protein site specific dye labeling.

PubMed

Thompson, Darren A; Evans, Eric G B; Kasza, Tomas; Millhauser, Glenn L; Dawson, Philip E

2014-05-01

Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this acetophenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. © 2014 Wiley Periodicals, Inc.

Adapter Reagents for Protein Site Specific Dye Labeling

PubMed Central

Thompson, Darren A.; Evans, Eric G. B.; Kasza, Tomas; Millhauser, Glenn L.; Dawson, Philip E.

2016-01-01

Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this aceto-phenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. PMID:24599728

Passive micromixer using by convection and surface tension effects with air-liquid interface.

PubMed

Ju, Jongil; Warrick, Jay

2013-12-01

This article describes a passive micromixer that utilizes an air-liquid interface and surface tension effects to enhance fluid mixing via convection and Marangoni effects. Performance of the microfluidic component is tested within a passive-pumping-based device that consists of three microchannels connected in succession using passive micro-mixers. Mixing was quantified at 5 key points along the length of the device using microscope images of patterned streams of Alexa 488 fluorescent-dyed water and pure DI water flowing through the device. The passive micro-mixer mixed fluid 15-20 times more effectively than diffusion between laminar flow streams alone and is a novel micro-mixer embodiment that provides an additional strategy for removing external components from microscale devices for simpler, autonomous operation.

Passive micromixer using by convection and surface tension effects with air-liquid interface

PubMed Central

Ju, Jongil; Warrick, Jay

2014-01-01

This article describes a passive micromixer that utilizes an air-liquid interface and surface tension effects to enhance fluid mixing via convection and Marangoni effects. Performance of the microfluidic component is tested within a passive-pumping-based device that consists of three microchannels connected in succession using passive micro-mixers. Mixing was quantified at 5 key points along the length of the device using microscope images of patterned streams of Alexa 488 fluorescent-dyed water and pure DI water flowing through the device. The passive micro-mixer mixed fluid 15–20 times more effectively than diffusion between laminar flow streams alone and is a novel micro-mixer embodiment that provides an additional strategy for removing external components from microscale devices for simpler, autonomous operation. PMID:25104979

Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time

PubMed Central

Hohng, Sungchul

2010-01-01

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy. PMID:20808851

Microbial strengthening of loose sand.

PubMed

Banagan, B L; Wertheim, B M; Roth, M J S; Caslake, L F

2010-08-01

To test whether the addition of Flavobacterium johnsoniae could increase the strength of saturated Ottawa 30 sand. A box model was built that simulates groundwater-like flow through a main sand compartment. Strength tests were performed at seven locations and at two depths, 10.8 and 20.3 cm below the top of the tank, using a vane shear device before and after the addition of bacteria. After the addition of Fl. johnsoniae, sand samples were obtained from multiple sampling ports on the vertical sides of the box model. The presence of a bacterial biofilm was confirmed by staining these sand samples with SYTO-9 and Alexa Fluor 633 and viewing with a confocal microscope. The average shear strength increases after the addition of Fl. johnsoniae were 15.2-87.5%, depending on the experimental conditions. Flavobacterium johnsoniae caused a statistically significant increase in the strength of saturated Ottawa 30 sand. Biofilm-forming bacteria can increase the shear strength of saturated sand. The addition of biofilm-forming bacteria to a building site may be an alternate method to mitigate the effects of liquefaction.

Effect of fluorescent particle size on the modulation efficiency of ultrasound-modulated fluorescence.

PubMed

Liu, Yuan; Yuan, Baohong; Vignola, Joseph

2012-01-01

To investigate whether the size of fluorescent particles affects the modulation efficiency of ultrasound-modulated fluorescence (UMF), we measured UMF and DC (direct current) signals of the fluorescence emission from four different sized fluorescent particles: (1) three carboxylate-modified fluorescent microspheres (FM) with diameters of 20 nm, 200 nm, and 1.0 µm and (2) streptavidin-conjugated Alexa Fluor 647 with a diameter of approximately 5 nm. The UMF and DC signals were simultaneously measured using a broadband lock-in amplifier and a narrowband amplifier, respectively. The ratio of the UMF strength to the DC signal strength is defined as the modulation efficiency. This modulation efficiency was then used to evaluate the effects of fluorophore size and concentration. Results show that the modulation efficiency was improved by approximately a factor of two when the size of the fluorescent particles is increased from 5 nm to 1 µm. In addition, the linear relationship between the UMF strength and ultrasound pressure (observed in our previous study) were maintained regardless of the fluorescent particle sizes.

Effect of fluorescent particle size on the modulation efficiency of ultrasound-modulated fluorescence

PubMed Central

Liu, Yuan; Yuan, Baohong; Vignola, Joseph

2013-01-01

To investigate whether the size of fluorescent particles affects the modulation efficiency of ultrasound-modulated fluorescence (UMF), we measured UMF and DC (direct current) signals of the fluorescence emission from four different sized fluorescent particles: (1) three carboxylate-modified fluorescent microspheres (FM) with diameters of 20 nm, 200 nm, and 1.0 µm and (2) streptavidin-conjugated Alexa Fluor 647 with a diameter of approximately 5 nm. The UMF and DC signals were simultaneously measured using a broadband lock-in amplifier and a narrowband amplifier, respectively. The ratio of the UMF strength to the DC signal strength is defined as the modulation efficiency. This modulation efficiency was then used to evaluate the effects of fluorophore size and concentration. Results show that the modulation efficiency was improved by approximately a factor of two when the size of the fluorescent particles is increased from 5 nm to 1 µm. In addition, the linear relationship between the UMF strength and ultrasound pressure (observed in our previous study) were maintained regardless of the fluorescent particle sizes. PMID:24179476

Improvement of In Situ PCR by Optimization of PCR Cycle Number and Proteinase K Concentration: Localization of X Chromosome-Linked Phosphoglycerate Kinase-1 Gene in Mouse Reproductive Organs

PubMed Central

Hishikawa, Yoshitaka; An, Shucai; Yamamoto-Fukuda, Tomomi; Shibata, Yasuaki; Koji, Takehiko

2009-01-01

In situ polymerase chain reaction (in situ PCR), which can detect a few copies of genes within a cell by amplifying the target gene, was developed to better understand the biological functions of tissues. In this study, we optimized the protocol conditions for the detection of X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene in paraffin-embedded sections of mouse reproductive organs. The effects of various concentrations of proteinase K (PK) and PCR cycle numbers were examined. To label the amplified DNA, we used digoxigenin-dUTP (Dig), Cy-3-dUTP (Cy-3), or FluorX-dCTP (FluorX). The optimal concentration of PK was 50 µg/ml for the ovary and 10 µg/ml for the testis. Ten PCR cycles were optimal for Dig and 25 cycles were optimal for FluorX and Cy-3 in the ovary and testis. The signal-to-noise ratio of FluorX and Cy-3 for ovarian tissue was better than that of Dig. Using the above conditions, we detected 1–4 and 1–2 spots of pgk-1 in the nuclei of granulosa and germ cells, respectively. Our results indicate that in situ PCR is useful for detecting a specific gene in paraffin-embedded sections under optimized conditions of both PCR cycle number and PK concentration. PMID:19492023

Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury

PubMed Central

Mantilla, Carlos B.; Bailey, Jeffrey P.; Zhan, Wen-Zhi; Sieck, Gary C.

2012-01-01

Following cervical spinal cord injury at C2 (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10 μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability. PMID:22227062

Kiss-and-Run Is a Significant Contributor to Synaptic Exocytosis and Endocytosis in Photoreceptors

PubMed Central

Wen, Xiangyi; Saltzgaber, Grant W.; Thoreson, Wallace B.

2017-01-01

Accompanying sustained release in darkness, rod and cone photoreceptors exhibit rapid endocytosis of synaptic vesicles. Membrane capacitance measurements indicated that rapid endocytosis retrieves at least 70% of the exocytotic membrane increase. One mechanism for rapid endocytosis is kiss-and-run fusion where vesicles briefly contact the plasma membrane through a small fusion pore. Release can also occur by full-collapse in which vesicles merge completely with the plasma membrane. We assessed relative contributions of full-collapse and kiss-and-run in salamander photoreceptors using optical techniques to measure endocytosis and exocytosis of large vs. small dye molecules. Incubation with small dyes (SR101, 1 nm; 3-kDa dextran-conjugated Texas Red, 2.3 nm) loaded rod and cone synaptic terminals much more readily than larger dyes (10-kDa Texas Red, 4.6 nm; 10-kDa pHrodo, 4.6 nm; 70-kDa Texas Red, 12 nm) consistent with significant uptake through 2.3–4.6 nm fusion pores. By using total internal reflection fluorescence microscopy (TIRFM) to image individual vesicles, when rods were incubated simultaneously with Texas Red and AlexaFluor-488 dyes conjugated to either 3-kDa or 10-kDa dextran, more vesicles loaded small molecules than large molecules. Using TIRFM to detect release by the disappearance of dye-loaded vesicles, we found that SR101 and 3-kDa Texas Red were released from individual vesicles more readily than 10-kDa and 70-kDa Texas Red. Although 10-kDa pHrodo was endocytosed poorly like other large dyes, the fraction of release events was similar to SR101 and 3-kDa Texas Red. We hypothesize that while 10-kDa pHrodo may not exit through a fusion pore, release of intravesicular protons can promote detection of fusion events by rapidly quenching fluorescence of this pH-sensitive dye. Assuming that large molecules can only be released by full-collapse whereas small molecules can be released by both modes, our results indicate that 50%–70% of release from rods involves kiss-and-run with 2.3–4.6 nm fusion pores. Rapid retrieval of vesicles by kiss-and-run may limit membrane disruption of release site function during ongoing release at photoreceptor ribbon synapses. PMID:28979188

The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

PubMed Central

Varisli, Omer; Scott, Hollie; Agca, Cansu; Agca, Yuksel

2013-01-01

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10–12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4°C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P<0.05). Sperm motility increased as cooling rate was increased for both strains (P<0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70 °C /min and 100 °C /min cooling rate improved post-thaw motility of rat sperm. PMID:23727068

Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation.

PubMed

Hégarat, Nadia; Novopashina, Darya; Fokina, Alesya A; Boutorine, Alexandre S; Venyaminova, Alya G; Praseuth, Danièle; François, Jean-Christophe

2014-03-01

Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2'-deoxycytidine and 5-propynyl-2'-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells. © 2014 FEBS.

Sildenafil (Viagra(®)) prevents and restores LPS-induced inflammation in astrocytes.

PubMed

de Santana Nunes, Ana Karolina; Rapôso, Catarina; Björklund, Ulrika; da Cruz-Höfling, Maria Alice; Peixoto, Christina Alves; Hansson, Elisabeth

2016-09-06

Astrocytes are effectively involved in the pathophysiological processes in the central nervous system (CNS) and may contribute to or protect against development of inflammatory and degenerative diseases. Sildenafil is a potent and selective phosphodiesterase-5 (PDE-5) inhibitor, which induces cyclic GMP accumulation. However, the mechanisms of actions on glial cells are not clear. The aim of the present work is to evaluate the role of sildenafil in lipopolysaccharide (LPS)-stimulated astrocytes. The cytoskeleton integrity and Ca(2+) waves were assessed as indicators of inflammatory state. Two main groups were done: (A) one prevention and (B) one restoration. Each of these groups: A1: control; A2: LPS for 24h; A3: sildenafil 1 or 10μM for 4h and then sildenafil 1 or 10μM+LPS for 24h. B1: control; B2: LPS for 24h; B3: LPS for 24h and then LPS+sildenafil 1 or 10μM for 24h. Cytoskeleton integrity was analyzed through GFAP immunolabeling and actin labeling with an Alexa 488-conjugated phalloidin probe. Calcium responses were assessed through a Ca(2+)-sensitive fluorophore Fura-2/AM. The results show that both preventive and restorative treatments with sildenafil (in both concentrations) reduced the Ca(2+) responses in intensity and induced a more organized actin fiber pattern, compared to LPS treated cells. This work demonstrated for the first time that astrocytes are a key part of the sildenafil protective effects in the CNS. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Phenotypic heterogeneity in the endothelium of the human vortex vein system.

PubMed

Yu, Paula K; Tan, Priscilla E Z; Cringle, Stephen J; McAllister, Ian L; Yu, Dao-Yi

2013-10-01

The vortex vein system is the drainage pathway for the choroidal circulation and serves an important function in the effective drainage of the exceptionally high blood flow from the choroidal circulation. As there are only 4-6 vortex veins, a large volume of blood must be drained from many choroidal veins into each individual vortex vein. The vortex vein system must also cope with passing through tissues of different rigidity and significant pressure gradient as it transverses from the intrao-cular to the extra-ocular compartments. However, little is known about how the vortex vein system works under such complex situations in both physiological and pathological condition. Endothelial cells play a vital role in other vascular systems, but they have not been studied in detail in the vortex vein system. The purpose of this study is to characterise the intracellular structures and morphology in both the intra-and extra-ocular regions of the human vortex vein system. We hypothesise the presence of endothelial phenotypic heterogeneity through the vortex vein system. The inferior temporal vortex vein system from human donor eyes were obtained and studied histologically using confocal microscopy. The f-actin cytoskeleton and nuclei were labelled using Alexa Fluor conjugated Phalloidin and YO-PRO-1. Eight regions of the vortex vein system were examined with the venous endothelium studied in detail with quantitative data obtained for endothelial cell and nuclei size and shape. Significant endothelial phenotypic heterogeneity was found throughout the vortex vein system with the most obvious differences observed between the ampulla and its downstream regions. Variation in the distribution pattern of smooth muscle cells, in particular the absence of smooth muscle cells around the ampulla, was noted. Our results suggest the presence of significantly different haemodynamic forces in different regions of the vortex vein system and indicate that the vortex vein system may play important roles in regulation of the choroidal circulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Wide-field depth-sectioning fluorescence microscopy using projector-generated patterned illumination

NASA Astrophysics Data System (ADS)

Delica, Serafin; Mar Blanca, Carlo

2007-10-01

We present a simple and cost-effective wide-field, depth-sectioning, fluorescence microscope utilizing a commercial multimedia projector to generate excitation patterns on the sample. Highly resolved optical sections of fluorescent pollen grains at 1.9 μm axial resolution are constructed using the structured illumination technique. This requires grid excitation patterns to be scanned across the sample, which is straightforwardly implemented by creating slideshows of gratings at different phases, projecting them onto the sample, and synchronizing camera acquisition with slide transition. In addition to rapid dynamic pattern generation, the projector provides high illumination power and spectral excitation selectivity. We exploit these properties by imaging mouse neural cells in cultures multistained with Alexa 488 and Cy3. The spectral and structural neural information is effectively resolved in three dimensions. The flexibility and commercial availability of this light source is envisioned to open multidimensional imaging to a broader user base.

Antibody-directed targeting of lysostaphin adsorbed onto polylactide nanoparticles increases its antimicrobial activity against S. aureus in vitro

NASA Astrophysics Data System (ADS)

Satishkumar, R.; Vertegel, A. A.

2011-12-01

The objective of this paper was to study the effect of antibody-directed targeting of S. aureus by comparing the activities of lysostaphin conjugated to biodegradable polylactide nanoparticles (NPs) in the presence and in the absence of co-immobilized anti-S. aureus antibody. Lysostaphin-antibody-NP conjugates were synthesized through physical adsorption at different enzyme:antibody:NP ratios. The synthesized enzyme-NP conjugates were characterized by means of dynamic light scattering and zeta potential analysis, and the total protein binding yield on the NPs was characterized using Alexa Fluor 350 and 594 dyes for the S. aureus antibody and lysostaphin respectively. We observed enhanced antimicrobial activity for both enzyme-coated and enzyme-antibody-coated NPs for lysostaphin coatings corresponding to ~ 40% of the initial monolayer and higher compared to the free enzyme case (p < 0.05). At the highest antibody coating concentration, bacterial lysis rates for antibody-coated samples were significantly higher than for lysostaphin-coated samples lacking the antibody (p < 0.05). Such enzyme-NP conjugates thus have the potential for becoming novel therapeutic agents for treating antibiotic-resistant S. aureus infections.

Highly fluorescent resorcinarene cavitand nanocapsules with efficient renal clearance

NASA Astrophysics Data System (ADS)

Mahadevan, Kalpana; Patthipati, Venkata Suresh; Han, Sangbum; Swanson, R. James; Whelan, Eoin C.; Osgood, Christopher; Balasubramanian, Ramjee

2016-08-01

Nanomaterial based imaging approaches hold substantial promise in addressing current diagnostic and therapeutic challenges. One of the key requirements for the successful clinical translation of nanomaterials is their complete clearance from the body within a reasonable time period preferably via the renal filtration route. This article describes the synthesis of highly fluorescent, water soluble, resorcinarene cavitand nanocapsules and demonstrates their effective renal clearance in mice. The synthesis and functionalization of nanocapsules was accomplished in a one-pot operation via thiol-ene reactions without involving self-assembly, sacrificial templates or emulsions. Water soluble resorcinarene cavitand nanocapsules obtained by this approach were covalently functionalized with Alexa Fluor 750. Highly fluorescent nanocapsules with hydrodynamic diameters of 122 nm and 68 nm and extinction coefficients of 1.3 × 109 M-1 cm-1 and 1.5 × 108 M-1 cm-1 respectively were prepared by varying the reaction conditions. The in vivo biodistribution and clearance of these nanocapsules in mice followed by whole-body fluorescence imaging showed that they were both cleared renally within a few hours. Given the inherent encapsulation capabilities of nanocapsules, the renal clearance demonstrated in this work opens up new opportunities for their theranostic applications especially for targeting and treating the urinary tract.

Real-time visualization of macromolecule uptake by epidermal Langerhans cells in living animals.

PubMed

Frugé, Rachel E; Krout, Colleen; Lu, Ran; Matsushima, Hironori; Takashima, Akira

2012-03-01

As a skin-resident member of the dendritic cell family, Langerhans cells (LCs) are generally regarded to function as professional antigen-presenting cells. Here we report a simple method to visualize the endocytotic activity of LCs in living animals. BALB/c mice received subcutaneous injection of FITC-conjugated dextran (DX) probes into the ear skin and were then examined under confocal microscopy. Large numbers of FITC(+) epidermal cells became detectable 12-24 hours after injection as background fluorescence signals began to disappear. Most (>90%) of the FITC(+) epidermal cells expressed Langerin, and >95% of Langerin(+) epidermal cells exhibited significant FITC signals. To assess intracellular localization, Alexa Fluor 546-conjugated DX probes were locally injected into IAβ-enhanced green fluorescent protein (EGFP) knock-in mice and Langerin-EGFP-diphtheria toxin receptor mice--three dimensional rotation images showed close association of most of the internalized DX probes with major histocompatibility complex (MHC) class II molecules, but not with Langerin molecules. These observations support the current view that LCs constantly sample surrounding materials, including harmful and innocuous antigens, at the environmental interface. Our data also validate the potential utility of the newly developed imaging approach to monitor LC function in wild-type animals.

In Vivo Dual Fluorescence Imaging to Detect Joint Destruction.

PubMed

Cho, Hongsik; Bhatti, Fazal-Ur-Rehman; Lee, Sangmin; Brand, David D; Yi, Ae-Kyung; Hasty, Karen A

2016-10-01

Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near-infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP-1 and MMP-13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro-orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune-reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis. © 2016 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Distinguishing between Protein Dynamics and Dye Photophysics in Single-Molecule FRET Experiments

PubMed Central

Chung, Hoi Sung; Louis, John M.; Eaton, William A.

2010-01-01

Abstract Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small α/β protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers—donor10/acceptor57 and donor57/acceptor10—which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol–coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects—a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity. PMID:20159166

Distinguishing between protein dynamics and dye photophysics in single-molecule FRET experiments.

PubMed

Chung, Hoi Sung; Louis, John M; Eaton, William A

2010-02-17

Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small alpha/beta protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers-donor(10)/acceptor(57) and donor(57)/acceptor(10)-which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol-coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects-a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single-lipid tracking on nanoscale membrane buds: The effects of curvature on lipid diffusion and sorting.

PubMed

Woodward, Xinxin; Stimpson, Eric E; Kelly, Christopher V

2018-05-29

Nanoscale membrane curvature in cells is critical for endocytosis/exocytosis and membrane trafficking. However, the biophysical ramifications of nanoscale membrane curvature on the behavior of lipids remain poorly understood. Here, we created an experimental model system of membrane curvature at a physiologically-relevant scale and obtained nanoscopic information on single-lipid distributions and dynamics. Supported lipid bilayers were created over 50 and 70 nm radius nanoparticles to create membrane buds. Single-molecule localization microscopy was performed with diverse mixtures of fluorescent and non-fluorescent lipids. Variations in lipid acyl tales length, saturation, head-group, and fluorescent labeling strategy were tested while maintaining a single fluid lipid phase throughout the membrane. Monte Carlo simulations were used to fit our experimental results and quantify the effects of curvature on the lipid diffusion and sorting. Whereas varying the composition of the non-fluorescent lipids yielded minimal changes to the curvature effects, the labeling strategy of the fluorescent lipids yielded highly varying effects of curvature. Most conditions yield single-population Brownian diffusion throughout the membrane; however, curvature-induced lipid sorting, slowing, and aggregation were observed in some conditions. Head-group labeled lipids such as DPPE-Texas Red and POPE-Rhodamine diffused >2.4× slower on the curved vs. the planar membranes; tail-labeled lipids such as NBD-PPC, TopFluor-PPC, TopFluor-PIP2, DiIC 12 , and DiIC 18 displayed no significant changes in diffusion due to the membrane curvature. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo. Copyright © 2018. Published by Elsevier B.V.

Cryptic Translocation Identification in Human and Mouse using Several Telomeric Multiplex FISH (TM-FISH) Strategies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henegariu, O; Artan, S; Greally, J M

2003-08-19

Experimental data published in recent years showed that up to 10% of all cases with mild to severe idiopathic mental retardation may result from small rearrangements of the subtelomeric regions of human chromosomes. To detect such cryptic translocations, we developed a ''telomeric'' multiplex FISH assay, using a set of previously published and commercially available subtelomeric probes. This set of probes includes 41 cosmid/PAC/P1 clones located from less than 100kb to about 1 Mb from the end of the chromosomes. Similarly, a published mouse probe set, comprised of BACs hybridizing to the closest known marker toward the centromere and telomere ofmore » each mouse chromosome, was used to develop a mouse-specific ''telomeric'' M-FISH. Three different combinatorial labeling strategies were used to simultaneously detect all human sub-telomeric regions on one slide. The simplest approach uses only three fluors, and can be performed in laboratories lacking sophisticated imaging equipment or personnel highly trained in cytogenetics. A standard fluorescence microscope equipped with only three filters is sufficient. Fluor-dUTPs and labeled probes can be custom-made, thus dramatically reducing costs. Images can be prepared using generic imaging software (Adobe Photoshop), and analysis performed by simple visual inspection.« less

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

PubMed

Mayrhofer, Corina; Krieger, Sigurd; Allmaier, Günter; Kerjaschki, Dontscho

2006-01-01

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.

PubMed

Kapustin, Alexander N; Chatrou, Martijn L L; Drozdov, Ignat; Zheng, Ying; Davidson, Sean M; Soong, Daniel; Furmanik, Malgorzata; Sanchis, Pilar; De Rosales, Rafael Torres Martin; Alvarez-Hernandez, Daniel; Shroff, Rukshana; Yin, Xiaoke; Muller, Karin; Skepper, Jeremy N; Mayr, Manuel; Reutelingsperger, Chris P; Chester, Adrian; Bertazzo, Sergio; Schurgers, Leon J; Shanahan, Catherine M

2015-04-10

Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention. © 2015 American Heart Association, Inc.

Photochemical activation of MH3-B1/rGel: a HER2-targeted treatment approach for ovarian cancer

PubMed Central

Bull-Hansen, Bente; Berstad, Maria B.; Berg, Kristian; Cao, Yu; Skarpen, Ellen; Fremstedal, Ane Sofie; Rosenblum, Michael G.; Peng, Qian; Weyergang, Anette

2015-01-01

HER2-targeted therapy has been shown to have limited efficacy in ovarian cancer despite frequent overexpression of this receptor. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, currently undergoing clinical evaluation. In the present project we studied the application of PCI in combination with the HER2-targeted recombinant fusion toxin, MH3-B1/rGel, for the treatment of ovarian cancer. The SKOV-3 cell line, resistant to trastuzumab- and MH3-B1/rGel- monotherapy, was shown to respond strongly to PCI of MH3-B1/rGel to a similar extent as observed for the treatment-sensitive SK-BR-3 breast cancer cells. Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells. This was shown by the positive Pearson's correlation coefficient between Alexa488-labeled MH3-B1/rGel and Lysotracker in SKOV-3 cells in contrast to the negative Pearson's correlation coefficient in SK-BR-3 cells. The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts. Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines. The presented results warrant future development of PCI in combination with MH3-B1/rGel as a novel therapeutic approach in preclinical models of ovarian cancer. PMID:26002552

Effect of alcohol on the structure of cytochrome C: FCS and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Amin, Md. Asif; Halder, Ritaban; Ghosh, Catherine; Jana, Biman; Bhattacharyya, Kankan

2016-12-01

Effect of ethanol on the size and structure of a protein cytochrome C (Cyt C) is investigated using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. For FCS studies, Cyt C is covalently labeled with a fluorescent probe, alexa 488. FCS studies indicate that on addition of ethanol, the size of the protein varies non-monotonically. The size of Cyt C increases (i.e., the protein unfolds) on addition of alcohol (ethanol) up to a mole fraction of 0.2 (44.75% v/v) and decreases at higher alcohol concentration. In order to provide a molecular origin of this structural transition, we explore the conformational free energy landscape of Cyt C as a function of radius of gyration (Rg) at different compositions of water-ethanol binary mixture using MD simulations. Cyt C exhibits a minimum at Rg ˜ 13 Å in bulk water (0% alcohol). Upon increasing ethanol concentration, a second minimum appears in the free energy surface with gradually larger Rg up to χEtOH ˜ 0.2 (44.75% v/v). This suggests gradual unfolding of the protein. At a higher concentration of alcohol (χEtOH > 0.2), the minimum at large Rg vanishes, indicating compaction. Analysis of the contact map and the solvent organization around protein indicates a preferential solvation of the hydrophobic residues by ethanol up to χEtOH = 0.2 (44.75% v/v) and this causes the gradual unfolding of the protein. At high concentration (χEtOH = 0.3 (58% v/v)), due to structural organization in bulk water-ethanol binary mixture, the extent of preferential solvation by ethanol decreases. This causes a structural transition of Cyt C towards a more compact state.

