Scanning microarray slides.

PubMed

Ares, Manuel

2014-02-01

Here we describe some practical concerns surrounding the scanning of microarray slides that have been hybridized with fluorescent dyes. We use a laser scanner that has two lasers, each set to excite a different fluor, and separate detectors to capture emission from each fluor. The laser passes over an address (position on the scanned surface) and the detectors capture photons emitted from each address. Two superimposed image files are written that carry intensities for each channel for each pixel of the image scan. These are the raw data. Image analysis software is used to identify and summarize the intensities of the pixels that make up each spot. After comparison to background pixels, the processed intensity levels representing the gene expression measurements are associated with the identity of each spot.

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

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.

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.

REMOVAL OF SYNTHETIC ORGANIC CHEMICAL CONTAMINANTS IN DRINKING WATER: RASCO, INC. ADVANCED SIMULTANEOUS OXIDATION PROCESS (ASOP)

EPA Science Inventory

The RASco, Inc. ASOP Drinking Water Treatment Module was tested at NSF’s Laboratory for the reduction of the following chemicals of concern: aldicarb, benzene, carbofuran, chloroform, dichlorvos, dicrotophos, methomyl, mevinphos, nicotine, oxamyl, paraquat, phorate, sodium fluor...

Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

PubMed Central

Gilabert-Oriol, Roger; Thakur, Mayank; von Mallinckrodt, Benedicta; Bhargava, Cheenu; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F.; Fuchs, Hendrik; Weng, Alexander

2014-01-01

Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM. PMID:24859158

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

Cytology of infection of apple leaves by Diplocarpon mali

USDA-ARS?s Scientific Manuscript database

Diplocarpon mali, the causal agent of Marssonina leaf blotch of apple, causes severe defoliation during the growing season. Little information is available on the mode of infection and infection process. In this study, the infection strategies of D. mali in apple leaves were investigated using fluor...

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.

Volume Labeling with Alexa-Fluor Dyes and Surface Functionalization of Highly Sensitive Fluorescent SiO2 Nanoparticles

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

Wang, Wei; Foster, Carmen M; Morrell-Falvey, Jennifer L

2013-01-01

A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and amore » polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or free surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.« less

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

Volume labeling with Alexa Fluor dyes and surface functionalization of highly sensitive fluorescent silica (SiO2) nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Wei; Nallathamby, Prakash D.; Foster, Carmen M.; Morrell-Falvey, Jennifer L.; Mortensen, Ninell P.; Doktycz, Mitchel J.; Gu, Baohua; Retterer, Scott T.

2013-10-01

A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems. Electronic supplementary information (ESI) available: Cell culture preparation for dose/response imaging experiments. See DOI: 10.1039/c3nr02639f

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-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

PubMed Central

Schaub, Franz X; Reza, Md Shamim; Flaveny, Colin A; Li, Weimin; Musicant, Adele M; Hoxha, Sany; Guo, Min; Cleveland, John L; Amelio, Antonio L

2015-01-01

Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer (BRET) that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bi-functional nature of these LumiFluor reporters enables greatly improved spatio-temporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, non-invasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes. PMID:26424696

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.

Tethered Hsp90 Inhibitors Carrying Optical or Radioiodinated Probes Reveal Selective Internalization of Ectopic Hsp90 in Malignant Breast Tumor Cells

PubMed Central