A novel broad specificity fucosidase capable of core α1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes.

PubMed

Vainauskas, Saulius; Kirk, Charlotte H; Petralia, Laudine; Guthrie, Ellen P; McLeod, Elizabeth; Bielik, Alicia; Luebbers, Alex; Foster, Jeremy M; Hokke, Cornelis H; Rudd, Pauline M; Shi, Xiaofeng; Taron, Christopher H

2018-06-22

Exoglycosidases are often used for detailed characterization of glycan structures. Bovine kidney α-fucosidase is commonly used to determine the presence of core α1-6 fucose on N-glycans, an important modification of glycoproteins. Recently, several studies have reported that removal of core α1-6-linked fucose from N-glycans labeled with the reactive N-hydroxysuccinimide carbamate fluorescent labels 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) and RapiFluor-MS is severely impeded. We report here the cloning, expression and biochemical characterization of an α-fucosidase from Omnitrophica bacterium (termed fucosidase O). We show that fucosidase O can efficiently remove α1-6- and α1-3-linked core fucose from N-glycans. Additionally, we demonstrate that fucosidase O is able to efficiently hydrolyze core α1-6-linked fucose from N-glycans labeled with any of the existing NHS-carbamate activated fluorescent dyes.

Evaluation of four affibody-based near-infrared fluorescent probes for optical imaging of epidermal growth factor receptor positive tumors.

PubMed

Qi, Shibo; Miao, Zheng; Liu, Hongguang; Xu, Yingding; Feng, Yaqing; Cheng, Zhen

2012-06-20

The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, ZEGFR:1907, has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors. The Affibody analogue, Ac-Cys-ZEGFR:1907, was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probes-Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, SR680-ZEGFR:1907, and 800CW-ZEGFR:1907. The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (KD) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors. The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 possessed high binding affinity in low nanomolar range (43.6 ± 8.4 and 28.3 ± 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of interest (ROI) analysis of images. Ex vivo studies further demonstrated that the four probes had high tumor uptakes. Particularly, Cy5.5-ZEGFR:1907 and Alex680-ZEGFR:1907 displayed higher tumor-to-normal tissue ratios than those of the other two probes. This work demonstrates that Affibody proteins can be modified with different NIR fluorescent dyes and used for imaging of EGFR expressing tumors. Different NIR fluorescent dyes have variable impact on the in vitro binding and in vivo performance of the resulting Affibody based probes. Therefore, selection of an appropriate NIRF label is important for optical probe development. The probes developed are promising for further tumor imaging applications and clinical translation. Particularly, Alex680-ZEGFR:1907 and Cy5.5-ZEGFR:1907 are excellent candidates as EGFR-targeted probes for optical imaging.

Colorful protein-based fluorescent probes for collagen imaging.

PubMed

Aper, Stijn J A; van Spreeuwel, Ariane C C; van Turnhout, Mark C; van der Linden, Ardjan J; Pieters, Pascal A; van der Zon, Nick L L; de la Rambelje, Sander L; Bouten, Carlijn V C; Merkx, Maarten

2014-01-01

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity.

PubMed

Sindbert, Simon; Kalinin, Stanislav; Nguyen, Hien; Kienzler, Andrea; Clima, Lilia; Bannwarth, Willi; Appel, Bettina; Müller, Sabine; Seidel, Claus A M

2011-03-02

In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.

Extension of Light-Harvesting Ability of Photosynthetic Light-Harvesting Complex 2 (LH2) through Ultrafast Energy Transfer from Covalently Attached Artificial Chromophores.

PubMed

Yoneda, Yusuke; Noji, Tomoyasu; Katayama, Tetsuro; Mizutani, Naoto; Komori, Daisuke; Nango, Mamoru; Miyasaka, Hiroshi; Itoh, Shigeru; Nagasawa, Yutaka; Dewa, Takehisa

2015-10-14

Introducing appropriate artificial components into natural biological systems could enrich the original functionality. To expand the available wavelength range of photosynthetic bacterial light-harvesting complex 2 (LH2 from Rhodopseudomonas acidophila 10050), artificial fluorescent dye (Alexa Fluor 647: A647) was covalently attached to N- and C-terminal Lys residues in LH2 α-polypeptides with a molar ratio of A647/LH2 ≃ 9/1. Fluorescence and transient absorption spectroscopies revealed that intracomplex energy transfer from A647 to intrinsic chromophores of LH2 (B850) occurs in a multiexponential manner, with time constants varying from 440 fs to 23 ps through direct and B800-mediated indirect pathways. Kinetic analyses suggested that B800 chromophores mediate faster energy transfer, and the mechanism was interpretable in terms of Förster theory. This study demonstrates that a simple attachment of external chromophores with a flexible linkage can enhance the light harvesting activity of LH2 without affecting inherent functions of energy transfer, and can achieve energy transfer in the subpicosecond range. Addition of external chromophores, thus, represents a useful methodology for construction of advanced hybrid light-harvesting systems that afford solar energy in the broad spectrum.

Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis

NASA Technical Reports Server (NTRS)

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems.

Disruption of the Actin Cytoskeleton Results in the Promotion of Gravitropism in Inflorescence Stems and Hypocotyls of Arabidopsis1

PubMed Central

Yamamoto, Kazuyoshi; Kiss, John Z.

2002-01-01

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems. PMID:11842170

Image-guided intraocular injection using multimodality optical coherence tomography and fluorescence confocal scanning laser ophthalmoscopy in rodent ophthalmological models

NASA Astrophysics Data System (ADS)

Terrones, Benjamin D.; Benavides, Oscar R.; Leeburg, Kelsey C.; Mehanathan, Sankarathi B.; Levine, Edward M.; Tao, Yuankai K.

2018-02-01

Intraocular injections are routinely performed for delivery of anti-VEGF and anti-inflammatory therapies in humans. While these injections are also performed in mice to develop novel models of ophthalmic diseases and screen novel therapeutics, the injection location and volume are not well-controlled and reproducible. We overcome limitations of conventional injections methods by developing a multimodality, long working distance, non-contact optical coherence tomography (OCT) and fluorescence confocal scanning laser ophthalmoscopy (cSLO) system for retinal imaging before and after injections. Our OCT+cSLO system combines a custom-built spectraldomain OCT engine (875+/-85 nm) with 125 kHz line-rate with a modified commercial cSLO with a maximum frame-rate of 30 fps (512 x 512 pix.). The system was designed for an overlapping OCT+cSLO field-of-view of 1.1 mm with a 7.76 mm working distance to the pupil. cSLO excitation light sources and filters were optimized for simultaneous GFP and tdTomato imaging. Lateral resolution was 3.02 µm for OCT and 2.74 μm for cSLO. Intravitreal injections of 5%, 10%, and 20% intralipid with Alex Fluor 488 were manually injected intraocularly in C57BL/6 mice. Post-injection imaging showed structural changes associated with retinal puncture, including the injection track, a retinal elevation, and detachment of the posterior hyaloid. OCT enables quantitative analysis of injection location and volumes whereas complementary cSLO improves specificity for identifying fluorescently labeled injected compounds and transgenic cells. The long working distance of our non-contact OCT+cSLO system is uniquely-suited for concurrent imaging with intraocular injections and may be applied for imaging of ophthalmic surgical dynamics and real-time image-guided injections.

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

NASA Astrophysics Data System (ADS)

Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

2015-10-01

DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers. Electronic supplementary information (ESI) available: Synthesis of CdSe/CdS/ZnS core/shell/shell QDs. Sequences of primers used for amplifying the promoter regions in bisulfate-modified DNA. Comparison of detected methylation levels in different gene promoters using the QD-based FRET method versus bisulfite pyrosequencing. Methylation levels of the RASSF1A gene in one pair of NT and cancer samples as indicated by pyrosequencing. Theoretical calculation of the Förster distance R0. See DOI: 10.1039/c5nr04956c

The role of protease-activated receptor-2 on pulmonary neutrophils in the innate immune response to cockroach allergen

PubMed Central

2012-01-01

Background Serine proteases in German cockroach (GC) have been shown to mediate allergic airway inflammation through the activation of protease activated receptor (PAR)-2. Neutrophils play an important role in regulating the innate immune response, and are recruited into the airways following GC frass exposure. As such, we investigated the role of PAR-2 in airway neutrophil recruitment, activation and cytokine production following allergen exposure. Methods Wild type and PAR-2-deficient mice were administered a single intratracheal instillation of PBS or GC frass and neutrophil recruitment, expression of PAR-2, CD80, CD86, and MHC class II were assessed by flow cytometry and levels of tumor necrosis factor (TNF)α was assessed by ELISA. Uptake of AlexaFluor 405-labeled GC frass by neutrophils was performed by flow cytometry. Results Neutrophil recruitment in the lung and airways following GC frass exposure was significantly decreased in PAR-2-deficient mice compared to wild type mice. GC frass exposure increased the level of PAR-2 on pulmonary neutrophils and increased numbers of PAR-2-positive neutrophils were found in the lungs; however PAR-2 did not play a role in meditating allergen uptake. Comparing wild type and PAR-2-deficient mice, we found that a single exposure to GC frass increased levels of CD80 and CD86 on pulmonary neutrophils, an effect which was independent of PAR-2 expression. Neutrophils isolated from the whole lungs of naïve PAR-2-deficient mice treated ex vivo with GC frass produced significantly less TNFα than in similarly treated wild type neutrophils. Lastly, neutrophils were isolated from the bronchoalveolar lavage fluid of wild type and PAR-2-deficient mice following a single intratracheal exposure to GC frass. Airway neutrophils from PAR-2-deficient mice released substantially decreased levels of TNFα, suggesting a role for PAR-2 in neutrophil-derived cytokine production. Conclusions Together these data suggest PAR-2 expression can be upregulated on lung neutrophils following allergen exposure and the consequence is altered release of TNFα which could drive the early innate immune response. PMID:22954301

Human Macrophages and Dendritic Cells Can Equally Present MART-1 Antigen to CD8+ T Cells after Phagocytosis of Gamma-Irradiated Melanoma Cells

PubMed Central

Barrio, María Marcela; Abes, Riad; Colombo, Marina; Pizzurro, Gabriela; Boix, Charlotte; Roberti, María Paula; Gélizé, Emmanuelle; Rodriguez-Zubieta, Mariana

2012-01-01

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine. Other clinical trials have used IFN-gamma activated macrophage killer cells (MAK) to treat cancer patients. However, the clinical use of MAK has been based on their direct tumoricidal activity rather than on their ability to act as antigen-presenting cells to stimulate an adaptive antitumor response. Thus, in the present work, we compared the fate of MART-1 after phagocytosis of gamma-irradiated cells by clinical grade DC or MAK as well as the ability of these cells to cross present MART-1 to CD8+ T cells. Using a high affinity antibody against MART-1, 2A9, which specifically stains melanoma tumors, melanoma cell lines and normal melanocytes, the expression level of MART-1 in melanoma cell lines could be related to their ability to stimulate IFN-gamma production by a MART-1 specific HLA-A*0201-restricted CD8+ T cell clone. Confocal microscopy with Alexa Fluor®647-labelled 2A9 also showed that MART-1 could be detected in tumor cells attached and/or fused to phagocytes and even inside these cells as early as 1 h and up to 24 h or 48 h after initiation of co-cultures between gamma-irradiated melanoma cells and MAK or DC, respectively. Interestingly, MART-1 was cross-presented to MART-1 specific T cells by both MAK and DC co-cultured with melanoma gamma-irradiated cells for different time-points. Thus, naturally occurring MART-1 melanoma antigen can be taken-up from dying melanoma cells into DC or MAK and both cell types can induce specific CD8+ T cell cross-presentation thereafter. PMID:22768350

Extracellular delivery induced by ultrasound and microbubbles in cells

NASA Astrophysics Data System (ADS)

Hussein, Farah; Antonescu, Costin; Karshafian, Raffi

2017-03-01

Ultrasound and microbubble treatment (USMB) can enhance the intracellular uptake of molecules, which otherwise would be excluded from the cell, through USMB-mediated transient membrane disruption and through enhanced endocytosis. However, the effect of USMB on the outward movement of molecules from cells is not well understood. This study investigates the effects of USMB on the release of molecules from various cellular compartments including cytoplasm, lysosomes, and recycling endosomes. In vitro ARPE-19 (RPE henceforth) cells were loaded with Alexa fluor-labeled transferrin as a marker for recycling endosomes, LAMP-1 antibody was used to detect the fusion of lysosomes with the plasma membrane, GFP-transfected RPE cells were used to examine the release of GFP from the cytoplasm, and 7-AAD was used to assess cell viability. Subsequently, cells were exposed to USMB (106 cells/mL, 300 kPa peak negative pressure, 1 min treatment duration, and 20 µL/mL Definity microbubbles). Following USMB, the release of the fluorescent markers was examined at 1.5, 11.5, and 21.5 minutes from the start of USMB. The mean fluorescent intensity (MFI) of untreated and USMB treated samples were measured using flow cytometry. USMB increased the extracellular delivery of GFP molecules from the cytoplasm; the MFI in USMB treated GFP-transfected RPE cells decreased by 17% in viable cells and this MFI decreased by 70% in non-viable cells. This could be due to diffusion of GFP through the membrane disruptions induced by USMB. Additionally, the MFI of viable cells stained with LAMP-1 antibody increased by 50% and this increase was 15 folds in the non-viable cells indicating lysosome exocytosis as a mechanism for membrane repair. Furthermore, the MFI of cells loaded with fluorescent transferrin decreased by 22% after USMB treatment in viable cells, indicating a significant increase in transferrin recycling to the cell membrane. However, the increased recycling was not statistically significant in the non-viable cells. This indicates that the increase in transferrin recycling was through an active mechanism that was triggered or enhanced by USMB. It was concluded from this study that USMB enhances the release of molecules from the cytoplasm, lysosomes, and recycling endosomes.

Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate

PubMed Central

Berguig, Geoffrey Y.; Convertine, Anthony J.; Shi, Julie; Palanca-Wessels, Maria Corinna; Duvall, Craig L.; Pun, Suzie H.; Press, Oliver W.; Stayton, Patrick S.

2012-01-01

Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody-poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-releasing dynamics of the polymer-mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alex Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH-distribution of the HD39/SA-polymer conjugates were quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH values experienced by the conjugates were also characterized as a function of time by flow cytometry. PPAA was shown to strongly alter the intracellular trafficking kinetics compared to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 hours only 11% of the HD39/SA-PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast the average intracellular pH of HD39/SA alone dropped from pH 6.7 ± 0.2 at 1 hour to pH 5.6 ± 0.5 after 3 hours and pH 4.7 ± 0.6 after 6 hours. Conjugation of the control PMAA to HD39/SA showed an average pH drop similar to HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 hours, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA-PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody-polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time. PMID:23075320

Elastin Shapes Small Molecule Distribution in Lymph Node Conduits.

PubMed

Lin, Yujia; Louie, Dante; Ganguly, Anutosh; Wu, Dequan; Huang, Peng; Liao, Shan

2018-05-01

The spatial and temporal Ag distribution determines the subsequent T cell and B cell activation at the distinct anatomical locations in the lymph node (LN). It is well known that LN conduits facilitate small Ag distribution in the LN, but the mechanism of how Ags travel along LN conduits remains poorly understood. In C57BL/6J mice, using FITC as a fluorescent tracer to study lymph distribution in the LN, we found that FITC preferentially colocalized with LN capsule-associated (LNC) conduits. Images generated using a transmission electron microscope showed that LNC conduits are composed of solid collagen fibers and are wrapped with fibroblastic cells. Superresolution images revealed that high-intensity FITC is typically colocalized with elastin fibers inside the LNC conduits. Whereas tetramethylrhodamine isothiocyanate appears to enter LNC conduits as effectively as FITC, fluorescently-labeled Alexa-555-conjugated OVA labels significantly fewer LNC conduits. Importantly, injection of Alexa-555-conjugated OVA with LPS substantially increases OVA distribution along elastin fibers in LNC conduits, indicating immune stimulation is required for effective OVA traveling along elastin in LN conduits. Finally, elastin fibers preferentially surround lymphatic vessels in the skin and likely guide fluid flow to the lymphatic vessels. Our studies demonstrate that fluid or small molecules are preferentially colocalized with elastin fibers. Although the exact mechanism of how elastin fibers regulate Ag trafficking remains to be explored, our results suggest that elastin can be a potentially new target to direct Ag distribution in the LN during vaccine design. Copyright © 2018 by The American Association of Immunologists, Inc.

On-site remediation shakes out

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morris, G.D.L.

1996-01-03

Consolidation in several segments of the environmental services industry is being driven by continued sluggishness. Process engineering and construction company Fluor Daniel (Irvine, CA) has reached a definitive agreement to acquire a controlling interest in environmental remediation firm Groundwater Technology Inc.. Fluor Daniel will combine its environmental services unit with GTI. Fluor Daniel/GTI will be headquartered in Norwood and remain a publicly traded entity. The transaction is subject to approval by GTI`s shareholders and other customer conditions and is expected to be completed in the next few months. Fluor Daniel/GTI will operate as a subsidiary of Fluor Daniel and willmore » become one of the world`s largest on-site remediation companies. David Myers, president of Fluor Daniel`s environmental services unit, will become chairman, while Walter C. Barber, chairman and CEO of GTI, will continue as the company`s president and CEO. Certain Fluor Daniel environmental business components, specifically the Department of Energy management and operations business, will not be included in the transaction and will operate separately.« less

FluorMODgui V3.0: A graphic user interface for the spectral simulation of leaf and canopy chlorophyll fluorescence

NASA Astrophysics Data System (ADS)

Zarco-Tejada, P. J.; Miller, J. R.; Pedrós, R.; Verhoef, W.; Berger, M.

2006-06-01

The FluorMODgui Graphic User Interface (GUI) software package developed within the frame of the FluorMOD project Development of a Vegetation Fluorescence Canopy Model is presented in this manuscript. The FluorMOD project was launched in 2002 by the European Space Agency (ESA) to advance the science of vegetation fluorescence simulation through the development and integration of leaf and canopy fluorescence models based on physical methods. The design of airborne or space missions dedicated to the measurement of solar-induced chlorophyll fluorescence using remote-sensing instruments require physical methods for quantitative feasibility analysis and sensor specification studies. The FluorMODgui model developed as part of this project is designed to simulate the effects of chlorophyll fluorescence at leaf and canopy levels using atmospheric inputs, running the leaf model, FluorMODleaf, and the canopy model, FluorSAIL, independently, through a coupling scheme, and by a multiple iteration protocol to simulate changes in the viewing geometry and atmospheric characteristics. Inputs for the FluorMODleaf model are the number of leaf layers, chlorophyll a+ b content, water equivalent thickness, dry matter content, fluorescence quantum efficiency, temperature, species type, and stoichiometry. Inputs for the FluorSAIL canopy model are a MODTRAN-4 6-parameter spectra or measured direct horizontal irradiance and diffuse irradiance spectra, a soil reflectance spectrum, leaf reflectance & transmittance spectra and a excitation-fluorescence response matrix in upward and downward directions (all from FluorMODleaf), 2 PAR-dependent coefficients for the fluorescence response to light level, relative azimuth angle and viewing zenith angle, canopy leaf area index, leaf inclination distribution function, and a hot spot parameter. Outputs available in the 400-1000 nm spectral range from the graphical user interface, FluorMODgui, are the leaf spectral reflectance and transmittance, and the canopy reflectance, with and without fluorescence effects. In addition, solar and sky irradiance on the ground, radiance with and without fluorescence on the ground, and top-of-atmosphere (TOA) radiances for bare soil and surroundings same as target are also produced. The models and documentation regarding the FluorMOD project can be downloaded at http://www.ias.csic.es/fluormod.

Metal Ion Sensor with Catalytic DNA in a Nanofluidic Intelligent Processor

DTIC Science & Technology

2011-12-01

attributed to decreased diffusion and less active DNAzyme complex because of pore constraints. Uncleavable Alexa546 intensity is shown in gray ...is shown in gray , cleavable fluorescein in green, and the ratio of Fl/Alexa in red. Error bars represent one standard deviation of four independent...higher concentrations inhibiting cleaved fragment release. Uncleavable Alexa 546 intensity is shown in gray , cleavable fluorescein in green, and the

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone.

PubMed

Smirnova, Daria V; Samsonova, Jeanne V; Ugarova, Natalia N

2016-01-01

The sensitive BRET system for the homogeneous immunoassay of a low-molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)-the "red" mutant with λmax.em = 590 nm (RedLuc) and the "green" mutant with λmax.em = 550 nm (GreenLuc)-were tested as the donors. The water-soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase-progesterone (Luc-Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF-Ab) were developed. Both conjugates retained their functional properties, had high antigen-antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL(-1) . © 2015 The American Society of Photobiology.

Rapid multiple immunofluorescent staining for the simultaneous detection of cytokeratin and vimentin in the cytology of canine tumors.

PubMed

Sawa, Mariko; Yabuki, Akira; Kohyama, Moeko; Miyoshi, Noriaki; Yamato, Osamu

2018-06-01

Immunocytochemistry (ICC) is utilized as an advanced technique in veterinary cytology. In tumor diagnosis, cytokeratin and vimentin are markers used to distinguish the origin of tumor cells. Standard enzyme-based ICC has limitations in clinical use; and therefore, more convenient and reliable methods are needed. The purpose of this study was to develop a rapid multiple immunofluorescent (RMIF) detection method for dual cytokeratin and vimentin staining on cytology slides in dogs. Air-dried smear samples from solid tumors and sediments of pleural effusions were prepared from dogs (n = 14) that were admitted to the Veterinary Teaching Hospital, Kagoshima University, Japan. Mouse monoclonal anti-human cytokeratin (AE1/AE3) and rabbit monoclonal anti-human vimentin (SP20) antibodies were used as primary antibodies, followed by staining with Alexa Fluor-conjugated secondary antibodies. Staining using the RMIF method was compared with enzyme-based ICC staining. Rapid multiple immunofluorescent immunostaining was clear and specific in the evaluated smears, whereas the enzyme-based ICC showed nonspecific signals. By using the RMIF staining method, epithelial cells, mesenchymal cells, and mesothelial cells could be classified on a single smear of a pleural effusion. In smears of lymph nodes with epithelial tumor metastases, the RMIF method successfully detected metastatic epithelial tumor cells. The RMIF method might be a useful tool for diagnostic cytology in veterinary medicine. © 2018 American Society for Veterinary Clinical Pathology.

An aposymbiotic primary coral polyp counteracts acidification by active pH regulation

NASA Astrophysics Data System (ADS)

Ohno, Yoshikazu; Iguchi, Akira; Shinzato, Chuya; Inoue, Mayuri; Suzuki, Atsushi; Sakai, Kazuhiko; Nakamura, Takashi

2017-01-01

Corals build their skeletons using extracellular calcifying fluid located in the tissue-skeleton interface. However, the mechanism by which corals control the transport of calcium and other ions from seawater and the mechanism of constant alkalization of calcifying fluid are largely unknown. To address these questions, we performed direct pH imaging at calcification sites (subcalicoblastic medium, SCM) to visualize active pH upregulation in live aposymbiotic primary coral polyps treated with HCl-acidified seawater. Active alkalization was observed in all individuals using vital staining method while the movement of HPTS and Alexa Fluor to SCM suggests that certain ions such as H+ could diffuse via a paracellular pathway to SCM. Among them, we discovered acid-induced oscillations in the pH of SCM (pHSCM), observed in 24% of polyps examined. In addition, we discovered acid-induced pH up-regulation waves in 21% of polyps examined, which propagated among SCMs after exposure to acidified seawater. Our results showed that corals can regulate pHSCM more dynamically than was previously believed. These observations will have important implications for determining how corals regulate pHSCM during calcification. We propose that corals can sense ambient seawater pH via their innate pH-sensitive systems and regulate pHSCM using several unknown pH-regulating ion transporters that coordinate with multicellular signaling occurring in coral tissue.

Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro

PubMed Central

Wang, Guo-Qi; Li, Tong-Tong; Li, Zhi-Rui; Zhang, Li-Cheng

2016-01-01

Objective. To investigate the effect of negative pressure conditions induced by NPWT on P. aeruginosa. Methods. P. aeruginosa was cultured in a Luria–Bertani medium at negative pressure of −125 mmHg for 24 h in the experimental group and at atmospheric pressure in the control group. The diameters of the colonies of P. aeruginosa were measured after 24 h. ELISA kit, orcinol method, and elastin-Congo red assay were used to quantify the virulence factors. Biofilm formation was observed by staining with Alexa Fluor® 647 conjugate of concanavalin A (Con A). Virulence-regulated genes were determined by quantitative RT-PCR. Results. As compared with the control group, growth of P. aeruginosa was inhibited by negative pressure. The colony size under negative pressure was significantly smaller in the experimental group than that in the controls (p < 0.01). Besides, reductions in the total amount of virulence factors were observed in the negative pressure group, including exotoxin A, rhamnolipid, and elastase. RT-PCR results revealed a significant inhibition in the expression level of virulence-regulated genes. Conclusion. Negative pressure could significantly inhibit the growth of P. aeruginosa. It led to a decrease in the virulence factor secretion, biofilm formation, and a reduction in the expression level of virulence-regulated genes. PMID:28074188

Fluor Hanford, Inc. Groundwater and Technical Integration Support (Master Project) Quality Assurance Management Plan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fix, N. J.

The scope of the Fluor Hanford, Inc. Groundwater and Technical Integration Support (Master Project) is to provide technical and integration support to Fluor Hanford, Inc., including operable unit investigations at 300-FF-5 and other groundwater operable units, strategic integration, technical integration and assessments, remediation decision support, and science and technology. This Quality Assurance Management Plan provides the quality assurance requirements and processes that will be followed by the Fluor Hanford, Inc. Groundwater and Technical Integration Support (Master Project).

Proton transfer bis-benzazole fluors and their use in scintillator detectors

DOEpatents

Kauffman, Joel M.

1994-01-01

A novel class of proton transfer, bis-benzazole, fluorescent compounds, i.e., fluors, is disclosed. The novel fluors include substituted or unsubstituted 1,4-bis(2-benzazolyl)-2-hydroxybenzenes and 1,4-bis(2-benzazolyl)-2-amidobenzenes wherein the benzazolyl group may be benzoxazolyl, benzimidazolyl, benzothiazolyl, and the like. The benzazolyl groups may be substituted with one or more alkyl groups to improve solubility in organic matrix materials such as solvents, monomers, resins, polymers, and the like. The novel fluors may be used in the manufacture of fluorescent coatings, objects, scintillators, light sources and the like. The novel fluors are particularly useful for radiation-hard, solid scintillators for the detection and measurement of high energy particles and radiation.

Proton transfer bis-benzazole fluors and their use in scintillator detectors

DOEpatents

Kauffman, J.M.

1994-03-29

A novel class of proton transfer, bis-benzazole, fluorescent compounds, i.e., fluors, is disclosed. The novel fluors include substituted or unsubstituted 1,4-bis(2-benzazolyl)-2-hydroxybenzenes and 1,4-bis(2-benzazolyl)-2-amidobenzenes wherein the benzazolyl group may be benzoxazolyl, benzimidazolyl, benzothiazolyl, and the like. The benzazolyl groups may be substituted with one or more alkyl groups to improve solubility in organic matrix materials such as solvents, monomers, resins, polymers, and the like. The novel fluors may be used in the manufacture of fluorescent coatings, objects, scintillators, light sources and the like. The novel fluors are particularly useful for radiation-hard, solid scintillators for the detection and measurement of high energy particles and radiation.

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

PubMed Central

Baldo, Cristiani; Jamora, Colin; Yamanouye, Norma; Zorn, Telma M.; Moura-da-Silva, Ana M.

2010-01-01

Background Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption. Methodology/Principal Findings In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues. Conclusions/Significance These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology. PMID:20614020

Structural Heterogeneity and Quantitative FRET Efficiency Distributions of Polyprolines through a Hybrid Atomistic Simulation and Monte Carlo Approach

PubMed Central

Hoefling, Martin; Lima, Nicola; Haenni, Dominik; Seidel, Claus A. M.; Schuler, Benjamin; Grubmüller, Helmut

2011-01-01

Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies. PMID:21629703

Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles.

PubMed

Richert, Ludovic; Humbert, Nicolas; Larquet, Eric; Girerd-Chambaz, Yves; Manin, Catherine; Ronzon, Frédéric; Mély, Yves

2016-10-01

Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.

77 FR 23269 - Determination That FUNDUSCEIN-25 (fluorescein sodium injection), 25%, and AK-FLUOR (fluorescein...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-04-18

...] Determination That FUNDUSCEIN-25 (fluorescein sodium injection), 25%, and AK-FLUOR (fluorescein sodium injection... FUNDUSCEIN-25 (fluorescein sodium injection), 25%, and AK-FLUOR (fluorescein sodium injection), 25%, were not... abbreviated new drug applications (ANDAs) for fluorescein sodium injection, 25%, if all other legal and...

Office of Inspector General audit report on Fluor Daniel Fernald`s use of temporary services subcontractors

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1998-04-01

The Department of Energy (Department) and Fluor Daniel Fernald (Fluor Daniel) implemented two work force restructurings at the Fernald Environmental Management Project between Fiscal Years (FY) 1994 and 1996. During the restructurings, the Department`s cost for temporary service subcontracts increased from $2.8 million to $9.8 million annually. The objective of this audit was to determine whether Fluor Daniel utilized temporary service agreements in an economical and efficient manner and in accordance with the policy and goals of the Department`s Work Force Restructuring Program.

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat.

PubMed

Xu, Dongsheng; Cui, Jingjing; Wang, Jia; Zhang, Zhiyun; She, Chen; Bai, Wanzhu

2018-04-12

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Flow cytometry: a promising technique for the study of silicone oil-induced particulate formation in protein formulations.

PubMed

Ludwig, D Brett; Trotter, Joseph T; Gabrielson, John P; Carpenter, John F; Randolph, Theodore W

2011-03-15

Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also used to investigate the effects of silicone oil emulsions on the stability of BSA, lysozyme, abatacept, and trastuzumab formulations containing surfactant, sodium chloride, or sucrose. To aid in particle characterization, the fluorescence detection capabilities of flow cytometry were exploited by staining silicone oil with BODIPY 493/503 and model proteins with Alexa Fluor 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. The addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations. Copyright © 2010 Elsevier Inc. All rights reserved.

NIR to NIR upconversion in KYb2F7: RE3+ (RE = Tm, Er) nanoparticles for biological imaging

NASA Astrophysics Data System (ADS)

Pedraza, F.; Yust, B.; Tsin, A.; Sardar, D.

2014-03-01

Until recently, many contrast agents widely used in biological imaging have absorbed and emitted in the visible region, limiting their usefulness for deeper tissue imaging. In order to push the boundaries of deep tissue imaging with non-ionizing radiation, contrast agents in the near infrared (NIR) regime, which is not strongly absorbed or scattered by most tissues, are being sought after. Upconverting nanoparticles (UCNPs) are attractive candidates since their upconversion emission is tunable with a very narrow bandwidth and they do not photobleach or blink. The upconversion produced by the nanoparticles can be tailored for NIR to NIR by carefully choosing the lanthanide dopants and dopant ratios such as KYb2F7: RE3+ (RE = Tm, Er). Spectroscopic characterization was done by analyzing absorption, fluorescence, and quantum yield data. In order to study the toxicity of the nanoparticles Monkey Retinal Endothelial Cells (MREC) were cultivated in 24 well plates and then treated with nanoparticles at different concentrations in triplicate to obtain the optimal concentration for in vivo experiments. It will be shown that these UCNPs do not elicit a strong toxic response such as quantum dots and some noble metal nanoparticles. 3-D optical slices of nanoparticle treated fibroblast cells were imaged using a confocal microscope where the nucleus and cytoplasm were stained with DAPI and Alexa Fluor respectively. These results presented support the initial assumption, which suggests that KYb2F7: RE3+ would be excellent candidates for NIR contrast agents.

In situ roughening of polymeric microstructures.

PubMed

Shadpour, Hamed; Allbritton, Nancy L

2010-04-01

A method to perform in situ roughening of arrays of microstructures weakly adherent to an underlying substrate was presented. SU8, 1002F, and polydimethylsiloxane (PDMS) microstructures were roughened by polishing with a particle slurry. The roughness and the percentage of dislodged or damaged microstructures was evaluated as a function of the roughening time for both SU8 and 1002F structures. A maximal RMS roughness of 7-18 nm for the surfaces was obtained within 15-30 s of polishing with the slurry. This represented a 4-9 fold increase in surface roughness relative to that of the native surface. Less than 0.8% of the microstructures on the array were removed or damaged after 5 min of polishing. Native and roughened arrays were assessed for their ability to support fibronectin adhesion and cell attachment and growth. The quantity of adherent fibronectin was increased on roughened arrays by two-fold over that on native arrays. Cell adhesion to the roughened surfaces was also increased compared to native surfaces. Surface roughening with the particle slurry also improved the ability to stamp molecules onto the substrate during microcontact printing. Roughening both the PDMS stamp and substrate resulted in up to a 20-fold improvement in the transfer of BSA-Alexa Fluor 647 from the stamp to the substrate. Thus roughening of micrometer-scale surfaces with a particle slurry increased the adhesion of biomolecules as well as cells to microstructures with little to no damage to largescale arrays of the structures.

In-Situ Roughening of Polymeric Microstructures

PubMed Central

Shadpour, Hamed; Allbritton, Nancy L.

2010-01-01

A method to perform in-situ roughening of arrays of microstructures weakly adherent to an underlying substrate was presented. SU8, 1002F, and polydimethylsiloxane (PDMS) microstructures were roughened by polishing with a particle slurry. The roughness and the percentage of dislodged or damaged microstructures was evaluated as a function of the roughening time for both SU8 and 1002F structures. A maximal RMS roughness of 7-18 nm for the surfaces was obtained within 15 to 30 s of polishing with the slurry. This represented a 4-9 fold increase in surface roughness relative to that of the native surface. Less than 0.8% of the microstructures on the array were removed or damage after 5 min of polishing. Native and roughened arrays were assessed for their ability to support fibronectin adhesion and cell attachment and growth. The quantity of adherent fibronectin was increased on roughened arrays by two-fold over that on native arrays. Cell adhesion to the roughened surfaces was also increased compared to native surfaces. Surface roughening with the particle slurry also improved the ability to stamp molecules onto the substrate during microcontact printing. Roughening both the PDMS stamp and substrate resulted in up to a 20-fold improvement in the transfer of BSA-Alexa Fluor 647 from the stamp to the substrate. Thus roughening of micron-scale surfaces with a particle slurry increased the adhesion of biomolecules as well as cells to microstructures with little to no damage to large scale arrays of the structures. PMID:20423129

Cinema Catalogue.

ERIC Educational Resources Information Center

Lubovisk, Git, Ed.

This catalog lists some 14,600 books, magazines, photos and other material related to the film industry that are available from Larry Edmunds Bookshop Inc., Hollywood, Calif. The 488-page catalog is divided into eight sections. The first and largest section is labelled "Literature of the Cinema" and lists books in categories from aesthetics and…

Cone outer segment and Müller microvilli pericellular matrices provide binding domains for interphotoreceptor retinoid-binding protein (IRBP).

PubMed

Garlipp, Mary Alice; Gonzalez-Fernandez, Federico

2013-08-01

The close packing of vertebrate photoreceptors presents a challenge to the exchange of molecules between the outer segments, retinal pigmented epithelium (RPE), and Müller glia. An extracellular hyaluronan scaffold separates these cells while soluble interphotoreceptor matrix (IPM) proteins traffic visual cycle retinoids, fatty acids, and other molecules between them. In the IPM, retinoids and fatty acids are carried by interphotoreceptor retinoid-binding protein (IRBP). The fact that much of the retina's IRBP can be extracted by saline wash has led to the notion that IRBP does not bind to the retina, but freely distributes itself within the subretinal space. In this study, we challenge this idea by asking if there are specialized IPM domains that bind IRBP, perhaps facilitating its ability to target delivery/uptake of its ligands. Xenopus is an ideal animal model to study the role of the IPM in RPE-photoreceptor interactions. Here, we took advantage of the large size of its photoreceptors, ability to detach the retina in light, sustainability of the retina in short term organ culture, and the availability of recombinant full-length Xenopus IRBP and antisera directed against Xenopus IRBP. We compared the distribution of wash resistant native IRBP, and that of IRBP-Alexa 647 binding in Xenopus retina. IRBP and cone opsin were localized using anti-Xenopus IRBP serum, and monoclonal COS-1 respectively. Cone matrix sheath proteoglycans were localized with wheat germ agglutinin (WGA), and diffuse IPM proteoglycans with peanut agglutinin (PNA). Wholemounts and frozen sections were compared by immunofluorescence from retinas detached under Ringer's followed by additional washes, or detached directly under 4% paraformaldehyde without Ringer's wash. Undetached Lowicryl embedded retinas were subjected to IRBP immunogold electron microscopy (EM). Immunogold labeled a diffuse network of filamentous structures, and a separate distinct flocculant material directly coating the outer segments, filling the rod periciliary ridge, and associated with Müller microvilli. By immunofluorescence, Ringer's wash removed most of the diffuse IRBP, but not that coating the outer segments. IRBP-Alexa 647 bound to the cone outer segments and Müller villi region, and comparably less to rod outer segments. Co-incubation with unlabeled IRBP markedly reduced this binding; ovalbumin-Alexa 647 and Alexa 647 dye alone showed no binding. Our data suggest that the pericellular matrix of the cone outer segments and Müller microvilli provide specialized domains that facilitate IRBP's functions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

NASA Astrophysics Data System (ADS)

Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary; Benkovic, Stephen J.; Hammes, Gordon G.

2002-10-01

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Mid-Infrared Fiber Lasers (Les fibres laser infrarouge moyen)

DTIC Science & Technology

2010-09-01

Marcel Poulain, Université de Rennes / Le Verre Fluoré, France Fluoride Fiber Sources: Problems and Prospects Prof. Marcel Poulain from Rennes...University and Le Verre Fluoré, France, presented the problems and prospects of fluoride glass fiber sources. After some comments on pioneering...wavelength, etc. can be adjusted. Le Verre Fluoré has developed a commercialized supercontinuum source emitting from ∼0.7 µm to ∼4 µm Issues concerning

Design Compliance Matrices to ANSI and OSHA

DOE Office of Scientific and Technical Information (OSTI.GOV)

BENDIXSEN, R.B.

2000-04-03

U.S. Department of Energy Letter 98-SFD-028 requested Fluor Daniel Hanford, Inc. to provide clarifications as to compliance with ANSI 57.1, 57.2, 57.9, and 29 CFR 1910.179 (OSHA), in the form of an item-by-item compliance matrix, for the CSB. This Supporting Document contains Fluor Daniel, Inc.'s response for use by Fluor Daniel Hanford, Inc. regarding the clarifications requested by the U.S. Department of Energy.

NAVIGATING A QUALITY ROUTE TO A NATIONAL SAFETY AWARD

DOE Office of Scientific and Technical Information (OSTI.GOV)

PREVETTE SS

Deming quality methodologies applied to safety are recognized with the National Safety Council's annual Robert W. Campbell Award. Over the last ten years, the implementation of Statistical Process Control and quality methodologies at the U.S. Department of Energy's Hanford Site have contributed to improved safety. Improvements attributed to Statistical Process Control are evidenced in Occupational Safety and Health records and documented through several articles in Quality Progress and the American Society of Safety Engineers publication, Professional Safety. Statistical trending of safety, quality, and occurrence data continues to playa key role in improving safety and quality at what has been calledmore » the world's largest environmental cleanup project. DOE's Hanford Site played a pivotal role in the nation's defense beginning in the 1940s, when it was established as part of the Manhattan Project. After more than 50 years of producing material for nuclear weapons, Hanford, which covers 586 square miles in southeastern Washington state, is now focused on three outcomes: (1) Restoring the Columbia River corridor for multiple uses; (2) Transitioning the central plateau to support long-term waste management; and (3) Putting DOE assets to work for the future. The current environmental cleanup mission faces challenges of overlapping technical, political, regulatory, environmental, and cultural interests. From Oct. 1, 1996 through Sept. 30, 2008, Fluor Hanford was a prime contractor to the Department of Energy's Richland Operations Office. In this role, Fluor Hanford managed several major cleanup activities that included dismantling former nuclear-processing facilities, cleaning up the Site's contaminated groundwater, retrieving and processing transuranic waste for shipment and disposal off-site, maintaining the Site's infrastructure, providing security and fire protection, and operating the Volpentest HAMMER Training and Education Center. On October 1,2008, a transition occurred that changed Fluor's role at Hanford. Fluor's work at Hanford was split in two with the technical scope being assumed by the CH2M HILL Plateau Remediation Company (CHPRC) CHPRC is now spearheading much of the cleanup work associated with former nuclear-processing facilities, contaminated groundwater, and transuranic waste. Fluor is an integrated subcontractor to CH PRC in this effort. In addition, at the time of this writing, while the final outcome is being determined for the new Mission Support Contract, Fluor Hanford has had its contract extended to provide site-wide services that include security, fire protection, infrastructure, and operating the HAMMER facility. The emphasis has to be on doing work safely, delivering quality work, controlling costs, and meeting deadlines. Statistical support is provided by Fluor to the PRC, within Fluor Hanford, and to a third contractor, Washington Closure Hanford, which is tasked with cleaning up approximately 210 square miles designated as the Columbia River corridor along the outer edge of the Hanford Site. The closing months of Fluor Hanford's 12 year contract were busy, characterized by special events that capped its work as a prime cleanup contractor, transitions of work scope and personnel, and the completion numerous activities. At this time, Fluor's work and approach to safety were featured in state and national forums. A 'Blockbuster' presentation at the Washington State Governor's Industrial Safety Conference in September 2008 featured Fluor Hanford's Chief Operating Officer, a company Safety Representative, and me. Simultaneously, an award ceremony in Anaheim, Calif. recognized Fluor Hanford as the winner of the 2008 Robert W. Campbell Award. The Robert W. Campbell Award is co-sponsored by Exxon Mobil Corporation and the National Safety Council. Named after a pioneer of industrial safety, the Campbell Award recognizes organizations that demonstrate how integration of environmental, health and safety (EHS) management into business operations is a cornerstone of their corporate success. Fluor Hanford received the award for corporations with more than 1,000 employees. Campbell Award winners undergo rigorous assessments that include site visits and comprehensive evaluations of their commitment to, and implementation of, EHS practices. Award winners work with an international partnership of 21 organizations to develop case studies that illustrate their superior EHS programs and best practices, for use by top business and engineering schools worldwide. Quality methodologies in place at Fluor Hanford played a key role in the award process. Fluor Hanford's integrated use of Statistical Process Control and Pareto Charts for analyzing and displaying EHS performance were viewed favorably by the award judges.« less

Comparison of Measurements and FluorMOD Simulations for Solar Induced Chlorophyll Fluorescence and Reflectance of a Corn Crop under Nitrogen Treatments [SIF and Reflectance for Corn

NASA Technical Reports Server (NTRS)

Middleton, Elizabeth M.; Corp, Lawrence A.; Campbell, Petya K. E.

2007-01-01

The FLuorescence Explorer (FLEX) satellite concept is one of six semifinalist mission proposals selected in 2006 for pre-Phase studies by the European Space Agency (ESA). The FLEX concept proposes to measure passive solar induced chlorophyll fluorescence (SIF) of terrestrial ecosystems. A new spectral vegetation Fluorescence Model (FluorMOD) was developed to include the effects of steady state SIF on canopy reflectance. We used our laboratory and field measurements previously acquired from foliage and canopies of corn (Zea mays L.) under controlled nitrogen (N) fertilization to parameterize and evaluate FluorMOD. Our data included biophysical properties, fluorescence (F) and reflectance spectra for leaves; reflectance spectra of canopies and soil; solar irradiance; plot-level leaf area index; and canopy SIF emissions determined using the Fraunhofer Line Depth principal for the atmospheric telluric oxygen absorption features at 688 nm (O2-beta) and 760 nm (O2-alpha). FluorMOD simulations implemented in the default "look-up-table" mode did not reproduce the observed magnitudes of leaf F, canopy SIF, or canopy reflectance. However, simulations for all of these parameters agreed with observations when the default FluorMOD information was replaced with measurements, although N treatment responses were underestimated. Recommendations were provided to enhance FluorMOD's potential utility in support of SIF field experiments and studies of agriculture and ecosystems.

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT

PubMed Central

Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun

2017-01-01

Background Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. Methodology/principal findings The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). Conclusions/significance The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV. PMID:29267277

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

PubMed

Um, Jihye; Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun; Park, Jun-Sun; Kim, Su Yeon

2017-12-01

Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

Health information on internet: quality, importance, and popularity of persian health websites.

PubMed

Samadbeik, Mahnaz; Ahmadi, Maryam; Mohammadi, Ali; Mohseni Saravi, Beniamin

2014-04-01

The Internet has provided great opportunities for disseminating both accurate and inaccurate health information. Therefore, the quality of information is considered as a widespread concern affecting the human life. Despite the increasingly substantial growth in the number of users, Persian health websites and the proportion of internet-using patients, little is known about the quality of Persian medical and health websites. The current study aimed to first assess the quality, popularity and importance of websites providing Persian health-related information, and second to evaluate the correlation of the popularity and importance ranking with quality score on the Internet. The sample websites were identified by entering the health-related keywords into four most popular search engines of Iranian users based on the Alexa ranking at the time of study. Each selected website was assessed using three qualified tools including the Bomba and Land Index, Google PageRank and the Alexa ranking. The evaluated sites characteristics (ownership structure, database, scope and objective) really did not have an effect on the Alexa traffic global rank, Alexa traffic rank in Iran, Google PageRank and Bomba total score. Most websites (78.9 percent, n = 56) were in the moderate category (8 ≤ x ≤ 11.99) based on their quality levels. There was no statistically significant association between Google PageRank with Bomba index variables and Alexa traffic global rank (P > 0.05). The Persian health websites had better Bomba quality scores in availability and usability guidelines as compared to other guidelines. The Google PageRank did not properly reflect the real quality of evaluated websites and Internet users seeking online health information should not merely rely on it for any kind of prejudgment regarding Persian health websites. However, they can use Iran Alexa rank as a primary filtering tool of these websites. Therefore, designing search engines dedicated to explore accredited Persian health-related Web sites can be an effective method to access high-quality Persian health websites.

Determination of lymph flow in murine oral mucosa using depot clearance of near-infrared-labeled albumin.

PubMed

Papadakou, Panagiota; Karlsen, Tine Veronica; Wiig, Helge; Berggreen, Ellen

2015-10-01

The lymphatic vessels are playing an important role in inflammation since they return extravasated fluid, proteins, and cells back into the circulation and regulate immune cell trafficking. The oral mucosa, including gingiva, is well supplied with lymphatic vessels and is frequently challenged with inflammatory insults. Lymphatic vessels in gingiva protect against periodontal disease development, but quantification of lymph flow in this area has so far never been performed, due to lack of reliable methods. Mice of FVB strain (n=17) were anesthetized with isoflurane and placed on a jaw retraction board allowing the mouth to be kept open and stable. Albumin conjugated with Alexa680-fluorochrome (with or without LPS from Porphyromonas gingivalis) was injected superficially in oral mucosa mesio-buccal to the left first molar in each mouse. 60 min post-injection the mouse was transferred to an OptixMX3 optical imager where the total fluorescence was measured in the posterior facial area. The measurements continued further every 60 min for 7h for each mouse. The mice were awake and active between measurements. The in vivo washout of Alexa680-albumin was calculated using the natural logarithm of the relative values creating a negative slope for each mouse. Statistical analysis of variance was performed. The injection and distribution site for tracer was verified with India ink and shown to be in the interstitium below the oral mucosal epithelium, in an area well supplied with initial lymphatic vessels. Washout of the tracer Alexa680-albumin was log-linear, and the basal lymph flow calculated from depot clearance averaged -0.28 ± 0.08%/min (n=8). The clearance was significantly faster (-0.30 ± 0.08%/min, n=9) in acutely inflamed oral mucosa (p=0.0326). We developed a method that can successfully quantify the lymph flow in oral mucosa in steady state conditions and under acute perturbation. By use of this method, new information about the lymphatic function in oral mucosa during physiological and pathological conditions can be achieved. Copyright © 2015 Elsevier B.V. All rights reserved.

Sliding enhances fluid and solute transport into buried articular cartilage contacts.

PubMed

Graham, B T; Moore, A C; Burris, D L; Price, C

2017-12-01

Solutes and interstitial water are naturally transported from cartilage by load-induced interstitial fluid pressures. Fluid and solute recovery during joint articulation have been primarily attributed to passive diffusion and mechanical 'pumping' from dynamic loading. This paper tests if the sliding action of articulation is a significant and independent driver of fluid and solute transport in cartilage. The large osteochondral samples utilized in the present study preserve the convergent wedges necessary for physiological hydrodynamics. Following static load-induced fluid exudation and prior to sliding, a fluorescent solute (AlexaFluor 633) was added to the lubricant bath. In situ confocal microscopy was used to quantify the transport of solute from the bath into the buried stationary contact area (SCA) during sliding. Following static exudation, significant reductions in friction and strain during sliding at 60 mm/s were accompanied by significant solute transport into the inaccessible center of the buried contact; no such transport was detected for the 0- or 1 mm/s sliding conditions. The results suggest that external hydrodynamic pressures from sliding induced advective flows that carried solutes from the bath toward the center of contact. These results provide the first direct evidence that the action of sliding is a significant contributor to fluid and solute recovery by cartilage. Furthermore, they indicate that the sliding-induced transport of solutes into the buried interface was orders of magnitude greater than that attributable to diffusion alone, a result with critical implications for disease prevention and tissue engineering. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Integrated smartphone imaging of quantum dot photoluminescence and Förster resonance energy transfer

NASA Astrophysics Data System (ADS)

Petryayeva, Eleonora; Algar, W. Russ

2015-06-01

Smartphones and other mobile devices are emerging as promising analytical platforms for point-of-care diagnostics, particularly when combined with nanotechnology. For example, we have shown that the optical properties of semiconductor quantum dots (QDs) are well suited to photoluminescence (PL) detection with a smartphone camera. However, this previous work has utilized an external excitation source for interrogation of QD PL. In this proceeding, we demonstrate that the white-light LED photographic flashes built into smartphones can be optically filtered to yield blue light suitable for excitation of QD PL. Measurements were made by recording video with filtered flash illumination and averaging the frames of the video to obtain images with good signal-to-background ratios. These images permitted detection of green-emitting and red-emitting QDs at levels comparable to those possible with excitation using an external long-wave UV lamp. The optical properties of QDs proved to be uniquely suited to smartphone PL imaging, exhibiting emission that was 1-2 orders magnitude brighter than that of common fluorescent dyes under the same conditions. Excitation with the smartphone flash was also suitable for imaging of FRET between green-emitting QD donors and Alexa Fluor 555 (A555) fluorescent dye acceptors. No significant difference in FRET imaging capability was observed between excitation with the smartphone flash and a long-wave UV lamp. Although the smartphone flash did have some disadvantages compared to an external UV lamp, these disadvantages are potentially offset by the benefit of having excitation and detection integrated into the smartphone.

Properties of coatings on RFID p-Chips that support plasmonic fluorescence enhancement in bioassays

PubMed Central

Rich, Ryan; Li, Ji; Fudala, Rafal; Gryczynski, Zygmunt; Gryczynski, Ignacy; Mandecki, Wlodek

2012-01-01

Microtransponders (RFID p-Chips) derivatized with silver island film (SIF) have previously seen success as a platform for the quantification of low-abundance biomolecules in nucleic acid-based assays and immunoassays. In this study, we further characterized the morphology of the SIF as well as the polymer matrix enveloping it by scanning electron microscopy (SEM). The polymer was a two-layer silane-based matrix engulfing the p-Chip and SIF. Through a series of SEM and confocal fluorescence microscopy experiments we found the depth of the polymer matrix to be 1–2 µm. The radiative effects of the SIF/polymer layer were assessed by fluorescence lifetime imaging (FLIM) of p-Chips coated with the polymer to which a fluorophore (Alexa Fluor 555) was conjugated. FLIM images showed an 8.7-fold increase in fluorescence intensity and an increased rate of radiative decay, the latter of which is associated with improved photostability and both of which are linked to plasmonic enhancement by the SIF. Plasmonic enhancement was found to extend uniformly across the p-Chip and, interestingly, to a depth of about 1.2 µm. The substantial depth of enhancement suggests that the SIF/polymer layer constitutes a three-dimensional matrix that is accessible to solvent and small molecules such as fluorescent dyes. Finally, we confirmed that no surface-enhanced Raman scattering (SERS) is seen from the SIF/polymer combination. The analysis provides a possible mechanism by which the SIF/polymer-coated p-Chips allow a highly sensitive immunoassay and, as a result, leads to an improved bioassay platform. PMID:22960796

Characterization of a novel ultra low refractive index material for biosensor application

PubMed Central

Memisevic, Jasenka; Korampally, Venumadhav; Gangopadhyay, Shubhra; Grant, Sheila A.

2009-01-01

Nanoporous materials can provide significant benefits to the field of biosensors. Their size and porous structure makes them an ideal tool for improving sensor performance. This study characterized a novel ultra low index of refraction nanoporous organosilicate (NPO) material for use as an optical platform for fluorescence-based optical biosensors. While serving as the low index cladding material, the novel coating based on organosilicate nanoparticles also provides an opportunity for a high surface area coating that can be utilized for immobilizing biological probes. Biological molecules were immobilized onto NPO, which was spin-coated on silicon and glass substrates. The biological molecule was composed of Protein A conjugated to AlexaFluor 546 fluorophore and then immobilized onto the NPO substrate via silanization. Sample analysis consisted of spectrofluorometry, FT-IR spectroscopy, scanning electron microscopy, contact angle measurement and ellipsometry. The results showed the presence of emission peaks at 574 nm, indicating that the immobilization of Protein A to the NPO material is possible. When compared to Si and glass substrates not coated with NPO, the results showed a 100X and 10X increase in packing density with the NPO coated films respectively. Ellipsometric analysis, FT-IR, contact angle, and SEM imaging of the surface immobilized NPO films suggested that while the surface modifications did induce some damage, it did not incur significant changes to its unique characteristics, i.e., pore structure, wettability and index of refraction. It was concluded that NPO films would be a viable sensor substrate to enhance sensitivity and improve sensor performance. PMID:20161155

Novel fluorescence nanobubbles for contrast-enhanced ultrasound imaging in rabbit VX2 hepatocellular carcinoma model

NASA Astrophysics Data System (ADS)

Yu, Houqiang; Wang, Wei; He, Xiaoling; Zhou, Qibing; Ding, Mingyue

2017-03-01

Ultrasound contrast agents (UCAs) such as SonoVue or Optison have been used widely in clinic for contrast-enhanced vascular imaging. However, microbubbles UCAs display limitations in tumor-targeted imaging due to the large sizes, nanoscaled UCAs has consequently attracted increasing attentions. In this work, we synthesized nanobubbles (NBs) by ultrasonic cavitation method, then a fluorescent marker of Alexa Fluor 680 was conjugated to the shell in order to observe the localization of NBs in tumor tissue. Measurement of fundamental characteristics showed that the NBs had homogeneous distribution of mean diameter of 267.9 +/- 19.2 nm and polydispersity index of 0.410 +/- 0.056. To assess in vivo tumor-selectivity of NBs, we established the rabbits VX2 hepatocellular carcinoma model though surgical implantation method. After the rabbits were intravenous administered of NBs, contrast-enhanced sonograms was observed in the surrounding of VX2 tumor, which showed there are rich capillaries in the tumor periphery. We additionally investigated the toxic of the NBs by hematoxylin-eosin staining. The results indicated that the NBs is a biocompatible non-toxic lipid system. Furthermore, the VX2 tumors and major organs were analyzed using ex vivo fluorescence imaging to confirm the targeted selectivity of NBs, and the results verified that the NBs were capable of targeting VX2 tumor. Confocal laser scanning microscopy examination showed that the NBs can traverse the VX2 tumor capillaries and target to the hepatocellular carcinoma tumor cells. All these results suggested that the newly prepared NBs have a potential application in molecular imaging and tumor-targeting therapy.

Terbutaline causes immobilization of single β2-adrenergic receptor-ligand complexes in the plasma membrane of living A549 cells as revealed by single-molecule microscopy

NASA Astrophysics Data System (ADS)

Sieben, Anne; Kaminski, Tim; Kubitscheck, Ulrich; Häberlein, Hanns

2011-02-01

G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β2-adrenergic receptors (β2AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β2ARs are constitutively immobile. About 2/3 of the β2ARs moved with a diffusion constant of D2 = 0.03+/-0.001 μm2/s and about 17% were diffusing five-fold faster (D3 = 0.15+/-0.02 μm2/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ1 = 77+/-1 ms and τ2 = 388+/-11 ms. Agonistic stimulation of the β2AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β2AR mobility suggesting that different receptor dynamics characterize different receptor states.

34. DOOR AND WINDOW DETAILS. INEEL DRAWING NUMBER 200063300287106358. FLUOR ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

34. DOOR AND WINDOW DETAILS. INEEL DRAWING NUMBER 200-0633-00-287-106358. FLUOR NUMBER 5775-CPP-633-A-8. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

33. ROOF PLAN AND DETAILS. INEEL DRAWING NUMBER 200063300287106357. FLUOR ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

33. ROOF PLAN AND DETAILS. INEEL DRAWING NUMBER 200-0633-00-287-106357. FLUOR NUMBER 5775-CPP-633-A-7. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

PubMed

Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

2016-02-15

Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated following U50,488H and etorphine respectively. Thus KOPR internalization requires receptor phosphorylation above a certain threshold, and higher-order KOPR phosphorylation may be disproportionally important. © 2016 Authors; published by Portland Press Limited.

Using Intelligent Personal Assistants for Second Language Learning: A Case Study of Alexa

ERIC Educational Resources Information Center

Dizon, Gilbert

2017-01-01

The proliferation of smartphones has given rise to intelligent personal assistants (IPAs), software that helps users accomplish day-to-day tasks. However, little is known about IPAs in the context of second language (L2) learning. Therefore, the primary objectives of this case study were twofold: to assess the ability of Amazon's IPA, Alexa, to…

Fluor Daniel Hanford contract standards/requirements identification document

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennett, G.L.

1997-04-24

This document, the Standards/Requirements Identification Document (S/RID) for the Fluor Daniel Hanford Contract, represents the necessary and sufficient requirements to provide an adequate level of protection of the worker, public health and safety, and the environment.

Kappa Opioid Receptor Activation of p38 MAPK Is GRK3- and Arrestin-dependent in Neurons and Astrocytes*

PubMed Central

Bruchas, Michael R.; Macey, Tara A.; Lowe, Janet D.; Chavkin, Charles

2007-01-01

AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (γ-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism. PMID:16648139

Calcium transport into the cells of the sea urchin larva in relation to spicule formation

PubMed Central

Vidavsky, Netta; Addadi, Sefi; Schertel, Andreas; Ben-Ezra, David; Shpigel, Muki; Addadi, Lia; Weiner, Steve

2016-01-01

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium. PMID:27791140

Calcium transport into the cells of the sea urchin larva in relation to spicule formation.

PubMed

Vidavsky, Netta; Addadi, Sefi; Schertel, Andreas; Ben-Ezra, David; Shpigel, Muki; Addadi, Lia; Weiner, Steve

2016-10-24

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.

Real-time water and wastewater quality monitoring using LED-based fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Bridgeman, John; Zakharova, Yulia

2016-04-01

In recent years there have been a number of attempts to design and introduce into water management tools that are capable of measuring organic and microbial matter in real time and in situ. This is important, as the delivery of safe water to customers, and the discharge of good quality effluent to rivers are primary concerns to water undertakers. A novel, LED-based portable fluorimeter 'Duo Fluor' has been designed and constructed at the University of Birmingham to monitor the quality of (waste)water continuously and in real time, and its performance has been assessed in a range of environments. To be of use across a range of environments, special attention must be paid to two crucially important characteristics of such instruments, i.e. their sensitivity and robustness. Thus, the objectives of this study were: 1. To compare the performance (in terms of their sensitivity and robustness) of the Duo Fluor and two other commercial fluorescence devices in laboratory conditions. 2. To assess the performance of the Duo Fluor in situ, in real time at a 450,000PE WwTW. Initially, the impact of quinine sulphate (QS), a highly fluorescent alkaloid with high quantum fluorescence yield, on peak T fluorescence in environmental waters was examined for the Duo Fluor and two commercially available, chamber-based fluorimeters, (F1) and (F2). The instruments' responses to three scenarios were assessed: 1. Deionised water (DW) spiked with QS (from 0.05 to 0.4 mg/L); 2. Environmental water (pond water, PW) spiked with QS (from 0.05 to 0.4 mg/L); 3. Different water samples from various environmental source. The results show that the facility to amend gain settings and the suitable choice of gain are crucial to obtaining reliable data on both peaks T and C in a wide range of water types. The Duo Fluor offers both of these advantages whilst commercially available instruments currently do not. The Duo Fluor was subsequently fixed at the final effluent (FE) discharge point of a WwTW and FE was pumped into the cuvette. The instruments, along with other sensors, were checked twice a week. The Duo Fluor successfully collected data continuously from 10th August 2015 to 7th September 2015 (~500 hrs). Data analysis showed that the Duo Fluor demonstrated very good correlation with the other instruments; albeit, some commercially available instruments measure only peak T or peak C, whereas Duo Fluor measures both. The results also identified rainfall events in the catchment, which manifested themselves via decreases in both peak C and peak, suggesting that a dilution of organic and microbial matter had occurred.

Health Information on Internet: Quality, Importance, and Popularity of Persian Health Websites

PubMed Central

Samadbeik, Mahnaz; Ahmadi, Maryam; Mohammadi, Ali; Mohseni Saravi, Beniamin

2014-01-01

Background: The Internet has provided great opportunities for disseminating both accurate and inaccurate health information. Therefore, the quality of information is considered as a widespread concern affecting the human life. Despite the increasingly substantial growth in the number of users, Persian health websites and the proportion of internet-using patients, little is known about the quality of Persian medical and health websites. Objectives: The current study aimed to first assess the quality, popularity and importance of websites providing Persian health-related information, and second to evaluate the correlation of the popularity and importance ranking with quality score on the Internet. Materials and Methods: The sample websites were identified by entering the health-related keywords into four most popular search engines of Iranian users based on the Alexa ranking at the time of study. Each selected website was assessed using three qualified tools including the Bomba and Land Index, Google PageRank and the Alexa ranking. Results: The evaluated sites characteristics (ownership structure, database, scope and objective) really did not have an effect on the Alexa traffic global rank, Alexa traffic rank in Iran, Google PageRank and Bomba total score. Most websites (78.9 percent, n = 56) were in the moderate category (8 ≤ x ≤ 11.99) based on their quality levels. There was no statistically significant association between Google PageRank with Bomba index variables and Alexa traffic global rank (P > 0.05). Conclusions: The Persian health websites had better Bomba quality scores in availability and usability guidelines as compared to other guidelines. The Google PageRank did not properly reflect the real quality of evaluated websites and Internet users seeking online health information should not merely rely on it for any kind of prejudgment regarding Persian health websites. However, they can use Iran Alexa rank as a primary filtering tool of these websites. Therefore, designing search engines dedicated to explore accredited Persian health-related Web sites can be an effective method to access high-quality Persian health websites. PMID:24910795

Lithium-bearing fluor-arfvedsonite from Hurricane Mountain, New Hampshire: A crystal-chemical study

USGS Publications Warehouse

Hawthorne, F.C.; Oberti, R.; Ottolini, L.; Foord, E.E.

1996-01-01

The structures of two crystals of Li-bearing fluor-arfvedsonite (1) (K0.32Na0.68)Na2(Li0.48Fe 2+2.83Mn2+0.10Zn 0.06Fe3+1.46Ti0.07) (Si7.88Al0.12)O22[Fu1.15(OH) 0.85] and (2) (K0.25Na0.75)Na2(Li0.48Fe 2+2.84Mn2+0.11Zn 0.05Fe3+1.45Ti0.07)(Si 7.89Al0.11)O22[F1.35(OH) 0.65] from a granitic pegmatite, Hurricane Mountain, New Hampshire, have been refined to R indices of 1.5(1.6)% based on 1380(1387) reflections measured with MoK?? X-radiation. The unit cell parameters are (1) a 9.838(4), b 17.991(6), c 5.315(2) A??, 103.78(3)??, V 913.7 A??3 and (2) a 9.832(3), b 17.990(7), c 5.316(3) A??, ?? 103.79(3)??, V 913.2 A??3. Site-scattering refinement shows Li to be completely ordered at the M(3) site in these crystals. The amphibole composition is intermediate between fluor-arfvedsonite and fluor-ferro-leakeite with a small component (???10%) of fluor-ferro-ferri-nybo??ite. These amphibole crystals project into miarolitic cavities in a pegmatitic phase of a riebeckite granite. The early-crystallizing amphibole is close to fluor-ferro-leakeite in composition, but becomes progressively depleted in Li and F as crystals project out into miarolitic cavities; the final amphibole to crystallize is a fibrous Li-poor riebeckite. Li plays a significant role in late-stage fractionation involving the crystallization of alkali amphibole in peralkaline granitic environments.

Office of Inspector General audit report on Project Hanford management contract costs and performance

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1998-11-01

On August 6, 1996, the Richland Operations Office (Richland) awarded the Project Hanford Management Contract (Management Contract) to Fluor Daniel Hanford, Inc. (Fluor Daniel). This performance-based, 5-year contract to support cleanup of the Department of Energy`s (DOE) Hanford Site (Hanford) contained performance goals or expectations related to the stabilization, transition, and diversification of the Tri-Cities` economy near Hanford in southeastern Washington. One of these economic goals was that Fluor Daniel and its major subcontractors would help generate 3,000 new, non-Hanford, private sector jobs that would help stabilize and diversify the Tri-Cities` economy. The contract specifically called for Fluor Daniel tomore » help generate 200 jobs, establish an investment fund, and bring 6 new growth-oriented enterprise companies to the Tri-Cities by the end of Fiscal Year (FY) 1997. The objective of the audit was to determine whether Richland was making adequate progress in stabilizing and diversifying the economy of the Tri-Cities by creating 3,000 new, non-Hanford jobs within 5 years. Accordingly, the author examined the progress made in FY 1997, which was the first year of the Management Contract. Richland and Fluor Daniel are at risk of not meeting the Management Contract`s goals of stabilizing and diversifying the economy of the Tri-Cities because most of the new jobs created during FY 1997 were not comparable to Hanford jobs and, thus, may not sustain long-term economic goals. Therefore, Fluor Daniel has not met its expectations in the first year and is not making adequate progress toward meeting the Management Contract`s overall economic goals.« less

CHARA/FLUOR updates and performance

NASA Astrophysics Data System (ADS)

Mérand, Antoine; Coudé du Foresto, Vincent; Kellerer, Aglaé; ten Brummelaar, Theo; Reess, Jean-Michel; Ziegler, Denis

2006-06-01

In 2002, the Fiber Linked Unit for Optical Recombination (FLUOR) has been moved from the Infrared Optical Telescope Array (IOTA) to the CHARA Array. We present here the main upgrades that followed the installation, the new features installed, including spectral dispersion, and the current capabilities of the instrument.

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

PubMed

Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

2004-12-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

PubMed Central

Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

2004-01-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

Human serum albumin hydropersulfide is a potent reactive oxygen species scavenger in oxidative stress conditions such as chronic kidney disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shibata, Akitomo; Ishima, Yu; Ikeda, Mayumi

Recently, hydropersulfide (RSSH) was found to exist in mammalian tissues and fluids. Cysteine hydropersulfide can be found in free cysteine residues as well as in proteins, and it has potent antioxidative activity. Human serum albumin (HSA) is the most abundant protein in mammalian serum. HSA possesses a free thiol group in Cys-34 that could be a site for hydropersulfide formation. HSA hydropersulfide of high purity as a positive control was prepared by treatment of HSA with Na{sub 2}S. The presence of HSA hydropersulfide was confirmed by spectroscopy and ESI-TOFMS analysis where molecular weights of HSA hydropersulfide by increments of approximatelymore » 32 Da (Sulfur atom) were detected. The fluorescent probe results showed that Alexa Fluor 680 conjugated maleimide (Red-Mal) was a suitable assay and bromotrimethylammoniumbimane bromide appeared to be a selective reagent for hydropersulfide. The effect of oxidative stress related disease on the existence of albumin hydropersulfides was examined in rat 5/6 nephrectomy model of chronic kidney disease (CKD). Interestingly, the level of hydropersulfides in rat 5/6 nephrectomy model serum was decreased by a uremic toxin that increases oxidative stress in rat 5/6 nephrectomy model. Furthermore, we demonstrated that the levels of HSA hydropersulfide in human subjects were reduced in CKD but restored by hemodialysis using Red-Mal assay. We conclude that HSA hydropersulfide could potentially play an important role in biological anti-oxidative defense, and it is a promising diagnostic and therapeutic marker of oxidative diseases. - Highlights: • Hydropersulfide can behave as potent antioxidants. • We firstly detected human serum albumin hydropersulfide in healthy subjects. • Human serum albumin hydropersulfide in human subjects were reduced in chronic kidney disease but restored by hemodialysis.« less

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

PubMed

Carulli, Giovanni; Marini, Alessandra; Sammuri, Paola; Domenichini, Cristiana; Ottaviano, Virginia; Pacini, Simone; Petrini, Mario

2015-01-01

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

Compact, integrable, and long life time Raman multiline UV-Vis source based on hypocycloid core Kagome HC-PCF

NASA Astrophysics Data System (ADS)

Chafer, M.; Lekiefs, Q.; Gorse, A.; Beaudou, B.; Debord, B.; Gérôme, F.; Benabid, F.

2017-02-01

Raman-gas filled HC-PCF has proved to be an outstanding Raman-convertor, as illustrated by the generation of more than 5 octaves wide Raman comb using a hydrogen-filled Kagome HC-PCF pumped with high power picosecond-laser, or the generation of multiline Raman-source in the UV-Vis using a very compact system pumped with micro-chip laser. Whilst these demonstrations are promising, a principal challenge for the industrialization of such a Raman source is its lifetime as the H2 diffusion through silica is high enough to leak out from the fiber within only a few months. Here, we report on a HC-PCF based Raman multiline source with a very long life-span. The system consists of hydrogen filled ultra-low loss HC-PCF contained in highly sealed box, coined CombBox, and pumped with a 532 nm micro-chip laser. This combination is a turnkey multiline Raman-source with a "shoe box" size. The CombBox is a robust and compact component that can be integrated and pumped with any common pulsed laser. When pumped with a 32 mW average power and 1 ns frequency-doubled Nd:Yag microchip laser, this Raman-source generates 24 lines spanning from 355 to 745 nm, and a peak power density per line of 260 mW/nm for the strongest lines. Both the output power and the spectrum remained constant over its monitoring duration of more than six months. The spectrum of this multiline laser superimposes with no less than 17 absorption peaks of fluorescent dyes from the Alexa Fluor family used as biological markers.

Hyperspectral fluorescence imaging with multi wavelength LED excitation

NASA Astrophysics Data System (ADS)

Luthman, A. Siri; Dumitru, Sebastian; Quirós-Gonzalez, Isabel; Bohndiek, Sarah E.

2016-04-01

Hyperspectral imaging (HSI) can combine morphological and molecular information, yielding potential for real-time and high throughput multiplexed fluorescent contrast agent imaging. Multiplexed readout from targets, such as cell surface receptors overexpressed in cancer cells, could improve both sensitivity and specificity of tumor identification. There remains, however, a need for compact and cost effective implementations of the technology. We have implemented a low-cost wide-field multiplexed fluorescence imaging system, which combines LED excitation at 590, 655 and 740 nm with a compact commercial solid state HSI system operating in the range 600 - 1000 nm. A key challenge for using reflectance-based HSI is the separation of contrast agent fluorescence from the reflectance of the excitation light. Here, we illustrate how it is possible to address this challenge in software, using two offline reflectance removal methods, prior to least-squares spectral unmixing. We made a quantitative comparison of the methods using data acquired from dilutions of contrast agents prepared in well-plates. We then established the capability of our HSI system for non-invasive in vivo fluorescence imaging in small animals using the optimal reflectance removal method. The HSI presented here enables quantitative unmixing of at least four fluorescent contrast agents (Alexa Fluor 610, 647, 700 and 750) simultaneously in living mice. A successful unmixing of the four fluorescent contrast agents was possible both using the pure contrast agents and with mixtures. The system could in principle also be applied to imaging of ex vivo tissue or intraoperative imaging in a clinical setting. These data suggest a promising approach for developing clinical applications of HSI based on multiplexed fluorescence contrast agent imaging.

Anesthetic Sevoflurane Causes Rho-Dependent Filopodial Shortening in Mouse Neurons.

PubMed

Zimering, Jeffrey H; Dong, Yuanlin; Fang, Fang; Huang, Lining; Zhang, Yiying; Xie, Zhongcong

2016-01-01

Early postnatal anesthesia causes long-lasting learning and memory impairment in rodents, however, evidence for a specific neurotoxic effect on early synaptogenesis has not been demonstrated. Drebrin A is an actin binding protein whose localization in dendritic protrusions serves an important role in dendritic spine morphogenesis, and is a marker for early synaptogenesis. We therefore set out to investigate whether clinically-relevant concentrations of anesthetic sevoflurane, widely- used in infants and children, alters dendritic morphology in cultured fetal day 16 mouse hippocampal neurons. After 7 days in vitro, mouse hippocampal neurons were exposed to four hours of 3% sevoflurane in 95% air/5% CO2 or control condition (95% air/5% CO2). Neurons were fixed in 4% paraformaldehyde and stained with Alexa Fluor555-Phalloidin, and/or rabbit anti-mouse drebrin A/E antibodies which permitted subcellular localization of filamentous (F)-actin and/or drebrin immunoreactivity, respectively. Sevoflurane caused acute significant length-shortening in filopodia and thin dendritic spines in days-in-vitro 7 neurons, an effect which was completely rescued by co-incubating neurons with ten micromolar concentrations of the selective Rho kinase inhibitor Y27632. Filopodia and thin spine recovered in length two days after sevoflurane exposure. Yet cluster-type filopodia (a precursor to synaptic filopodia) were persistently significantly decreased in number on day-in-vitro 9, in part owing to preferential localization of drebrin immunoreactivity to dendritic shafts versus filopodial stalks. These data suggest that sevoflurane induces F-actin depolymerization leading to acute, reversible length-shortening in dendritic protrusions through a mechanism involving (in part) activation of RhoA/Rho kinase signaling and impairs localization of drebrin A to filopodia required for early excitatory synapse formation.

NIR-emitting molecular-based nanoparticles as new two-photon absorbing nanotools for single particle tracking

NASA Astrophysics Data System (ADS)

Daniel, J.; Godin, A. G.; Clermont, G.; Lounis, B.; Cognet, L.; Blanchard-Desce, M.

2015-07-01

In order to provide a green alternative to QDs for bioimaging purposes and aiming at designing bright nanoparticles combining both large one- and two-photon brightness, a bottom-up route based on the molecular engineering of dedicated red to NIR emitting dyes that spontaneously form fluorescent organic nanoparticles (FONs) has been implemented. These fully organic nanoparticles built from original quadrupolar dyes are prepared using a simple, expeditious and green protocol that yield very small molecular-based nanoparticles (radius ~ 7 nm) suspension in water showing a nice NIR emission (λem=710 nm). These FONs typically have absorption coefficient more than two orders larger than popular NIR-emitting dyes (such as Alexa Fluor 700, Cy5.5 ….) and much larger Stokes shift values (i.e. up to over 5500 cm-1). They also show very large two-photon absorption response in the 800-1050 nm region (up to about 106 GM) of major promise for two-photon excited fluorescence microscopy. Thanks to their brightness and enhanced photostability, these FONs could be imaged as isolated nanoparticles and tracked using wide-field imaging. As such, thanks to their size and composition (absence of heavy metals), they represent highly promising alternatives to NIR-emitting QDs for use in bioimaging and single particle tracking applications. Moreover, efficient FONs coating was achieved by using a polymeric additive built from a long hydrophobic (PPO) and a short hydrophilic (PEO) segment and having a cationic head group able to interact with the highly negative surface of FONs. This electrostatically-driven interaction promotes both photoluminescence and two-photon absorption enhancement leading to an increase of two-photon brightness of about one order of magnitude. This opens the way to wide-field single particle tracking under two-photon excitation

In-vivo fluorescence detection of breast cancer growth factor receptors by fiber-optic probe

NASA Astrophysics Data System (ADS)

Bustamante, Gilbert; Wang, Bingzhi; DeLuna, Frank; Sun, LuZhe; Ye, Jing Yong

2018-02-01

Breast cancer treatment options often include medications that target the overexpression of growth factor receptors, such as the proto-oncogene human epidermal growth factor receptor 2 (HER2/neu) and epidermal growth factor receptor (EGFR) to suppress the abnormal growth of cancerous cells and induce cancer regression. Although effective, certain treatments are toxic to vital organs, and demand assurance that the pursued receptor is present at the tumor before administration of the drug. This requires diagnostic tools to provide tumor molecular signatures, as well as locational information. In this study, we utilized a fiber-optic probe to characterize in vivo HER2 and EGFR overexpressed tumors through the fluorescence of targeted dyes. HER2 and EGFR antibodies were conjugated with ICG-Sulfo-OSu and Alexa Fluor 680, respectively, to tag BT474 (HER2+) and MDA-MB-468 (EGFR+) tumors. The fiber was inserted into the samples via a 30-gauge needle. Different wavelengths of a supercontinuum laser were selected to couple into the fiber and excite the corresponding fluorophores in the samples. The fluorescence from the dyes was collected through the same fiber and quantified by a time-correlated single photon counter. Fluorescence at different antibody-dye concentrations was measured for calibration. Mice with subcutaneous HER2+ and/or EGFR+ tumors received intravenous injections of the conjugates and were later probed at the tumor sites. The measured fluorescence was used to distinguish between tumor types and to calculate the concentration of the antibody-dye conjugates, which were detectable at levels as low as 40 nM. The fiber-optic probe presents a minimally invasive instrument to characterize the molecular signatures of breast cancer in vivo.

The assembly of GM1 glycolipid- and cholesterol-enriched raft-like membrane microdomains is important for giardial encystation.

PubMed

De Chatterjee, Atasi; Mendez, Tavis L; Roychowdhury, Sukla; Das, Siddhartha

2015-05-01

Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Simultaneous assessment of glomerular filtration and barrier function in live zebrafish

PubMed Central

Kotb, Ahmed M.; Müller, Tobias; Xie, Jing; Anand-Apte, Bela; Endlich, Nicole

2014-01-01

The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish. PMID:25298528

Comparison of Dextran Perfusion and GSI-B4 Isolectin Staining in a Mouse Model of Oxygen-induced Retinopathy.

PubMed

Huang, Shaofen; Liang, Jiajian; Yam, Gary Hin-Fai; Lu, Zhihao; Pang, Chi Pui; Chen, Haoyu

2015-06-01

Oxygen-induced retinopathy (OIR) is a robust and widely used animal model for the study of retinal neovascularization (NV). Dextran perfusion and Griffonia simplicifolia isolectin B4 (GSI-B4) staining are two common methods for examining the occurrence and extent of OIR. This study provides a quantitative comparison of the two for OIR detection. At postnatal day 7 (PN7), fifteen C57BL/6J mice were exposed to a 75% hyperoxic condition for 5 days and then returned to room air conditions. At PN17, the mice received intravitreal injection of GSI-B4 Alexa Fluor 568 conjugate. After 10 hours, they were infused with FITC-dextran conjugate via the left ventricle. Retinal flat mounts were photographed by confocal microscopy. Areas with fluorescent signals and the total retinal areas were quantified by Image J software. Both GSI-B4 and dextran detected the peripheral neovascular area. The mean hyper fluorescence area was 0.33 ± 0.14% of whole retinal area determined by GSI-B4 staining and 0.25 ± 0.28% determined by dextran perfusion. The difference between the two measures was 0.08% (95% CI:-0.59%, 0.43%). The Pearson correlation coefficient between the two methods was 0.386,P =0.035. The mean coincidence rates were 14.3 ± 13.4% and 24.9 ± 18.5% for GSI-B4 and dextran staining, respectively. Both methods can complement each other in demonstrating and quantitatively evaluating retinal NV. A poor agreement was found between the two methods; GSI-B4 isolectin was more effective than FITC-dextran perfusion in evaluating the extent of retinal NV in a mouse model of OIR.

RESOLFT nanoscopy with photoswitchable organic fluorophores

NASA Astrophysics Data System (ADS)

Kwon, Jiwoong; Hwang, Jihee; Park, Jaewan; Han, Gi Rim; Han, Kyu Young; Kim, Seong Keun

2015-12-01

Far-field optical nanoscopy has been widely used to image small objects with sub-diffraction-limit spatial resolution. Particularly, reversible saturable optical fluorescence transition (RESOLFT) nanoscopy with photoswitchable fluorescent proteins is a powerful method for super-resolution imaging of living cells with low light intensity. Here we demonstrate for the first time the implementation of RESOLFT nanoscopy for a biological system using organic fluorophores, which are smaller in size and easier to be chemically modified. With a covalently-linked dye pair of Cy3 and Alexa647 to label subcellular structures in fixed cells and by optimizing the imaging buffer and optical parameters, our RESOLFT nanoscopy achieved a spatial resolution of ~74 nm in the focal plane. This method provides a powerful alternative for low light intensity RESOLFT nanoscopy, which enables biological imaging with small organic probes at nanoscale resolution.

Restoring public trust while tearing down site in rural Ohio

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schneider, Jerry; Wagner, Jeffrey; Connell, Judy

2007-07-01

In the mid-1980's, the impact of three decades of uranium processing near rural Fernald, Ohio, 18 miles northwest of Cincinnati, became the centre of national public controversy. When a series of incidents at the uranium foundry brought to light the years of contamination to the environment and surrounding farmland communities, local citizens' groups united and demanded a role in determining the plans for cleaning up the site. One citizens' group, Fernald Residents for Environmental Safety and Health (FRESH), formed in 1984 following reports that nearly 300 pounds of enriched uranium oxide had been released from a dust-collector system, and threemore » off-property wells south of the site were contaminated with uranium. For 22 years, FRESH monitored activities at Fernald and participated in the decision-making process with management and regulators. The job of FRESH ended on 19 January this year when the Fernald Site was declared clean of all nuclear contamination and open to public access. It marked the end of a remarkable turnaround in public confidence and trust that had attracted critical reports from around the world. When personnel from Fluor arrived in 1992, the management team thought it understood the issues and concerns of each stakeholder group, and was determined to implement the decommissioning scope of work aggressively, confident that stakeholders would agree with its plans. This approach resulted in strained relationships with opinion leaders during the early months of Fluor's contract. To forge better relationships, the U.S. Department of Energy (DOE) who owns the site, and Fluor embarked on three new strategies based on engaging citizens and interested stakeholder groups in the decision-making process. The first strategy was opening communication channels with site leadership, technical staff, and regulators. This strategy combined a strong public-information program with two-way communications between management and the community, soliciting and encouraging stakeholder participation early in the decision-making process. Fluor's public-participation strategy exceeded the 'check-the-box' approach common within the nuclear-weapons complex, and set a national standard that stands alone today. The second stakeholder-engagement strategy sprang from mending fences with the regulators and the community. The approach for disposing low-level waste was a 25-year plan to ship it off the site. Working with stakeholders, DOE and Fluor were able to convince the community to accept a plan to safely store waste permanently on site, which would save 15 years of cleanup and millions of dollars in cost. The third strategy addressed the potentially long delays in finalizing remedial action plans due to formal public comment periods and State and Federal regulatory approvals. Working closely with the U.S. and Ohio Environmental Protection Agencies (EPA) and other stakeholders, DOE and Fluor were able to secure approvals of five Records of Decision on time - a first for the DOE complex. Developing open and honest relationships with union leaders, the workforce, regulators and community groups played a major role in DOE and Fluor cleaning up and closing the site. Using lessons learned at Fernald, DOE was able to resolve challenges at other sites, including worker transition, labour disputes, and damaged relationships with regulators and the community. It took significant time early in the project to convince the workforce that their future lay in cleanup, not in holding out hope for production to resume. It took more time to repair relationships with Ohio regulators and the local community. Developing these relationships over the years required constant, open communications between site decision makers and stakeholders to identify issues and to overcome potential barriers. Fluor's open public-participation strategy resulted in stakeholder consensus of five remedial-action plans that directed Fernald cleanup. This strategy included establishing a public-participation program that emphasized a shared-decision making process and abandoned the government's traditional, non-participatory 'Decide, Announce, Defend' approach. Fernald's program became a model within the DOE complex for effective public participation. Fluor led the formation of the first DOE site-specific advisory board dedicated to remediation and closure. The board was successful at building consensus on critical issues affecting long-term site remediation, such as cleanup levels, waste disposal and final land use. Fluor created innovative public outreach tools, such as 'Cleanopoly', based on the Monopoly game, to help illustrate complex concepts, including risk levels, remediation techniques, and associated costs. These innovative tools helped DOE and Fluor gain stakeholder consensus on all cleanup plans. To commemorate the outstanding commitment of Fernald stakeholders to this massive environmental-restoration project, Fluor donated $20,000 to build the Weapons to Wetlands Grove overlooking the former 136-acre production area. The grove contains 24 trees, each dedicated to '[a] leader(s) behind the Fernald cleanup'. Over the years, Fluor, through the Fluor Foundation, also invested in educational and humanitarian projects, contributing nearly $2 million to communities in southwestern Ohio, Kentucky and Indiana. Further, to help offset the economic impact of the site's closing to the community, DOE and Fluor promoted economic development in the region by donating excess equipment and property to local schools and townships. This paper discusses the details of the public-involvement program - from inception through maturity - and presents some lessons learned that can be applied to other similar projects. (authors)« less

Halophilic life on Mars ?

NASA Astrophysics Data System (ADS)

Stan-Lotter, Helga; Fendrihan, Sergiu; Dornmayr-Pfaffenhuemer, Marion; Holzinger, Anita; Polacsek, Tatjana K.; Legat, Andrea; Grösbacher, Michael; Weigl, Andreas

2010-05-01

Background: The search for extraterrestrial life has been declared as a goal for the 21th century by several space agencies. Potential candidates are microorganisms on or in the surface of moons and planets, such as Mars. Extremely halophilic archaea (haloarchaea) are of astrobiological interest since viable strains have been isolated from million years old salt deposits (1) and halite has been found in Martian meteorites and in surface pools. Therefore, haloarchaeal responses to simulated and real space conditions were explored. Immuno assays for a potential Life Marker Chip experiment were developed with antisera against the universal enzyme ATP synthase. Methods: The focus of these studies was on the application of fluorescent probes since they provide strong signals, and detection devices are suitable for miniaturization. Viability of haloarchaeal strains (Halococcus dombrowskii and Halobacterium salinarum NRC-1) was probed with the LIVE/DEAD BacLight™ kit and the BacLight™ Bacterial Membrane Potential kit. Cyclobutane pyrimidine dimers (CPD) in the DNA, following exposure to simulated and real space conditions (UV irradiation from 200 - 400 nm; 18 months exposure on the International Space Station [ISS] within the ADAPT experiment by Dr. P. Rettberg), were detected with fluorescent Alexa-Fluor-488-coupled antibodies. Immuno assays with antisera against the A-ATPase subunits from Halorubrum saccharovorum were carried out with the highly sensitive Immun-Star ™ WesternC ™ chemiluminescent kit (Bio-Rad). Results: Using the LIVE/DEAD BacLight™ kit, the D37 (dose of 37% survival) for Hcc. dombrowskii and Hbt. salinarum NRC-1, following exposure to UV (200-400 nm) was about 400 kJ/m2, when cells were embedded in halite and about 1 kJ/m2, when cells were in liquid cultures. Fluorescent staining indicated a slightly higher cellular activity than that which was derived from the determination of colony forming units. Assessment of viability with the BacLight™ Bacterial Membrane Potential kit gave strong signals with Hcc. dombrowskii and the control microorganism E. coli; as expected, the uncoupler CCCP diminished the membrane potential. Reaction times were generally longer with Hcc. dombrowskii than with E. coli. Hcc. dombrowskii from the ISS experiment showed > 80% viable cells when judged with the LIVE/DEAD kit. CPD formation was detectable in about 3-5 % of the total cells. It is not yet known if growing cells of Hcc. dombrowskii were recovered from the ISS. ATPase subunits were detected in crude membrane preparations, in whole haloarchaeal and bacterial cells, and even in spores (from Geobacillus stearothermophilus), suggesting the usefulness of the ATP synthase as a molecular target for life detection. Conclusions: Fluorescent dyes provide strong signals, which are suitable for remote detection and are compatible with high ionic strength. The advantages of staining with fluorescent dyes are rapid results on membrane intactness, membrane potential, and the presence of certain biomolecules. But more data are needed for a better correlation to cellular viability. (1) Stan-Lotter H, Pfaffenhuemer M, Legat A, Busse H-J, Radax C, Gruber C (2002) Halococcus dombrowskii sp. nov., an archaeal isolate from a Permian alpine salt deposit. Int System Evol Microbiol 52, 1807-1814.

Development of Nanomedicines for Treatment of Posttraumatic Osteoarthritis

DTIC Science & Technology

2016-03-01

explants fixed in PFA and paraffin-embedded showed positive signal in P-Dex-Alexa groups (with high background in the PBS control groups), which validate...addition (IL1beta-treated group not shown). Fluorescence images of samples after PBS washes and before PFA fixation are shown in (B) and (E) for P-Alexa...647 and Control groups, respectively. Representative fluorescence images of samples after PFA fixation and washes are shown in (C) and (F) for P

SAFETY AT FLUOR HANFORD (A) CASE STUDY - PREPARED BY THUNDERBIRD SCHOOL OF GLOBAL MANAGEMENT

DOE Office of Scientific and Technical Information (OSTI.GOV)

ARNOLD LD

2009-09-25

By November of 1997, Fluor Hanford (Fluor) had been the site manager of the Hanford nuclear reservation for a year. The Hanford site had been established as part of the Manhattan Project in the 1940s that gave birth to the atomic bomb. Hanford produced two thirds of U.S. plutonium during the Cold War period. The Hanford site was half the size of Rhode Island and occupied 586 square miles in southeastern Washington State. The production of plutonium for more than 40 years left a huge legacy of chemical and radiological contamination: 80 square miles of contaminated groundwater; 2,300 tons ofmore » spent nuclear fuel stored in underwater basins; 20 tons of plutonium-laced contaminated materials; and 500 contaminated facilities. The cleanup involved a challenging combination of radioactive material handling within an infrastructure constructed in the 1940s and 1950s. The cleanup that began in 1988 was expected to take 30 years or more. Improving safety at Hanford had already proven to be a significant challenge. As the new site manager at Hanford, Fluor Hanford inherited lower- and mid-level managers and thousands of unionized employees, many of whom were second or third generation Hanford employees. These employees had seen many contractors come and go over the years. Some of the managers who had worked with the previous contractor saw Fluor's emphasis on safety as getting in the way of operations. Union-management relations were fractious. Hanford's culture was described as 'production driven-management told everyone what to do, and, if you didn't do it, there were consequences'. Worker involvement in designing and implementing safety programs was negligible. Fluor Hanford also was having trouble satisfying its client, the Department of Energy (DOE). The DOE did not see a clear path forward for performance improvements at Hanford. Clearly, major change was necessary, but how and where should it be implemented?« less

Calcium transport into the cells of the sea urchin larva in relation to spicule formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vidavsky, Netta; Addadi, Sefi; Schertel, Andreas

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence ofmore » vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.« less

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy

PubMed Central

Nahidiazar, Leila; Agronskaia, Alexandra V.; Broertjes, Jorrit; van den Broek, Bram; Jalink, Kees

2016-01-01

Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off (“blink”), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording. PMID:27391487

Calcium transport into the cells of the sea urchin larva in relation to spicule formation

DOE PAGES

Vidavsky, Netta; Addadi, Sefi; Schertel, Andreas; ...

2016-10-24

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence ofmore » vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.« less

Lithium-containing scintillators for thermal neutron, fast neutron, and gamma detection

DOEpatents

Zaitseva, Natalia P.; Carman, M. Leslie; Faust, Michelle A.

2016-03-01

In one embodiment, a scintillator includes a scintillator material; a primary fluor, and a Li-containing compound, where the Li-containing compound is soluble in the primary fluor, and where the scintillator exhibits an optical response signature for thermal neutrons that is different than an optical response signature for fast neutrons and gamma rays.

FluorWPS: A Monte Carlo ray-tracing model to compute sun-induced chlorophyll fluorescence of three-dimensional canopy

USDA-ARS?s Scientific Manuscript database

A model to simulate radiative transfer (RT) of sun-induced chlorophyll fluorescence (SIF) of three-dimensional (3-D) canopy, FluorWPS, was proposed and evaluated. The inclusion of fluorescence excitation was implemented with the ‘weight reduction’ and ‘photon spread’ concepts based on Monte Carlo ra...

Remote observations with FLUOR and the CHARA Array

NASA Astrophysics Data System (ADS)

Merand, Antoine; Birlan, Mirel; Lelu de Brach, Remi; Coudé du Foresto, Vincent

2004-10-01

Two years ago, the FLUOR interferometric beam combiner moved from IOTA (Infrared Optical Telescopes Array, Mount Hopkins, AZ) to the Center for High Angular Resolution Astronomy (CHARA) Array (Mount Wilson, CA). Apart from offering the largest baselines in the northern hemisphere, this array can be fully operated remotely to allow observations from a distant place. We present here the automations added to the FLUOR hardware, as well as software modifications made in order to allow us to observe from Paris Observatory. We required the remote service to be as reactive as local observations, implying frequent communications between the instrument and the remote observer. We took particular attention to the available bandwidth and reactivity imposed by the secured connection (Virtual Private Network). The first tests are presented.

Fluorophore-labeling of core-crosslinked polymeric micelles for multimodal in vivo and ex vivo optical imaging

PubMed Central

Shi, Yang; Kunjachan, Sijumon; Wu, Zhuojun; Gremse, Felix; Moeckel, Diana; van Zandvoort, Marc; Kiessling, Fabian; Storm, Gert; van Nostrum, Cornelus F.; Hennink, Wim E.; Lammers, Twan

2015-01-01

Aim To enable multimodal in vivo and ex vivo optical imaging of the biodistribution and tumor accumulation of core-crosslinked polymeric micelles (CCPM). Materials & Methods mPEG-b-p(HPMAm-Lac)-based polymeric micelles, core-crosslinked via cystamine and covalently labeled with two fluorophores (Dy-676/488) were synthesized. The CCPM were intravenously injected in CT26 tumor-bearing mice. Results Upon intravenous injection, the CCPM accumulated in CT26 tumors reasonably efficiently, with values reaching ~4 %ID at 24 hours. Ex vivo TPLSM confirmed efficient extravasation of the iCCPM out of tumor blood vessels and deep penetration into the tumor interstitium. Conclusions CCPM were labeled with multiple fluorophores, and they exemplify that combining different in vivo and ex vivo optical imaging techniques is highly useful for analyzing the biodistribution and tumor accumulation of nanomedicines. PMID:25929568

Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy.

PubMed

Prenner, Lars; Sieben, Anne; Zeller, Karin; Weiser, Dieter; Häberlein, Hanns

2007-05-01

Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

Alkyl Aryl Ether Bond Formation with PhenoFluor**

PubMed Central

Shen, Xiao; Neumann, Constanze N.; Kleinlein, Claudia; Claudia, Nathaniel W.; Ritter, Tobias

2015-01-01

An alkyl aryl ether bond formation reaction between phenols and primary and secondary alcohols with PhenoFluor has been developed. The reaction features a broad substrate scope and tolerates many functional groups, and substrates that are challenging for more conventional ether bond forming processes may be coupled. A preliminary mechanistic study indicates reactivity distinct from conventional ether bond formation. PMID:25800679

Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: implications for Alzheimer's disease.

PubMed

Thomas, Rhian S; Henson, Alex; Gerrish, Amy; Jones, Lesley; Williams, Julie; Kidd, Emma J

2016-07-18

Polymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer's disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME). Following depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression. The depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD.

Effects of Two Fluoride Varnishes and One Fluoride/Chlorhexidine Varnish on Streptococcus mutans and Streptococcus sobrinus Biofilm Formation in Vitro

PubMed Central

Pinar Erdem, Arzu; Sepet, Elif; Kulekci, Güven; Trosola, Sule Can; Guven, Yegane

2012-01-01

Aims: The aim of this study was to evaluate and to compare the effect of two fluoride varnishes and one fluoride/chlorhexidine varnish on Streptococcus mutans and Streptococcus sobrinus biofilm formation, in vitro. Study design: Standard acrylic discs were prepared and divided into groups based on the varnish applied to the disc surface: Fluor Protector, Bifluoride 12, and Fluor Protector + Cervitec (1:1). Untreated discs served as controls. In the study groups, biofilms of S. mutans and S. sobrinus were formed over 24 h, 48 h, and 5 days. The fluoride concentrations in the monospecies biofilms and viable counts of S. mutans and S. sobrinus were investigated. Results: In all study groups, a statistically significant increase in the viable number of S. mutans and S. sobrinus cells was observed between 24 h and 5 days. In both monospecies biofilms, the greatest antibacterial efficacy was detected in the Fluor Protector and Fluor Protector + Cervitec groups at 24 h. For all groups, the amount of fluoride released was highest during the first 24 h, followed by a significant decrease over the next 4 days. A negative correlation was detected between fluoride concentration and antibacterial effect in those groups with biofilms containing both species. Despite the release of high levels of fluoride, the greatest number of viable S. mutans and S. sobrinus cells was detected in the Bifluoride 12 group. Statistics: The data were analyzed using GraphPad Prism software (ver. 3). Conclusions: The Fluor Protector + Cervitec varnish exerted prolonged antibacterial effects on S. mutans and S. sobrinus biofilms compared to the other varnishes tested. PMID:22253559

Interest of a simple on-line screening registry for measuring ICU burden related to an influenza pandemic.

PubMed

Richard, Jean-Christophe Marie; Pham, Tài; Brun-Buisson, Christian; Reignier, Jean; Mercat, Alain; Beduneau, Gaëtan; Régnier, Bernard; Mourvillier, Bruno; Guitton, Christophe; Castanier, Matthias; Combes, Alain; Le Tulzo, Yves; Brochard, Laurent

2012-07-09

The specific burden imposed on Intensive Care Units (ICUs) during the A/H1N1 influenza 2009 pandemic has been poorly explored. An on-line screening registry allowed a daily report of ICU beds occupancy rate by flu infected patients (Flu-OR) admitted in French ICUs. We conducted a prospective inception cohort study with results of an on-line screening registry designed for daily assessment of ICU burden. Among the 108 centers participating to the French H1N1 research network on mechanical ventilation (REVA) - French Society of Intensive Care (SRLF) registry, 69 ICUs belonging to seven large geographical areas voluntarily participated in a website screening-registry. The aim was to daily assess the ICU beds occupancy rate by influenza-infected and non-infected patients for at least three weeks. Three hundred ninety-one critically ill infected patients were enrolled in the cohort, representing a subset of 35% of the whole French 2009 pandemic cohort; 73% were mechanically ventilated, 13% required extra corporal membrane oxygenation (ECMO) and 22% died. The global Flu-OR in these ICUs was only 7.6%, but it exceeded a predefined 15% critical threshold in 32 ICUs for a total of 103 weeks. Flu-ORs were significantly higher in University than in non-University hospitals. The peak ICU burden was poorly predicted by observations obtained at the level of large geographical areas. The peak Flu-OR during the pandemic significantly exceeded a 15% critical threshold in almost half of the ICUs, with an uneven distribution with time, geographical areas and between University and non-University hospitals. An on-line assessment of Flu-OR via a simple dedicated registry may contribute to better match resources and needs.

UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN.

PubMed

Park, Sungjin; Foote, Peter K; Krist, David T; Rice, Sarah E; Statsyuk, Alexander V

2017-10-06

Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes. To address this challenge, we developed novel chemical probes, ubiquitin C-terminal fluorescein thioesters UbMES and UbFluor, to qualitatively and quantitatively assess the activity of the RBR E3 ligase PARKIN in a simple experimental setup and in real time using fluorescence polarization. First, we confirmed that PARKIN does not require an E2 enzyme for substrate ubiquitination, lysine selection, and polyubiquitin chain formation. Second, we confirmed that UbFluor quantitatively detects naturally occurring activation states of PARKIN caused by Ser 65 phosphorylation (pPARKIN) and phosphorylated ubiquitin (pUb). Third, we showed that both pUb and the ubiquitin-accepting substrate contribute to maximal pPARKIN ubiquitin conjugation turnover. pUb enhances the transthiolation step, whereas the substrate clears the pPARKIN∼Ub thioester intermediate. Finally, we established that UbFluor can quantify activation or inhibition of PARKIN by structural mutations. These results demonstrate the feasibility of using UbFluor for quantitative studies of the biochemistry of RBR E3s and for high-throughput screening of small-molecule activators or inhibitors of PARKIN and other RBR E3 ligases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Dynamics of Dengue Virus (DENV)–Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions

PubMed Central

Woda, Marcia; Friberg, Heather; Currier, Jeffrey R.; Srikiatkhachorn, Anon; Macareo, Louis R.; Green, Sharone; Jarman, Richard G.; Rothman, Alan L.; Mathew, Anuja

2016-01-01

Background. The development of reagents to identify and characterize antigen-specific B cells has been challenging. Methods. We recently developed Alexa Fluor–labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. Results. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV+ class-switched memory B cells (IgD−CD27+ CD19+ cells) reached up to 8% during acute infection and early convalescence. AF DENV–labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38−CD27+) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. Conclusions. AF DENVs reveal changes in the phenotype of DENV serotype–specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination. PMID:27443614

Nassriya Water Treatment Plant Nassriya, Iraq. Sustainment Assessment

DTIC Science & Technology

2008-04-28

Progress During Construction 8 Status of the WTP at Time of Turnover 11 Site Assessment 13 Continuing Problems for the Nassriya WTP 27...Future Plans for the Nassriya WTP 35 Conclusions 37 Recommendations 38 Management Comments 39 Evaluation of Management Comments 39...Appendices A. Scope and Methodology 40 B. FluorAmec’s Organizational Structure for the Nassriya WTP 41 C. FluorAmec’s Nassriya WTP Staffing Plan 42 D

L-325 Sagebrush Habitat Mitigation Project: FY2008 Compensation Area Monitoring Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Durham, Robin E.; Sackschewsky, Michael R.

2008-09-30

This document provides a review and status of activities conducted in support of the Fluor Daniel Hanford Company (Fluor) Mitigation Action Plan (MAP) for Project L-325, Electrical Utility Upgrades. It includes time-zero monitoring results for planting activities conducted in January 2008, annual survival monitoring for all planting years (2007 and 2008), and recommendations for the successful completion of DOE habitat mitigation commitments for this project.

Collaboration in crisis and emergency management: Identifying the gaps in the case of storm 'Alexa'.

PubMed

Sawalha, Ihab Hanna Salman

2014-01-01

Failing to collaborate in crisis and emergency situations will increase the vulnerability of organisations and societies towards potential disasters. This paper highlights the significance of effective collaboration at different levels in times of crises. The case of snow storm 'Alexa', which hit Jordan in December 2013, was considered for the purpose of this research. The impact of Alexa raised many questions regarding the country's preparedness and the capacity of its infrastructure to maintain critical business functions across various industry sectors. First, should people individually take all the responsibility to manage crises and emergencies in order to protect themselves and their belongings? Secondly, should organisations join efforts with other organisations within the same or different sectors? Thirdly, should governments seek external collaboration for the ultimate goal of securing their economies? These issues are significant as they underline the element of collaboration. This paper contributes to the understanding of the role of collaboration in times of intense difficulty and loss of control. The proposition made by this research is that an effective collaborative process is positively associated with perceptions of improved disaster risk reduction practices.

New fluorescence correlation spectroscopy enabling direct observation of spatiotemporal dependence of diffusion constants as an evidence of anomalous transport in extracellular matrices.

PubMed

Masuda, Akiko; Ushida, Kiminori; Okamoto, Takayuki

2005-05-01

The potential of fluorescence correlation spectroscopy (FCS) is extended to enable the direct observation of anomalous subdiffusion (ASD) in inhomogeneous media that are of great importance particularly in many biological systems, such as membranes, cytoplasm, and extracellular matrices (ECMs). Because ASD can be confirmed by monitoring the spatiotemporal dependence of observable diffusion coefficients (D(obs)), the size of the effective confocal volume (V(eff)) for FCS sampling (sampling volume) was continuously changed on a scale of 300-500 nm using a motorized variable beam expander through which an illuminating laser beam passes. This new method, namely, sampling-volume-controlled (SVC)-FCS, was applied to the analysis of hyaluronan (HA) aqueous solutions where the D(obs) of light-emitting solute (Alexa 488) markedly changed, corresponding to the change in V(eff) (220-340 nm in the half-axis), because the network structure of HA of 7-33 nm (nanostructure) interferes with the material transport within it. The results indicate that moderate ASD may occur even in the presence of a small amount ( approximately 0.1 wt %) of HA in ECM. Because the change in D(obs) along with the traveling distance (the mean-square displacement) can be identified even in systems with no deformation of the autocorrelation function, this technique has a great potential for general applications to many biological systems in which ASD shows complex time and space dependences.

Sulfur mustard disrupts human α3β1-integrin receptors in concert with α6β4-integrin receptors and collapse of the keratin K5/K14 cytoskeleton

NASA Astrophysics Data System (ADS)

Werrlein, Robert J.; Braue, Catherine R.

2004-06-01

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a chemical warfare agent that produces persistent, incapacitating blisters of the skin. The lesions inducing vesication remain elusive, and there is no completely effective treatment. Using mulitphoton microscopy and immunofluorescent staining, we found that exposing human epidermal keratinocytes (HEK) and intact epidermis to SM (400 μm for 5 min) caused progressive collapse of the keratin (K5/K14) cytoskeleton and depletion of α6β integrins. We now report that SM causes concomitant disruption nad collapse of the basal cell's α3β1-integrin receptors. At 1 h postexposure, images of Alexa488-conjugated HEK/α3β1 integrins showed almost complete withdrawal and disappearance of retraction fibers and a progressive loss of polarized mobility. With stero imaging, in vitro expression of this SM effect was characterized by collapse and abutment of adjacent cell membranes. At 2 h postexposure, there was an average 13% dorso-ventral collapse of HEK membranes that paralleled progressive collapse of the K5/K14 cytoskeleton. α3β1 integrin, like α6β4 integrin, is a regulator of cytoskeletal assembly, a receptor for laminin 5 and a mediator of HEK attachment to the basement membrane. Our images indicate that SM disrupts these receptors. We suggest that the progressive disruption destabilizes and potentiates blistering of the epidermal-dermal junction.

Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.

PubMed

Tsuganezawa, Keiko; Shinohara, Yoshiyasu; Ogawa, Naoko; Tsuboi, Shun; Okada, Norihisa; Mori, Masumi; Yokoyama, Shigeyuki; Noda, Nobuo N; Inagaki, Fuyuhiko; Ohsumi, Yoshinori; Tanaka, Akiko

2013-10-01

The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.

A Versatile Click-Compatible Monolignol Probe to Study Lignin Deposition in Plant Cell Walls

PubMed Central

Pandey, Jyotsna L.; Wang, Bo; Diehl, Brett G.; Richard, Tom L.; Chen, Gong; Anderson, Charles T.

2015-01-01

Lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly. PMID:25884205

The CHPRC Columbia River Protection Project Quality Assurance Project Plan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fix, N. J.

Pacific Northwest National Laboratory researchers are working on the CHPRC Columbia River Protection Project (hereafter referred to as the Columbia River Project). This is a follow-on project, funded by CH2M Hill Plateau Remediation Company, LLC (CHPRC), to the Fluor Hanford, Inc. Columbia River Protection Project. The work scope consists of a number of CHPRC funded, related projects that are managed under a master project (project number 55109). All contract releases associated with the Fluor Hanford Columbia River Project (Fluor Hanford, Inc. Contract 27647) and the CHPRC Columbia River Project (Contract 36402) will be collected under this master project. Each projectmore » within the master project is authorized by a CHPRC contract release that contains the project-specific statement of work. This Quality Assurance Project Plan provides the quality assurance requirements and processes that will be followed by the Columbia River Project staff.« less

Transfer of PAMAM dendrimers across human placenta: prospects of its use as drug carrier during pregnancy.

PubMed

Menjoge, Anupa R; Rinderknecht, Amber L; Navath, Raghavendra S; Faridnia, Masoud; Kim, Chong J; Romero, Roberto; Miller, Richard K; Kannan, Rangaramanujam M

2011-03-30

Dendrimers offer significant potential as nanocarriers for targeted delivery of drugs and imaging agents. The objectives of this study were to evaluate the transplacental transport, kinetics and biodistribution of PAMAM dendrimers ex-vivo across the human placenta in comparison with antipyrine, a freely diffusible molecule, using dually perfused re-circulating term human placental lobules. The purpose of this study is to determine if dendrimers as drug carriers can be used to design drug delivery systems directed at selectively treating either the mother or the fetus. The transplacental transfers of fluorescently (Alexa 488) tagged PAMAM dendrimer (16 kDa) and antipyrine (188 Da) from maternal to fetal circulation were measured using HPLC/dual UV and fluorescent detector (sensitivity of 10 ng/mL for dendrimer and 100 ng/mL for antipyrine respectively). C(max) for the dendrimer-Alexa (DA) in maternal perfusate (T(max)=15 min) was 18 times higher than in the fetal perfusate and never equilibrated with the maternal perfusate during 5.5 h of perfusion (n=4). DA exhibited a measurable but low transplacental transport of 2.26±0.12 μg/mL during 5.5h, where the mean transplacental transfer was 0.84±0.11% of the total maternal concentration and the feto-maternal ratio as percent was 0.073%±0.02. The biochemical and physiological analysis of the placentae perfused with DA demonstrated normal function throughout the perfusion. The immunofluorescence histochemistry confirmed that the biodistribution of DA in perfused placenta was sparsely dispersed, and when noted was principally seen in the inter-villous spaces and outer rim of the villous branches. In a few cases, DA was found internalized and localized in nuclei and cytoplasm of syncytiotrophoblast and inside the villous core; however, DA was mostly absent from the villous capillaries. In conclusion, the PAMAM dendrimers exhibited a low rate of transfer from maternal to the fetal side across the perfused human placenta, which is similar to other investigations of large macromolecules, e.g., IgG. These overall findings suggest that entry of drugs conjugated to polymers, i.e., dendrimers, would be limited in their transfer across the human placenta when compared to smaller drug molecules alone, suggesting novel methods for selectively delivering therapeutics to the pregnant woman without significant transfer to the fetus, especially since the half life of the dendrimer in blood is relatively short. Copyright © 2010 Elsevier B.V. All rights reserved.

The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells.

PubMed

Law, Jacqueline P; Hirschkorn, Dale F; Owen, Rachel E; Biswas, Hope H; Norris, Philip J; Lanteri, Marion C

2009-12-01

Foxp3 is a key marker for CD4(+) regulatory T cells (T(regs)) and was used in developing a multiparameter flow cytometric panel to identify T(regs). Achieving reproducible staining and analysis first required optimization of Foxp3 staining. We present a comparative study of PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7 clones of anti-human-Foxp3 antibodies used in combination with five different fixation/permeabilization buffers. Staining for CD25, CD152, and CD127 was also compared between fixation/permeabilization treatments. Promising antibody/buffer combinations were tested in a panel of peripheral blood mononuclear cells from 10 individuals, and then on fresh versus frozen cells from four individuals. Finally, different fluorochromes coupled to two representative antibodies were compared to optimize separation of Foxp3(+) from Foxp3(-) events. Foxp3 gates were set using two gating strategies based on CD127(+)CD25(-) "non-T(regs)" or based on isotype controls. For Foxp3 staining, the best conditions for fixation/permeabilization were obtained using the eBioscience Foxp3, Imgenex, BioLegend, and BD Foxp3 buffers. Comparing results from 10 subjects, 259D/C7, PCH101, 236A/E7, and 206D antibodies yielded statistically higher levels of Foxp3 cells than those by 150D and 3G3 antibodies (mean = 6.9, 5.1, 4.7, and 3.7% compared with 1.7, and 0.3% of CD25(+)Foxp3(+) events within CD4(+) cells, respectively). Importantly, the "nonspecificity" of some antibodies observed with a Foxp3 gate based on isotype controls could be eliminated by setting the Foxp3 gate on "non-T(regs)". Better separation of Foxp3(+) and Foxp3(-) populations was observed using the PCH101 clone coupled to Alexa647 compared with FITC or the 259D/C7 clone coupled to PE compared with Alexa488 fluorochrome. Foxp3 staining can be highly variable and depends on the choice of antibody/buffer pair and the fluorochrome used. Selecting the correct population for setting the Foxp3 gate is critical to avoid including non-T(regs) in the Foxp3(+) gate. The experiments presented here will aid in optimization of flow cytometry staining panels to quantify T(reg) frequencies in humans.

Relationship between ornithine decarboxylase levels in anaplastic gliomas and progression-free survival in patients treated with DFMO-PCV chemotherapy.

PubMed

Levin, Victor A; Jochec, Jacob L; Shantz, Lisa M; Aldape, Kenneth D

2007-11-15

The purpose of this study was to assess the relationship between progression-free survival (PFS) in patients treated with DFMO + PCV (procarbazine, CCNU, vincristine) chemotherapy for malignant gliomas with tumor cell ornithine decarboxylase (ODC) activity. Formalin-fixed slides were obtained for study patients with anaplastic gliomas (AGs) and glioblastoma treated on protocol DM92-035. ODC levels were measured using an antibody to ODC coupled to Alexa 647 dye (Ab-ODC-Alexa 647). Ab-ODC-Alexa 647 intensity in transgenic murine hearts of differing ODC activity was used to calculate ODC activity in tumor cell nucleoplasm. In total, tumor specimens for 31 of 114 (27%) patients treated on the AG strata and 10 patients from the GBM strata were obtained. We found that tumor ODC level heterogeneity increased with increasing tumor malignancy. In a Cox proportional hazards model, PFS was found to be inversely related to median tumor ODC activity, with an unadjusted hazard ratio for median ODC group (>3.3 vs. 3.3 nmol/30 min/mug protein. Of AG tumors in which ODC activity was evaluated, 26% had ODC levels > 3.3 nmol/30 min/mug protein. This study shows that Ab-ODC-Alexa 647 fluorescence intensity can be used as a surrogate marker of ODC biochemical activity in AGs and can predict PFS to DFMO-based chemotherapy. (c) 2007 Wiley-Liss, Inc.

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor.

PubMed

Matulis, Daumantas; Kranz, James K; Salemme, F Raymond; Todd, Matthew J

2005-04-05

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

Microscopic observations of sonoporation mechanisms

NASA Astrophysics Data System (ADS)

Zeghimi, Aya; Escoffre, Jean-Michel; Bouakaz, Ayache

2017-03-01

Background Sonoporation promises a local gene/drug delivery with a high therapeutic efficacy and low toxicity level. However, the mechanisms orchestrating the molecules uptake are still unclear. Here, we investigate the effects of sonoporation on the plasma membrane of U-87 MG cells, either immediately or at different times post-sonoporation, using electron microscopy, and also the implication of cytoskeleton during the sonoporation process. Methods In our set-up, the U-87 MG cells were seeded on 18 mm diameter cover slips, placed in 24-well plates. The acoustic exposure conditions consisted of ultrasound pulses at 1 MHz, 1W/cm2 with duty cycle of 20% for 60 seconds. BR14® microbubbles were added to the cell medium before sonoporation at a microbubble/cell ratio of 5. These acoustic parameters were obtained as a result of a prior optimization experiments. Membrane permeabilization after sonoporation was assessed using SYTOX® Green dye (1 µM), as a model drug which does not cross the membrane of normal cells. The cell mortality was measured with propidium iodide staining. The alterations, on the plasma membrane, after sonoporation were monitored by scanning electron microscopy (SEM). The cell samples were processed immediately (0 min) and every 5 min up to 60 min post-sonoporation and coated by platinum sputtering (5 nm). For immunofluorescence experiments, the cells were fixed with 4% paraformaldehyde, and then incubated with TRITC-labeled Phalloidin, used to stain the actin cytoskeleton. Tubulin antibody Alexa Fluor® 555 conjugate was used to label the microtubules. Results Our results showed that immediately after ultrasound and microbubble exposure, dark and spherical structures appear on the plasma membrane. These structures have a diameter ranging from few nanometers to 160 nm. These structures are transient, since 15 min post-sonoporation, almost half of these structures disappeared. The decrease in the number of permeant structures is accentuated over time to be fully resorbed 60 min post-sonoporation, consequently the cells still metabolically active. Moreover, flow cytometry results show a positive correlation between membrane permeabilization and the number of these electron dense structures. Indeed, 60% of SYTOX® Green incorporation is achieved immediately after sonoporation, to decay over time and therefore as a function of the presence of these permeant structures on the cell membrane. These structures are named here "permeation structures". To define the nature of the TPS structures the cells were treated with Genistein, an inhibitor of caveolae-mediated endocytosis. Scanning Electron microscopy images showed a significant diminution of the number of TPS for cells incubated with Genistein, suggesting that a large part of these structures are caveolae still open. Moreover, immunofluorescence analysis showed a depolymerization of actin and tubulin cytoskeleton, immediately after sonoporation. This depolymerization is accompanied with a massive uptake of SYTOX® Green, while the use of cytochalasin D and nocodazole (inhibitors of actin and tubulin polymerization) induced a decrease in the percentage of SYTOX® Green incorporation. Conclusion In conclusion, our findings reveal the reversibility of sonoporation effects on the cell membrane, and show that the caveolae-mediated endocytosis is a dominant pathway involved in the sonoporation process of U-87 MG cells, with a probable involvement of other endocytic and non-endocytic pathways. Otherwise, the study of sonoporation on cytoskeleton gives evidence on the involvement of endocytosis during the sonoporation process (entry and transport of molecules).

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.

Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH 3) to nitrite (NO 2 ₋) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH 4 +-dependent O 2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide usingmore » a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.« less

Neurturin-deficient mice develop dry eye and keratoconjunctivitis sicca.

PubMed

Song, Xiu Jun; Li, De-Quan; Farley, William; Luo, Li Hui; Heuckeroth, Robert O; Milbrandt, Jeffrey; Pflugfelder, Stephen C

2003-10-01

Neurturin has been identified as a neurotrophic factor for parasympathetic neurons. Neurturin-deficient (NRTN(-/-)) mice have defective parasympathetic innervation of their lacrimal glands. This study was conducted to evaluate tear function and ocular surface phenotype in NRTN(-/-) mice. Determined by tail genomic DNA PCR, 25 NRTN(-/-) mice and 17 neurturin-normal (NRTN(+/+)) mice aged 6 weeks to 4 months were evaluated. Aqueous tear production, tear fluorescein clearance and corneal sensation were serially measured. Corneal permeability to AlexaFluor dextran (AFD; Molecular Probes, Eugene, OR) was measured by a fluorometric assay at 485 nm excitation and 530 nm emission. Histology was evaluated in PAS-stained sections. Mucin and HLA class II (IA) antigen were assessed by immunofluorescent staining. Tear IL-1beta was measured by ELISA, and tear matrix metalloproteinase (MMP)-9 by zymography. Gene expression in the corneal epithelia was analyzed by semiquantitative RT-PCR. In comparison to that in age-matched NRTN(+/+) mice, aqueous tear production, tear fluorescein clearance, and corneal sensation were significantly reduced in NRTN(-/-) mice, whereas corneal permeability to AFD was significantly increased. Immunoreactive MUC-4 and -5AC mucin and goblet cell density (P < 0.001) in the conjunctiva of NRTN(-/-) mice were lower than in NRTN(+/+) mice. The expression of MUC-1 and -4 mRNA by the corneal epithelium was reduced in NRTN(-/-) mice. There were a significantly greater number of IA antigen-positive conjunctival epithelial cells in NRTN(-/-) mice than NRTN(+/+) mice. Tear fluid IL-1beta and MMP-9 concentrations and the expression of IL-1beta, TNF-alpha, macrophage inflammatory protein (MIP)-2, cytokine-induced neutrophil chemoattractant (KC), and MMP-9 mRNA by the corneal epithelia were significantly increased in NRTN(-/-) mice, compared with NRTN(+/+) mice. Neurturin-deficient mice show phenotypic changes and ocular surface inflammation that mimic human keratoconjunctivitis sicca. This model supports the importance of a functional ocular surface-central nervous system-lacrimal gland sensory-autonomic neural network in maintaining ocular surface health and homeostasis.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

PubMed Central

Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris

2016-01-01

Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2−) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, and de novo protein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. PMID:26826234

Production and characterization of vaginal suppositories with propolis wax as active agent to prevent and treat Fluor albus

NASA Astrophysics Data System (ADS)

Farida, Siti; Azizah, Nurul; Hermansyah, Heri; Sahlan, Muhamad

2017-02-01

Based on the content contained in propolis wax especially antimicrobial function, it can be analyzed that propolis wax had superiority for Fluor albus. This research was conducted on two formulation of vaginal suppositories with base, supplementary and active agent as a fixed variable: 2% propolis wax (% w/w). Evaluation of this research were weight variation, melting time, consistency, irritation effect test and physical and chemical stability test (organoleptic, pH and polyphenol content).

Ground-Based Phase of Spaceflight Experiment "Biosignal" Using Autonomic Microflurimeter "Fluor-K"

NASA Astrophysics Data System (ADS)

Grigorieva, O. V.; Gal'chuk, S. V.; Rudimov, E. G.; Buravkova, L. B.

2013-02-01

The majority of flight experiments with the use of cell cultures and equipment like KUBIK and CRIOGEM carried out on board of the satellites (Bion, Foton) and ISS only allows the after-flight biosamples to be analyzed. As far as with few exceptions, the real-time cellular parameters registration for a long period is hard to be implemented. We developed the "Fluor-K" equipment - precision, small-sized, autonomous, two-channel, programmed fluorimeter. This device is designed for registration of differential fluorescent signal from organic and non-organic objects of microscale in small volumes (cellular organelles suspensions, animal and human cells, unicellular algae, bacteria, various fluorescent colloid solutions). Beside that, "Fluor-K" allows simultaneous detection of temperature. The ground-based tests of the device proved successful. The developed software can support experimental schedules while real-time data registration with the built-in storage device allows changes in selected parameters to be analyzed using wide range of fluorescent probes.

Progressively safer, cheaper demolition of Fernald

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robert Nichols; Norman Pennington

2000-09-29

Fluor Fernald, Inc. has been progressively improving Decontamination and Dismantlement (D&D) at the Department of Energy's Fernald Environmental Management Project by applying new technologies and better methodologies to the work. Demolition issues existed in the past that necessitated new or improved solutions to maintain worker safety, protect the environment and accomplish the work in a cost effective manner. Lessons learned from D&D of 80 structures has led to a systematic approach, which can be implemented in various D&D arenas. When facility production was halted, hold-up material and process residues remained in the process piping and components. Over 500,000 pounds ofmore » material was removed by workers who completed the tasks two years ahead of schedule, $7 million under budget and with an excellent safety record. This success was the result of detailed planning and irdision of lessons learned as work progressed from facility to facility. Work sequences were developed that reduced airborne contamination. Demolition of structures has been performed at Fernald by carefully selected and qualified subcontractors. Asbestos and lead abatement, equipment, piping and conduit removal, and structural demolition have been completed to progressively higher performance specifications developed by Fluor Fernald based on lessons learned during execution. Safety continues to be the primary consideration in performing potentially hazardous work. Technologies such as hydraulic shears have been developed and used to keep workers away from danger. A new technology, ''Cool Suits,'' has been demonstrated to help prevent heat stress when anti-contamination clothing is required in elevated temperature working conditions. For tall structures, implosion technologies have been employed with progressively improved results, Several other new technologies have been evaluated by Fluor Fernald and applied by subcontractors. The improved technologies included the oxy-gas torch, which uses gasoline instead of acetylene gas, and a vacuum system for asbestos removal of wall insulation. These new methods proved effective and beneficial. Fluor Fernald has integrated demolition activities with waste disposal requirements to enhance overall efficiency. The relatively straight steel configurations required for recycling, and waste acceptance criteria that dictate waste sizes are typically included in the subcontract specifications The progressive improvements by Fluor Fernald have led to cost savings and schedule acceleration without increased risk to workers or the environment. When Fluor Fernald came to the site in 1992, the remediation baseline reflected a completion schedule of 2020 and a cost of $7.2 billion. The current projection is 2008 and $4.2 billion.« less

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

PubMed

Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

2010-03-01

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

PubMed

Erlandsen, Stanley L; Jarroll, Edward; Wallis, Peter; van Keulen, Harry

2005-08-01

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice

NASA Astrophysics Data System (ADS)

Dobosz, Michael; Strobel, Steffen; Stubenrauch, Kay-Gunnar; Osl, Franz; Scheuer, Werner

2014-01-01

Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F( fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

Effects of perinatal asphyxia on rat striatal cytoskeleton.

PubMed

Saraceno, G E; Ayala, M V; Badorrey, M S; Holubiec, M; Romero, J I; Galeano, P; Barreto, G; Giraldez-Alvárez, L D; Kölliker-Fres, R; Coirini, H; Capani, F

2012-01-01

Perinatal asphyxia (PA) is a medical condition associated with a high short-term morbimortality and different long-term neurological diseases. In previous works, we have shown that neuronal and synaptic changes in rat striatum lead to ubi-protein accumulation in post-synaptic density (PSD) after six months of sub-severe PA. However, very little is known about the synaptic and related structural modifications induced by PA in young rats. In the present work, we studied neuronal cytoskeleton modifications in striatum induced by subsevere PA in 30-day-old rats. We observed a significant decrease in the number of neurons, in particular calbindin immunoreactive neurons after PA. In addition, it was also observed that actin cytoskeleton was highly modified in the PSD as well as an increment of F-actin staining by Phalloidin-alexa(488) in the striatum of PA rats. Using correlative fluorescence-electron microscopy photooxidation, we confirmed and extended confocal observations. F-actin staining augmentation was mostly related with an increment in the number of mushroom-shaped spines. Consistent with microscopic data, Western blot analysis revealed a β-actin increment in PSD in PA rats. On the other hand, MAP-2 immunostaining was decreased after PA, being NF-200 expression unmodified. Although neuronal death was observed, signs of generalized neurodegeneration were absent. Taken together these results showed early post-synaptic F-actin cytoskeleton changes induced by PA with slightly modifications in the other components of the neuronal cytoskeleton, suggesting that F-actin accumulation in the dendritic spines could be involved in the neuronal loss induced by PA. Copyright © 2011 Wiley Periodicals, Inc.

Integrated data analysis identifies potential inducers and pathways during the endothelial differentiation of bone-marrow stromal cells by DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine.

PubMed

Xu, Rui; Chen, Wenbin; Zhang, Zhifen; Qiu, Yang; Wang, Yong; Zhang, Bingchang; Lu, Wei

2018-05-30

Bone-Marrow Stromal Cells (BMSCs)-derived vascular endothelial cells (VECs) is regarded as an important therapeutic strategy for spinal cord injury, disc degeneration, cerebral ischemic disease and diabetes. The change in DNA methylation level is essential for stem cell differentiation. However, the DNA methylation related mechanisms underlying the endothelial differentiation of BMSCs are not well understood. In this study, DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC) significantly elevated the endothelial markers expression (CD31/PECAM1, CD105/ENG, eNOS and VE-cadherin), as well as promoted the capacity of angiogenesis on Matrigel. The result of Alexa 488-Ac-LDL uptake assay indicated that the differentiation ratio of BMSCs into VECs was 68.7% in 5-azaz-dC induced differentiation. And then we screened differentiation inducers with altered expression patterns and DNA methylation levels in four important families (VEGF, ANG, FGF and ETS). By integrating these data, five endothelial differentiation inducers (VEGFA, ANGPT2, FGF2, FGF9 and ETS1) which were directly upregulated by 5-aza-dC and five indirect factors (FGF1, FGF3, ETS2, ETV1 and ETV4) were identified. These data suggested that 5-aza-dC is an excellent chemical molecule for BMSCs differentiation into functional VECs and also provided essential clues for DNA methylation related signaling during 5-aza-dC induced endothelial differentiation of BMSCs. Copyright © 2018 Elsevier B.V. All rights reserved.

Alexa, Can I Trust You?

PubMed Central

Chung, Hyunji; Iorga, Michaela; Voas, Jeffrey; Lee, Sangjin

2017-01-01

Security diagnostics expose vulnerabilities and privacy threats that exist in commercial Intelligent Virtual Assistants (IVA) – diagnostics offer the possibility of securer IVA ecosystems. PMID:29213147

The occurrence of fluor-wagnerite in UHT granulites and its implications towards understanding fluid regimes in the evolution of deep crust: a case study from the Eastern Ghats Belt, India

NASA Astrophysics Data System (ADS)

Das, Kaushik; Tomioka, Naotaka; Bose, Sankar; Ando, Jun-ichi; Ohnishi, Ichiro

2017-06-01

We report the occurrence of a rare phosphate mineral, fluor-wagnerite (Mg1.91-1.94Fe0.06-0.07Ca<0.01) (P0.99-1.00O4)(OH0.02-0.17F0.98-0.83) from the Eastern Ghats Belt of India, an orogenic belt evolved during Meso- to Neoproterozoic time. The host rock, i.e. high- to ultrahigh temperature (UHT) granulites ( 1000 °C, 8-9 kbar) of the studied area was retrogressed after emplacement to mid-crustal level (800-850 °C, 6-6.5 kbar) as deduced from their pressure -temperature histories. Based on mineral chemical data and micro-Raman analyses, we document an unusual high Mg-F-rich chemistry of the F-wagnerite, which occur both in peak metamorphic porphyroblastic assemblages as well as in the retrograde matrix assemblage. Therefore, in absence of other common phosphates like apatite, fluor-wagnerite can act as an indicator for the presence of F-bearing fluids for rocks with high X Mg and/or fO2. The occurrence of F-rich minerals as monitors for fluid compositions has important implications for the onset of biotite dehydration melting and hence melt production in the deep crust. We propose that fluor-wagnerite can occur as an accessory mineral associated with F-rich fluids in lower-mid crustal rocks, and F in coexisting minerals should be taken into consideration when reconciling the petrogenetic grid of biotite-dehydration melting.

42 CFR 488.26 - Determining compliance.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Determining compliance. 488.26 Section 488.26... § 488.26 Determining compliance. (a) Additional rules for certification of compliance for SNFs and NFs are set forth in § 488.330. (b) The decision as to whether there is compliance with a particular...

Polyurethane/fluor-hydroxyapatite nanocomposite scaffolds for bone tissue engineering. Part I: morphological, physical, and mechanical characterization

PubMed Central

Asefnejad, Azadeh; Behnamghader, Aliasghar; Khorasani, Mohammad Taghi; Farsadzadeh, Babak

2011-01-01

In this study, new nano-fluor-hydroxyapatite (nFHA)/polyurethane composite scaffolds were fabricated for potential use in bone tissue engineering. Polyester urethane samples were synthesized from polycaprolactone, hexamethylene diisocyanate, and 1,4-butanediol as chain extender. Nano fluor-hydroxyapatite (nFHA) was successfully synthesized by sol-gel method. The solid–liquid phase separation and solvent sublimation methods were used for preparation of the porous composites. Mechanical properties, chemical structure, and morphological characteristics of the samples were investigated by compressive test, Fourier transform infrared, and scanning electron microscopy (SEM) techniques, respectively. The effect of nFHA powder content on porosity and pore morphology was investigated. SEM images demonstrated that the scaffolds were constituted of interconnected and homogeneously distributed pores. The pore size of the scaffolds was in the range 50–250 μm. The result obtained in this research revealed that the porosity and pore average size decreased and compressive modulus increased with nFHA percentage. Considering morphological, physical, and mechanical properties, the scaffold with a higher ratio of nFHA has suitable potential use in tissue regeneration. PMID:21289986

42 CFR 488.408 - Selection of remedies.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Selection of remedies. 488.408 Section 488.408... Compliance for Long-Term Care Facilities with Deficiencies § 488.408 Selection of remedies. (a) Categories of remedies. In this section, the remedies specified in § 488.406(a) are grouped into categories and applied...

42 CFR 498.3 - Scope and applicability.

Code of Federal Regulations, 2014 CFR

2014-10-01

... PROGRAM AND FOR DETERMINATIONS THAT AFFECT THE PARTICIPATION OF ICFs/IID AND CERTAIN NFs IN THE MEDICAID..., subpart D—for nursing facilities (NFs). (ii) Part 488, subpart E (§ 488.330(e))—for SNFs and NFs. (iii) Part 488, subpart E (§ 488.330(e)) and subpart F (§ 488.446)—for SNFs and NFs and their administrators...

42 CFR 498.3 - Scope and applicability.

Code of Federal Regulations, 2012 CFR

2012-10-01

... PROGRAM AND FOR DETERMINATIONS THAT AFFECT THE PARTICIPATION OF ICFs/IID AND CERTAIN NFs IN THE MEDICAID..., subpart D—for nursing facilities (NFs). (ii) Part 488, subpart E (§ 488.330(e))—for SNFs and NFs. (ii) Part 488, subpart E (§ 488.330(e)) and subpart F (§ 488.446)—for SNFs and NFs and their administrators...

42 CFR 498.3 - Scope and applicability.

Code of Federal Regulations, 2013 CFR

2013-10-01

... PROGRAM AND FOR DETERMINATIONS THAT AFFECT THE PARTICIPATION OF ICFs/IID AND CERTAIN NFs IN THE MEDICAID..., subpart D—for nursing facilities (NFs). (ii) Part 488, subpart E (§ 488.330(e))—for SNFs and NFs. (iii) Part 488, subpart E (§ 488.330(e)) and subpart F (§ 488.446)—for SNFs and NFs and their administrators...

42 CFR 498.3 - Scope and applicability.

Code of Federal Regulations, 2011 CFR

2011-10-01

... PROGRAM AND FOR DETERMINATIONS THAT AFFECT THE PARTICIPATION OF ICFs/MR AND CERTAIN NFs IN THE MEDICAID..., subpart D—for nursing facilities (NFs). (ii) Part 488, subpart E (§ 488.330(e))—for SNFs and NFs. (ii) Part 488, subpart E (§ 488.330(e)) and subpart F (§ 488.446)—for SNFs and NFs and their administrators...

Enhancing Endosomal Escape of Transduced Proteins by Photochemical Internalisation

PubMed Central

Mellert, Kevin; Lamla, Markus; Scheffzek, Klaus; Wittig, Rainer; Kaufmann, Dieter

2012-01-01

Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro. PMID:23285056

Enhancing endosomal escape of transduced proteins by photochemical internalisation.

PubMed

Mellert, Kevin; Lamla, Markus; Scheffzek, Klaus; Wittig, Rainer; Kaufmann, Dieter

2012-01-01

Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.

Popularity of Russian information sources of medical education.

PubMed

Vasilyeva, Irina V; Arseniev, Sergey B

2014-01-01

The aim of the present study is to analyze the popularity of information sources of medical educational sites <webmedinfo.ru>, medical information portal <meduniver.com>, medical portal for students <6years.net>, electronic library of medical literature <booksmed.com>, <medliter.ru> and <medbook.net.ru>. Three sites (<www.webmedinfo.ru>, <meduniver.com> and <6years.net>) provide sources of medical literature, educational videos, medical histories, medical papers and medical popular literature. And three other sites (<www.booksmed.com>, <www.medliter.ru> and <www.medbook.net.ru>) provide sources for electronic medical books on various subjects. Using on-line programs Alexa and Cy-pr we have analyzed the website's rating and identified the main data and time-varying data of the sites. Calculated Alexa Rank rating was determined for each site. Our study has shown that the most popular information sources of medical education among the six studied sites for Russian users is <meduniver.com>; the site <booksmed.com> is at the second place referring to the Alexa Rank rating and the site <webmedinfo.ru> is at the second place referring to the citation index in Yandex. The most popular medical site of electronic medical books is <booksmed.com>.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

PubMed Central

Kishen, Ria E. B.; Kluth, David C.; Bellamy, Christopher O. C.

2016-01-01

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches. PMID:27632367

Analysis of Molecular Movement Reveals Latticelike Obstructions to Diffusion in Heart Muscle Cells

PubMed Central

Illaste, Ardo; Laasmaa, Martin; Peterson, Pearu; Vendelin, Marko

2012-01-01

Intracellular diffusion in muscle cells is known to be restricted. Although characteristics and localization of these restrictions is yet to be elucidated, it has been established that ischemia-reperfusion injury reduces the overall diffusion restriction. Here we apply an extended version of raster image correlation spectroscopy to determine directional anisotropy and coefficients of diffusion in rat cardiomyocytes. Our experimental results indicate that diffusion of a smaller molecule (1127 MW fluorescently labeled ATTO633-ATP) is restricted more than that of a larger one (10,000 MW Alexa647-dextran), when comparing diffusion in cardiomyocytes to that in solution. We attempt to provide a resolution to this counterintuitive result by applying a quantitative stochastic model of diffusion. Modeling results suggest the presence of periodic intracellular barriers situated ∼1 μm apart having very low permeabilities and a small effect of molecular crowding in volumes between the barriers. Such intracellular structuring could restrict diffusion of molecules of energy metabolism, reactive oxygen species, and apoptotic signals, enacting a significant role in normally functioning cardiomyocytes as well as in pathological conditions of the heart. PMID:22385844

Characterization of cap-shaped silver particles for surface-enhanced fluorescence effects.

PubMed

Yamaguchi, Tetsuji; Kaya, Takatoshi; Takei, Hiroyuki

2007-05-15

Surface-enhanced fluorescence has potentially many desirable properties as an analytical method for medical diagnostics, but the effect observed so far is rather modest and only in conjunction with fluorophores with low quantum yields. Coupled with the fact that preparation of suitable surfaces at low costs has been difficult, this has limited its utilities. Here we report a novel method for forming uniform and reproducible surfaces with respectable enhancement ratios even for high-quantum-yield fluorophores. Formation of dense surface-adsorbed latex spheres on a flat surface via partial aggregation, followed by evaporation of silver, results in a film consisting of cap-shaped silver particles at high densities. Binding of fluorescence biomolecules, either through physisorption or antigen-antibody reaction, was performed, and enhancements close to 50 have been observed with fluorophores such as R-phycoerythrin and Alexa 546-labeled, bovine serum albumin, both of which have quantum yields around 0.8. We attribute this to the unique shape of the silver particle and the presence of abundant gaps among adjacent particles at high densities. The effectiveness of the new surface is also demonstrated with IL-6 sandwich assays.

42 CFR 488.312 - Consistency of survey results.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Consistency of survey results. 488.312 Section 488... Certification of Long-Term Care Facilities § 488.312 Consistency of survey results. CMS does and the survey... results and enforcement remedies. ...

38 CFR 4.88 - [Reserved

Code of Federal Regulations, 2012 CFR

2012-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2012-07-01 2012-07-01 false [Reserved] 4.88 Section 4.88 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88...

38 CFR 4.88 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2014-07-01 2014-07-01 false [Reserved] 4.88 Section 4.88 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88...

38 CFR 4.88 - [Reserved

Code of Federal Regulations, 2013 CFR

2013-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2013-07-01 2013-07-01 false [Reserved] 4.88 Section 4.88 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88...

38 CFR 4.88 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2010-07-01 2010-07-01 false [Reserved] 4.88 Section 4.88 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88...

38 CFR 4.88 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2011-07-01 2011-07-01 false [Reserved] 4.88 Section 4.88 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS SCHEDULE FOR RATING DISABILITIES Disability Ratings Infectious Diseases, Immune Disorders and Nutritional Deficiencies § 4.88...

Thermal and flow analysis of the Fluor Daniel, Inc., Nuclear Material Storage Facility renovation design (initial 30% effort of Title 1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinke, R.G.; Mueller, C.; Knight, T.D.

1998-03-01

The computational fluid dynamics code CFX4.2 was used to evaluate steady-state thermal-hydraulic conditions in the Fluor Daniel, Inc., Nuclear Material Storage Facility renovation design (initial 30% of Title 1). Thirteen facility cases were evaluated with varying temperature dependence, drywell-array heat-source magnitude and distribution, location of the inlet tower, and no-flow curtains in the drywell-array vault. Four cases of a detailed model of the inlet-tower top fixture were evaluated to show the effect of the canopy-cruciform fixture design on the air pressure and flow distributions.

Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells.

PubMed

Trache, Andreea; Meininger, Gerald A

2005-01-01

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

PubMed

Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

2014-05-01

Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

42 CFR 488.318 - Inadequate survey performance.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Inadequate survey performance. 488.318 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.318 Inadequate survey performance. (a) CMS considers survey...

42 CFR 488.318 - Inadequate survey performance.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Inadequate survey performance. 488.318 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.318 Inadequate survey performance. (a) CMS considers survey...

42 CFR 488.318 - Inadequate survey performance.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Inadequate survey performance. 488.318 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.318 Inadequate survey performance. (a) CMS considers survey...

42 CFR 488.318 - Inadequate survey performance.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Inadequate survey performance. 488.318 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.318 Inadequate survey performance. (a) CMS considers survey...

Neuroprotective effects of hypothermia on synaptic actin cytoskeletal changes induced by perinatal asphyxia.

PubMed

Muñiz, Javier; Romero, Juan; Holubiec, Mariana; Barreto, George; González, Janneth; Saint-Martin, Madeleine; Blanco, Eduardo; Carlos Cavicchia, Juan; Castilla, Rocío; Capani, Francisco

2014-05-14

Cerebral hypoxia-ischemia damages synaptic proteins, resulting in cytoskeletal alterations, protein aggregation and neuronal death. In the previous works, we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia that leads to ubi-protein accumulation. Recently, we also showed that, changes in F-actin organization could be related to early alterations induced by hypoxia in the Central Nervous System. However, little is known about effective treatment to diminish the damage. The main aim of this work is to study the effects of birth hypothermia on the actin cytoskeleton of neostriatal post-synaptic densities (PSD) in 60 days olds rats by immunohistochemistry, photooxidation and western blot. We used 2 different protocols of hypothermia: (a) intrahypoxic hypothermia at 15°C and (b) post-hypoxia hypothermia at 32°C. Consistent with previous data at 30 days, staining with phalloidin-Alexa(488) followed by confocal microscopy analysis showed an increase of F-actin fluorescent staining in the neostriatum of hypoxic animals. Correlative photooxidation electron microscopy confirmed these observations showing an increment in the number of mushroom-shaped F-actin staining spines in neostriatal excitatory synapses in rats subjected to hypoxia. In addition, western blot revealed β-actin increase in PSDs in hypoxic animals. The optic relative density measurement showed a significant difference between controls and hypoxic animals. When hypoxia was induced under hypothermic conditions, the changes observed in actin cytoskeleton were blocked. Post-hypoxic hypothermia showed similar answer but actin cytoskeleton modifications were not totally reverted as we observed at 15°C. These data suggest that the decrease of the body temperature decreases the actin modifications in dendritic spines preventing the neuronal death. Copyright © 2014 Elsevier B.V. All rights reserved.

Three-dimensional image study on the vascular structure after angiopoietin-1 transduction in isolated mouse pancreatic islets

NASA Astrophysics Data System (ADS)

He, Jing; Su, Dongming; Trucco, Massimo

2008-02-01

Angiopoietin-1 (Ang-1) is essential for remodeling the primitive vascular plexus during embryonic development and for reducing plasma leakage in inflammation of adult vasculature. However, the role for Ang-1 in maintenance of vascular stability in isolated pancreatic islets is not fully understood. In this study, we compared the difference of vascular morphology between Ang-1 treated (n=5) and control mouse islets (n=5) using both two- and three-dimensional optical image analysis. Isolated mouse islets were transduced with Ang-1 or Lac Z (control) vector at 37°C for 16 hours. Islets were incubated with both rat anti-CD31 antibody and rabbit anti-insulin antibody followed by incubation with Rhodamine-conjugated goat anti-rat IgG and Alexa-488 conjugated goat anti-rabbit IgG. Islets were viewed under a Nikon confocal microscope. Serial optical section images were captured and reconstructed using Nikon EZ-C1 software. Individual two-D and reconstructed three-D images were analyzed using MetaMorph Image Analysis software. Islet vascular density was determined. In two-D images, there was no significant difference of vascular density between the two groups. The vascular morphology didn't show any obvious differences in two-D images either. However, in the three-D images, we found higher vascular density and more vascular branches in the Ang-1 transducted islets and vascular dilation in control group. In conclusion, using three-D image analysis, Ang-1 displayed functions in maintenance of vascular stability and in stimulating growth of vascular branches in isolated mouse pancreatic islets. In order to study further the regeneration of different cell contents in the spherical pancreatic islet, three-D image analysis is an effective method to approach this goal.

Comparison of detection methods for cell surface globotriaosylceramide.

PubMed

Kim, Minji; Binnington, Beth; Sakac, Darinka; Fernandes, Kimberly R; Shi, Sheryl P; Lingwood, Clifford A; Branch, Donald R

2011-08-31

The cell surface-expressed glycosphingolipid (GSL), globotriaosylceramide (Gb(3)), is becoming increasingly important and is widely studied in the areas of verotoxin (VT)-mediated cytotoxicity, human immunodeficiency virus (HIV) infection, immunology and cancer. However, despite its diverse roles and implications, an optimized detection method for cell surface Gb(3) has not been determined. GSLs are differentially organized in the plasma membrane which can affect their availability for protein binding. To examine various detection methods for cell surface Gb(3), we compared four reagents for use in flow cytometry analysis. A natural ligand (VT1B) and three different monoclonal antibodies (mAbs) were optimized and tested on various human cell lines for Gb(3) detection. A differential detection pattern of cell surface Gb(3) expression, which was influenced by the choice of reagent, was observed. Two mAb were found to be suboptimal. However, two other methods were found to be useful as defined by their high percentage of positivity and mean fluorescence intensity (MFI) values. Rat IgM anti-Gb(3) mAb (clone 38-13) using phycoerythrin-conjugated secondary antibody was found to be the most specific detection method while the use of VT1B conjugated to Alexa488 fluorochrome was found to be the most sensitive; showing a rare crossreactivity only when Gb(4) expression was highly elevated. The findings of this study demonstrate the variability in detection of Gb(3) depending on the reagent and cell target used and emphasize the importance of selecting an optimal methodology in studies for the detection of cell surface expression of Gb(3). Copyright © 2011 Elsevier B.V. All rights reserved.

Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes.

PubMed

Lin, Guiting; Qiu, Xuefeng; Fandel, Thomas M; Albersen, Maarten; Wang, Zhong; Lue, Tom F; Lin, Ching-Shwun

2011-10-01

To investigate whether fluorochrome-conjugated phalloidin can delineate cavernous smooth muscle (CSM) cells and whether it can be combined with immunofluorescence (IF) staining to quantify erectile dysfunction (ED)-associated changes. ED was induced by cavernous nerve crush in rats. Penile tissues of control and ED rats were stained with Alexa-488-conjugated phalloidin and/or with antibodies against rat endothelial cell antigen (RECA), CD31, neuronal nitric oxide synthase (nNOS), and collagen-IV (Col-IV). Phalloidin was able to delineate CSM as composed of a circular and a longitudinal compartment. When combined with IF stain for CD31 or RECA, it helped the identification of the helicine arteries as covered by endothelial cells on both sides of the smooth muscle layer. When combined with IF stain for nNOS, it helped the identification that nNOS-positive nerves were primarily localized within the dorsal nerves and in the adventitia of dorsal arteries. When combined with IF stain for Col-IV, it helped identify that Col-IV was localized around smooth muscles and beneath the endothelium. Phalloidin also facilitated the quantitative analysis of ED-related changes in the penis. In rats with cavernous nerve injury, RECA or Col-IV expression did not change significantly, but CSM and nNOS nerve contents decreased significantly. Phalloidin stain improved penile histology, enabling the visualization of the circular and longitudinal compartments in the CSM. It also worked synergistically with IF stain, permitting the visualization of the dual endothelial covering in helicine arteries, and facilitating the quantification of ED-related histologic changes. Copyright © 2011 Elsevier Inc. All rights reserved.

Endomicroscopy and electromyography of neuromuscular junctions in situ

PubMed Central

Brown, Rosalind; Dissanayake, Kosala N; Skehel, Paul A; Ribchester, Richard R

2014-01-01

Objective Electromyography (EMG) is used routinely to diagnose neuromuscular dysfunction in a wide range of peripheral neuropathies, myopathies, and neuromuscular degenerative diseases including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Definitive neurological diagnosis may also be indicated by the analysis of pathological neuromuscular innervation in motor-point biopsies. Our objective in this study was to preempt motor-point biopsy by combining live imaging with electrophysiological analysis of slow degeneration of neuromuscular junctions (NMJs) in vivo. Methods We combined conventional needle electromyography with fiber-optic confocal endomicroscopy (CEM), using an integrated hand-held, 1.5-mm-diameter probe. We utilized as a test bed, various axotomized muscles in the hind limbs of anaesthetized, double-homozygous thy1.2YFP16: WldS mice, which coexpress the Wallerian-degeneration Slow (WldS) protein and yellow fluorescent protein (YFP) in motor neurons. We also tested exogenous vital stains, including Alexa488-α-bungarotoxin; the styryl pyridinium dye 4-Di-2-Asp; and a GFP conjugate of botulinum toxin Type A heavy chain (GFP-HcBoNT/A). Results We show that an integrated EMG/CEM probe is effective in longitudinal evaluation of functional and morphological changes that take place over a 7-day period during axotomy-induced, slow neuromuscular synaptic degeneration. EMG amplitude declined in parallel with overt degeneration of motor nerve terminals. EMG/CEM was safe and effective when nerve terminals and motor endplates were selectively stained with vital dyes. Interpretation Our findings constitute proof-of-concept, based on live imaging in an animal model, that combining EMG/CEM may be useful as a minimally invasive precursor or alternative to motor-point biopsy in neurological diagnosis and for monitoring local administration of potential therapeutics. PMID:25540801

12 CFR 48.8 - Capital requirements.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 12 Banks and Banking 1 2013-01-01 2013-01-01 false Capital requirements. 48.8 Section 48.8 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY RETAIL FOREIGN EXCHANGE TRANSACTIONS § 48.8 Capital requirements. A national bank offering or entering into retail forex transactions...

12 CFR 48.8 - Capital requirements.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 12 Banks and Banking 1 2014-01-01 2014-01-01 false Capital requirements. 48.8 Section 48.8 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY RETAIL FOREIGN EXCHANGE TRANSACTIONS § 48.8 Capital requirements. A national bank offering or entering into retail forex transactions...

12 CFR 48.8 - Capital requirements.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 12 Banks and Banking 1 2012-01-01 2012-01-01 false Capital requirements. 48.8 Section 48.8 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY RETAIL FOREIGN EXCHANGE TRANSACTIONS § 48.8 Capital requirements. A national bank offering or entering into retail forex transactions...

42 CFR 488.307 - Unannounced surveys.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Unannounced surveys. 488.307 Section 488.307 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.307 Unannounced surveys. (a) Basic rule. All standard surveys must be...

42 CFR 488.720 - Extended surveys.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Extended surveys. 488.720 Section 488.720 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.720 Extended surveys. (a) Purpose of survey. The purpose of an extended...

42 CFR 488.310 - Extended survey.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Extended survey. 488.310 Section 488.310 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.310 Extended survey. (a) Purpose of survey. The purpose of an extended...

42 CFR 488.307 - Unannounced surveys.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Unannounced surveys. 488.307 Section 488.307 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.307 Unannounced surveys. (a) Basic rule. All standard surveys must be...

42 CFR 488.307 - Unannounced surveys.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Unannounced surveys. 488.307 Section 488.307 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.307 Unannounced surveys. (a) Basic rule. All standard surveys must be...

42 CFR 488.308 - Survey frequency.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Survey frequency. 488.308 Section 488.308 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.308 Survey frequency. (a) Basic period. The survey agency must conduct...

42 CFR 488.307 - Unannounced surveys.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Unannounced surveys. 488.307 Section 488.307 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.307 Unannounced surveys. (a) Basic rule. All standard surveys must be...

42 CFR 488.710 - Standard surveys.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Standard surveys. 488.710 Section 488.710 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.710 Standard surveys. (a) For each HHA, the survey agency must conduct a...

42 CFR 488.715 - Partial extended surveys.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Partial extended surveys. 488.715 Section 488.715... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.715 Partial extended surveys. A partial extended survey is conducted...

42 CFR 488.11 - State survey agency functions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false State survey agency functions. 488.11 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.11 State survey agency functions. State and local agencies that have agreements under section 1864...

42 CFR 488.725 - Unannounced surveys.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Unannounced surveys. 488.725 Section 488.725 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.725 Unannounced surveys. (a) Basic rule. All HHA surveys must be unannounced...

42 CFR 488.715 - Partial extended surveys.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Partial extended surveys. 488.715 Section 488.715... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.715 Partial extended surveys. A partial extended survey is conducted...

42 CFR 488.310 - Extended survey.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Extended survey. 488.310 Section 488.310 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.310 Extended survey. (a) Purpose of survey. The purpose of an extended...

42 CFR 488.308 - Survey frequency.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Survey frequency. 488.308 Section 488.308 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.308 Survey frequency. (a) Basic period. The survey agency must conduct...

42 CFR 488.11 - State survey agency functions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false State survey agency functions. 488.11 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.11 State survey agency functions. State and local agencies that have agreements under section 1864...

42 CFR 488.310 - Extended survey.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Extended survey. 488.310 Section 488.310 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.310 Extended survey. (a) Purpose of survey. The purpose of an extended...

42 CFR 488.725 - Unannounced surveys.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Unannounced surveys. 488.725 Section 488.725 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.725 Unannounced surveys. (a) Basic rule. All HHA surveys must be unannounced...

42 CFR 488.11 - State survey agency functions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false State survey agency functions. 488.11 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.11 State survey agency functions. State and local agencies that have agreements under section 1864...

42 CFR 488.310 - Extended survey.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Extended survey. 488.310 Section 488.310 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.310 Extended survey. (a) Purpose of survey. The purpose of an extended...

42 CFR 488.307 - Unannounced surveys.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Unannounced surveys. 488.307 Section 488.307 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.307 Unannounced surveys. (a) Basic rule. All standard surveys must be...

42 CFR 488.308 - Survey frequency.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Survey frequency. 488.308 Section 488.308 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.308 Survey frequency. (a) Basic period. The survey agency must conduct...

42 CFR 488.710 - Standard surveys.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Standard surveys. 488.710 Section 488.710 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.710 Standard surveys. (a) For each HHA, the survey agency must conduct a...

42 CFR 488.308 - Survey frequency.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Survey frequency. 488.308 Section 488.308 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.308 Survey frequency. (a) Basic period. The survey agency must conduct...

42 CFR 488.310 - Extended survey.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Extended survey. 488.310 Section 488.310 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.310 Extended survey. (a) Purpose of survey. The purpose of an extended...

42 CFR 488.308 - Survey frequency.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Survey frequency. 488.308 Section 488.308 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.308 Survey frequency. (a) Basic period. The survey agency must conduct...

42 CFR 488.720 - Extended surveys.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Extended surveys. 488.720 Section 488.720 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.720 Extended surveys. (a) Purpose of survey. The purpose of an extended...

42 CFR 488.11 - State survey agency functions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false State survey agency functions. 488.11 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.11 State survey agency functions. State and local agencies that have agreements under section 1864...

42 CFR 488.406 - Available remedies.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Available remedies. 488.406 Section 488.406 Public... Long-Term Care Facilities with Deficiencies § 488.406 Available remedies. (a) General. In addition to the remedy of termination of the provider agreement, the following remedies are available: (1...

42 CFR 488.406 - Available remedies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Available remedies. 488.406 Section 488.406 Public... Long-Term Care Facilities with Deficiencies § 488.406 Available remedies. (a) General. In addition to the remedy of termination of the provider agreement, the following remedies are available: (1...

42 CFR 488.845 - Civil money penalties.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Civil money penalties. 488.845 Section 488.845 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Sanctions for Home Health Agencies With Deficiencies § 488.845 Civil money penalties. (a) Application. (1...

42 CFR 488.845 - Civil money penalties.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Civil money penalties. 488.845 Section 488.845 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Sanctions for Home Health Agencies With Deficiencies § 488.845 Civil money penalties. (a) Application. (1...

7 CFR 51.488 - Well formed.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 2 2014-01-01 2014-01-01 false Well formed. 51.488 Section 51.488 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Cantaloups 1 Definitions § 51.488 Well formed. Well formed means that the cantaloup has the normal shape characteristic of the variety. ...

7 CFR 51.488 - Well formed.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Well formed. 51.488 Section 51.488 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Cantaloups 1 Definitions § 51.488 Well formed. Well formed means that the cantaloup...

7 CFR 51.488 - Well formed.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Well formed. 51.488 Section 51.488 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Cantaloups 1 Definitions § 51.488 Well formed. Well formed means that the cantaloup...

7 CFR 51.488 - Well formed.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 2 2013-01-01 2013-01-01 false Well formed. 51.488 Section 51.488 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Cantaloups 1 Definitions § 51.488 Well formed. Well formed means that the cantaloup has the normal shape characteristic of the variety. ...

7 CFR 51.488 - Well formed.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Well formed. 51.488 Section 51.488 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Cantaloups 1 Definitions § 51.488 Well formed. Well formed means that the cantaloup...

42 CFR 488.314 - Survey teams.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Survey teams. 488.314 Section 488.314 Public Health... AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.314 Survey teams. (a) Team composition. (1) Surveys must be conducted by an...

42 CFR 488.314 - Survey teams.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Survey teams. 488.314 Section 488.314 Public Health... AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.314 Survey teams. (a) Team composition. (1) Surveys must be conducted by an...

42 CFR 488.312 - Consistency of survey results.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Consistency of survey results. 488.312 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.312 Consistency of survey results. CMS does and the survey...

42 CFR 488.305 - Standard surveys.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Standard surveys. 488.305 Section 488.305 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.305 Standard surveys. (a) For each SNF and NF, the State survey agency...

42 CFR 488.305 - Standard surveys.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Standard surveys. 488.305 Section 488.305 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.305 Standard surveys. (a) For each SNF and NF, the State survey agency...

42 CFR 488.314 - Survey teams.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Survey teams. 488.314 Section 488.314 Public Health... AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.314 Survey teams. (a) Team composition. (1) Surveys must be conducted by an...

42 CFR 488.7 - Validation survey.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Validation survey. 488.7 Section 488.7 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.7 Validation survey. (a) Basis for survey. CMS may require a survey of an accredited provider or supplier to...

42 CFR 488.7 - Validation survey.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Validation survey. 488.7 Section 488.7 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.7 Validation survey. (a) Basis for survey. CMS may require a survey of an accredited provider or supplier to...

42 CFR 488.312 - Consistency of survey results.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Consistency of survey results. 488.312 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.312 Consistency of survey results. CMS does and the survey...

42 CFR 488.314 - Survey teams.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Survey teams. 488.314 Section 488.314 Public Health... AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.314 Survey teams. (a) Team composition. (1) Surveys must be conducted by an...

42 CFR 488.305 - Standard surveys.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Standard surveys. 488.305 Section 488.305 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.305 Standard surveys. (a) For each SNF and NF, the State survey agency...

42 CFR 488.7 - Validation survey.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Validation survey. 488.7 Section 488.7 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.7 Validation survey. (a) Basis for survey. CMS may require a survey of an accredited provider or supplier to...

42 CFR 488.730 - Survey frequency and content.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Survey frequency and content. 488.730 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.730 Survey frequency and content. (a) Basic period. Each HHA must be...

42 CFR 488.305 - Standard surveys.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Standard surveys. 488.305 Section 488.305 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.305 Standard surveys. (a) For each SNF and NF, the State survey agency...

42 CFR 488.305 - Standard surveys.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Standard surveys. 488.305 Section 488.305 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.305 Standard surveys. (a) For each SNF and NF, the State survey agency...

42 CFR 488.312 - Consistency of survey results.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Consistency of survey results. 488.312 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.312 Consistency of survey results. CMS does and the survey...

42 CFR 488.312 - Consistency of survey results.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Consistency of survey results. 488.312 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.312 Consistency of survey results. CMS does and the survey...

42 CFR 488.7 - Validation survey.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Validation survey. 488.7 Section 488.7 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.7 Validation survey. (a) Basis for survey. CMS may require a survey of an accredited provider or supplier to...

42 CFR 488.7 - Validation survey.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Validation survey. 488.7 Section 488.7 Public...) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.7 Validation survey. (a) Basis for survey. CMS may require a survey of an accredited provider or supplier to...

42 CFR 488.730 - Survey frequency and content.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Survey frequency and content. 488.730 Section 488... (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Home Health Agencies § 488.730 Survey frequency and content. (a) Basic period. Each HHA must be...

42 CFR 488.314 - Survey teams.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Survey teams. 488.314 Section 488.314 Public Health... AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES Survey and Certification of Long-Term Care Facilities § 488.314 Survey teams. (a) Team composition. (1) Surveys must be conducted by an...

42 CFR 488.454 - Duration of remedies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Duration of remedies. 488.454 Section 488.454... Compliance for Long-Term Care Facilities with Deficiencies § 488.454 Duration of remedies. (a) Except as specified in paragraphs (b) and (d) of this section, alternative remedies continue until— (1) The facility...

42 CFR 488.454 - Duration of remedies.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Duration of remedies. 488.454 Section 488.454... Compliance for Long-Term Care Facilities with Deficiencies § 488.454 Duration of remedies. (a) Except as specified in paragraphs (b) and (d) of this section, alternative remedies continue until— (1) The facility...

42 CFR 488.64 - Remote facility variances for utilization review requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Remote facility variances for utilization review requirements. 488.64 Section 488.64 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Special Requirements § 488.64 Remote facility variances for utilization review requirements. (a...

42 CFR 488.64 - Remote facility variances for utilization review requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Remote facility variances for utilization review requirements. 488.64 Section 488.64 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Special Requirements § 488.64 Remote facility variances for utilization review requirements. (a...

42 CFR 488.64 - Remote facility variances for utilization review requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Remote facility variances for utilization review requirements. 488.64 Section 488.64 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Special Requirements § 488.64 Remote facility variances for utilization review requirements. (a...

42 CFR 488.64 - Remote facility variances for utilization review requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Remote facility variances for utilization review requirements. 488.64 Section 488.64 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Special Requirements § 488.64 Remote facility variances for utilization review requirements. (a...

42 CFR 488.835 - Temporary management.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Temporary management. 488.835 Section 488.835... Sanctions for Home Health Agencies With Deficiencies § 488.835 Temporary management. (a) Application. (1) CMS may impose temporary management of an HHA if it determines that an HHA has a condition-level...

42 CFR 488.415 - Temporary management.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Temporary management. 488.415 Section 488.415... Compliance for Long-Term Care Facilities with Deficiencies § 488.415 Temporary management. (a) Definition. Temporary management means the temporary appointment by CMS or the State of a substitute facility manager or...

42 CFR 488.835 - Temporary management.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Temporary management. 488.835 Section 488.835... Sanctions for Home Health Agencies With Deficiencies § 488.835 Temporary management. (a) Application. (1) CMS may impose temporary management of an HHA if it determines that an HHA has a condition-level...

42 CFR 488.415 - Temporary management.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Temporary management. 488.415 Section 488.415... Compliance for Long-Term Care Facilities with Deficiencies § 488.415 Temporary management. (a) Definition. Temporary management means the temporary appointment by CMS or the State of a substitute facility manager or...

42 CFR 488.415 - Temporary management.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Temporary management. 488.415 Section 488.415... Compliance for Long-Term Care Facilities with Deficiencies § 488.415 Temporary management. (a) Definition. Temporary management means the temporary appointment by CMS or the State of a substitute facility manager or...

42 CFR 488.415 - Temporary management.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Temporary management. 488.415 Section 488.415... Compliance for Long-Term Care Facilities with Deficiencies § 488.415 Temporary management. (a) Definition. Temporary management means the temporary appointment by CMS or the State of a substitute facility manager or...

42 CFR 488.425 - Directed inservice training.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Directed inservice training. 488.425 Section 488... Compliance for Long-Term Care Facilities with Deficiencies § 488.425 Directed inservice training. (a) Required training. CMS or the State agency may require the staff of a facility to attend an inservice...

42 CFR 488.425 - Directed inservice training.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Directed inservice training. 488.425 Section 488... Compliance for Long-Term Care Facilities with Deficiencies § 488.425 Directed inservice training. (a) Required training. CMS or the State agency may require the staff of a facility to attend an inservice...

42 CFR 488.425 - Directed inservice training.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Directed inservice training. 488.425 Section 488... Compliance for Long-Term Care Facilities with Deficiencies § 488.425 Directed inservice training. (a) Required training. CMS or the State agency may require the staff of a facility to attend an inservice...

42 CFR 488.425 - Directed inservice training.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Directed inservice training. 488.425 Section 488... Compliance for Long-Term Care Facilities with Deficiencies § 488.425 Directed inservice training. (a) Required training. CMS or the State agency may require the staff of a facility to attend an inservice...

42 CFR 488.425 - Directed inservice training.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Directed inservice training. 488.425 Section 488... Compliance for Long-Term Care Facilities with Deficiencies § 488.425 Directed inservice training. (a) Required training. CMS or the State agency may require the staff of a facility to attend an inservice...

42 CFR 488.444 - Civil money penalties: Settlement of penalties.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Civil money penalties: Settlement of penalties. 488.444 Section 488.444 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.444 Civil money penalties...

42 CFR 488.444 - Civil money penalties: Settlement of penalties.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Civil money penalties: Settlement of penalties. 488.444 Section 488.444 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.444 Civil money penalties...

42 CFR 488.444 - Civil money penalties: Settlement of penalties.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Civil money penalties: Settlement of penalties. 488.444 Section 488.444 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.444 Civil money penalties...

42 CFR 488.444 - Civil money penalties: Settlement of penalties.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Civil money penalties: Settlement of penalties. 488.444 Section 488.444 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.444 Civil money penalties...

42 CFR 488.444 - Civil money penalties: Settlement of penalties.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Civil money penalties: Settlement of penalties. 488.444 Section 488.444 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND... Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.444 Civil money penalties...

42 CFR 488.422 - State monitoring.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false State monitoring. 488.422 Section 488.422 Public... Long-Term Care Facilities with Deficiencies § 488.422 State monitoring. (a) A State monitor— (1... deficiencies on the last 3 consecutive standard surveys. (c) State monitoring is discontinued when— (1) The...

42 CFR 488.415 - Temporary management.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Temporary management. 488.415 Section 488.415... Compliance for Long-Term Care Facilities with Deficiencies § 488.415 Temporary management. (a) Definition. Temporary management means the temporary appointment by CMS or the State of a substitute facility manager or...

42 CFR 488.400 - Statutory basis.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Statutory basis. 488.400 Section 488.400 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Long-Term Care Facilities with Deficiencies § 488.400 Statutory basis. Sections 1819(h) and 1919(h) of...

A fluorescent approach for identifying P2X1 ligands

PubMed Central

Ruepp, Marc-David; Brozik, James A.; de Esch, Iwan J.P.; Farndale, Richard W.; Murrell-Lagnado, Ruth D.; Thompson, Andrew J.

2015-01-01

There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 106 M−1 s−1) and koff (0.136 s−1) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology’. PMID:26026951

A fluorescent approach for identifying P2X1 ligands.

PubMed

Ruepp, Marc-David; Brozik, James A; de Esch, Iwan J P; Farndale, Richard W; Murrell-Lagnado, Ruth D; Thompson, Andrew J

2015-11-01

There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 10(6) M(-1) s(-1)) and koff (0.136 s(-1)) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evaluation of Dynamic Changes in Rating of Russian Information Sources of Medical Education Sites.

PubMed

Vasilyeva, Irina V; Arseniev, Sergey B

2016-01-01

The aim of the present study is to analyze dynamic changes in the rating of information sources of medical literature in the sites of the following electronic libraries (<rsl.ru>, <rssi.ru>, <elibrary.ru>) and the rating of information sources for electronic medical books (<booksmed.com>, <medliter.ru> <medbook.net.ru>). While using the on-line programs Alexa and Cy-pr, we have analyzed their website's rating and identified basic data and time-varying site data obtained for fourteen months. Alexa Rank rating was calculated for each sitemonthly. Our study has shown that the most popular information sources of medical education among the six studied sites for Russian users is <elibrary.ru>; the site <rssi.ru> is at the second place.

Alexa, Siri, Cortana, and More: An Introduction to Voice Assistants.

PubMed

Hoy, Matthew B

2018-01-01

Voice assistants are software agents that can interpret human speech and respond via synthesized voices. Apple's Siri, Amazon's Alexa, Microsoft's Cortana, and Google's Assistant are the most popular voice assistants and are embedded in smartphones or dedicated home speakers. Users can ask their assistants questions, control home automation devices and media playback via voice, and manage other basic tasks such as email, to-do lists, and calendars with verbal commands. This column will explore the basic workings and common features of today's voice assistants. It will also discuss some of the privacy and security issues inherent to voice assistants and some potential future uses for these devices. As voice assistants become more widely used, librarians will want to be familiar with their operation and perhaps consider them as a means to deliver library services and materials.

Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

PubMed

Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2015-12-01

Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

42 CFR 488.60 - Special procedures for approving end stage renal disease facilities.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Special procedures for approving end stage renal disease facilities. 488.60 Section 488.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES Special Requirements § 488.60 Special procedures for approving end stage renal disease...

42 CFR 488.60 - Special procedures for approving end stage renal disease facilities.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Special procedures for approving end stage renal disease facilities. 488.60 Section 488.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES Special Requirements § 488.60 Special procedures for approving end stage renal disease...

42 CFR 488.442 - Civil money penalties: Due date for payment of penalty.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Civil money penalties: Due date for payment of penalty. 488.442 Section 488.442 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.442 Civil money...

42 CFR 488.442 - Civil money penalties: Due date for payment of penalty.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Civil money penalties: Due date for payment of penalty. 488.442 Section 488.442 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.442 Civil money...

42 CFR 488.442 - Civil money penalties: Due date for payment of penalty.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Civil money penalties: Due date for payment of penalty. 488.442 Section 488.442 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.442 Civil money...

42 CFR 488.442 - Civil money penalties: Due date for payment of penalty.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Civil money penalties: Due date for payment of penalty. 488.442 Section 488.442 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.442 Civil money...

42 CFR 488.442 - Civil money penalties: Due date for payment of penalty.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Civil money penalties: Due date for payment of penalty. 488.442 Section 488.442 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF... PROCEDURES Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.442 Civil money...

42 CFR 488.9 - Onsite observation of accreditation organization operations.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Onsite observation of accreditation organization operations. 488.9 Section 488.9 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) STANDARDS AND CERTIFICATION SURVEY, CERTIFICATION, AND ENFORCEMENT PROCEDURES General Provisions § 488.9 Onsite...

42 CFR 488.60 - Special procedures for approving end stage renal disease facilities.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Special procedures for approving end stage renal disease facilities. 488.60 Section 488.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES Special Requirements § 488.60 Special procedures for approving end stage renal disease...

42 CFR 488.60 - Special procedures for approving end stage renal disease facilities.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Special procedures for approving end stage renal disease facilities. 488.60 Section 488.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES Special Requirements § 488.60 Special procedures for approving end stage renal disease...

42 CFR 488.60 - Special procedures for approving end stage renal disease facilities.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Special procedures for approving end stage renal disease facilities. 488.60 Section 488.60 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES Special Requirements § 488.60 Special procedures for approving end stage renal disease...

42 CFR 488.28 - Providers or suppliers, other than SNFs and NFs, with deficiencies.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 5 2012-10-01 2012-10-01 false Providers or suppliers, other than SNFs and NFs, with deficiencies. 488.28 Section 488.28 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES General Provisions § 488.28 Providers or suppliers, other than SNFs and NFs, with...

42 CFR 488.28 - Providers or suppliers, other than SNFs and NFs, with deficiencies.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 5 2011-10-01 2011-10-01 false Providers or suppliers, other than SNFs and NFs, with deficiencies. 488.28 Section 488.28 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES General Provisions § 488.28 Providers or suppliers, other than SNFs and NFs, with...

42 CFR 488.28 - Providers or suppliers, other than SNFs and NFs, with deficiencies.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Providers or suppliers, other than SNFs and NFs, with deficiencies. 488.28 Section 488.28 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES General Provisions § 488.28 Providers or suppliers, other than SNFs and NFs, with...

42 CFR 488.438 - Civil money penalties: Amount of penalty.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Civil money penalties: Amount of penalty. 488.438... Enforcement of Compliance for Long-Term Care Facilities with Deficiencies § 488.438 Civil money penalties... review authority) finds that the basis for imposing a civil money penalty exists, as specified in § 488...

47 CFR 22.1007 - Channels for offshore radiotelephone systems.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 491.550 488.700 491.700 (3) These channels may be assigned for use by relay stations in systems where it would be impractical to provide offshore radiotelephone service without the use of relay stations... protection of life and property. Central Subscriber Central Subscriber 488.825 491.825 488.875 491.875 488...

47 CFR 22.1007 - Channels for offshore radiotelephone systems.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 491.550 488.700 491.700 (3) These channels may be assigned for use by relay stations in systems where it would be impractical to provide offshore radiotelephone service without the use of relay stations... protection of life and property. Central Subscriber Central Subscriber 488.825 491.825 488.875 491.875 488...

Zfp488 promotes oligodendrocyte differentiation of neural progenitor cells in adult mice after demyelination

PubMed Central

Soundarapandian, Mangala M.; Selvaraj, Vimal; Lo, U-Ging; Golub, Mari S.; Feldman, Daniel H.; Pleasure, David E.; Deng, Wenbin

2011-01-01

Basic helix-loop-helix transcription factors Olig1 and Olig2 critically regulate oligodendrocyte development. Initially identified as a downstream effector of Olig1, an oligodendrocyte-specific zinc finger transcription repressor, Zfp488, cooperates with Olig2 function. Although Zfp488 is required for oligodendrocyte precursor formation and differentiation during embryonic development, its role in oligodendrogenesis of adult neural progenitor cells is not known. In this study, we tested whether Zfp488 could promote an oligodendrogenic fate in adult subventricular zone (SVZ) neural stem/progenitor cells (NSPCs). Using a cuprizone-induced demyelination model in mice, we examined the effect of retrovirus-mediated Zfp488 overexpression in SVZ NSPCs. Our results showed that Zfp488 efficiently promoted the differentiation of the SVZ NSPCs into mature oligodendrocytes in vivo. After cuprizone-induced demyelination injury, Zfp488-transduced mice also showed significant restoration of motor function to levels comparable to control mice. Together, these findings identify a previously unreported role for Zfp488 in adult oligodendrogenesis and functional remyelination after injury. PMID:22355521

SCINTILLATOR COMPOSITION FOR COUNTERS AND METHOD OF MAKING

DOEpatents

Buck, W.L.; Swank, R.K.

1958-02-25

This patent deals with a new composition for plastic scintillators and the method of making them. This is accomplished by mixing a solvent, selected from the group consisting of styrene, methylstyrene where the methyl group is attached to the ring, and p-vinylbiphenyl with p-terphenyl as a primary fluor. Marked improvement in the fluorescent properties of this scintillator composition is obtained by incorporating as a second fluor, a small amount of a highly conjugated hydrocarbon having four phenyl groups such as quaterphenyl or 1,1,4,4- tetraphenyl-1,3-butadiene. It is advisable to use very pure monomers in this composition, and to carry out its preparation in the absence of air.

Fluor Daniel Hanford Inc. integrated safety management system phase 1 verification final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

PARSONS, J.E.

1999-10-28

The purpose of this review is to verify the adequacy of documentation as submitted to the Approval Authority by Fluor Daniel Hanford, Inc. (FDH). This review is not only a review of the Integrated Safety Management System (ISMS) System Description documentation, but is also a review of the procedures, policies, and manuals of practice used to implement safety management in an environment of organizational restructuring. The FDH ISMS should support the Hanford Strategic Plan (DOE-RL 1996) to safely clean up and manage the site's legacy waste; deploy science and technology while incorporating the ISMS theme to ''Do work safely''; andmore » protect human health and the environment.« less

42 CFR 488.432 - Civil money penalties: When a penalty is collected.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Civil money penalties: When a penalty is collected. 488.432 Section 488.432 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH... waives, in writing, its right to a hearing as specified in § 488.436, for a civil money penalty imposed...

42 CFR 488.28 - Providers or suppliers, other than SNFs, NFs, and HHAs with deficiencies.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 5 2013-10-01 2013-10-01 false Providers or suppliers, other than SNFs, NFs, and HHAs with deficiencies. 488.28 Section 488.28 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES General Provisions § 488.28 Providers or suppliers, other than SNFs, NFs, and HHAs with...

42 CFR 488.28 - Providers or suppliers, other than SNFs, NFs, and HHAs with deficiencies.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 5 2014-10-01 2014-10-01 false Providers or suppliers, other than SNFs, NFs, and HHAs with deficiencies. 488.28 Section 488.28 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES... ENFORCEMENT PROCEDURES General Provisions § 488.28 Providers or suppliers, other than SNFs, NFs, and HHAs with...

46 CFR 153.488 - Design and equipment for tanks carrying high melting point NLSs: Category B.

Code of Federal Regulations, 2010 CFR

2010-10-01

... HAZARDOUS MATERIALS Design and Equipment Design and Equipment for Pollution Control § 153.488 Design and... 46 Shipping 5 2010-10-01 2010-10-01 false Design and equipment for tanks carrying high melting point NLSs: Category B. 153.488 Section 153.488 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY...

46 CFR 153.488 - Design and equipment for tanks carrying high melting point NLSs: Category B.

Code of Federal Regulations, 2014 CFR

2014-10-01

... HAZARDOUS MATERIALS Design and Equipment Design and Equipment for Pollution Control § 153.488 Design and... 46 Shipping 5 2014-10-01 2014-10-01 false Design and equipment for tanks carrying high melting point NLSs: Category B. 153.488 Section 153.488 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY...

46 CFR 153.488 - Design and equipment for tanks carrying high melting point NLSs: Category B.

Code of Federal Regulations, 2013 CFR

2013-10-01

... HAZARDOUS MATERIALS Design and Equipment Design and Equipment for Pollution Control § 153.488 Design and... 46 Shipping 5 2013-10-01 2013-10-01 false Design and equipment for tanks carrying high melting point NLSs: Category B. 153.488 Section 153.488 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY...

46 CFR 153.488 - Design and equipment for tanks carrying high melting point NLSs: Category B.

Code of Federal Regulations, 2012 CFR

2012-10-01

... HAZARDOUS MATERIALS Design and Equipment Design and Equipment for Pollution Control § 153.488 Design and... 46 Shipping 5 2012-10-01 2012-10-01 false Design and equipment for tanks carrying high melting point NLSs: Category B. 153.488 Section 153.488 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY...

46 CFR 153.488 - Design and equipment for tanks carrying high melting point NLSs: Category B.

Code of Federal Regulations, 2011 CFR

2011-10-01

... HAZARDOUS MATERIALS Design and Equipment Design and Equipment for Pollution Control § 153.488 Design and... 46 Shipping 5 2011-10-01 2011-10-01 false Design and equipment for tanks carrying high melting point NLSs: Category B. 153.488 Section 153.488 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY...

Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.

PubMed

Cheng, Qing; Wållberg, Helena; Grafström, Jonas; Lu, Li; Thorell, Jan-Olov; Hägg Olofsson, Maria; Linder, Stig; Johansson, Katarina; Tegnebratt, Tetyana; Arnér, Elias S J; Stone-Elander, Sharon; Ahlzén, Hanna-Stina Martinsson; Ståhl, Stefan

2016-12-01

Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377-ST and ZTaq:3638-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with (11)C for in vivo PET studies. Kinetic scans with the (11)C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZEGFR:2377-ST-DyLight488. [Methyl-(11)C]-labeled ZEGFR:2377-ST-CH3 and ZTaq:3638-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-(11)C]-ZEGFR:2377-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since ZEGFR:2377 binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and (11)C-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.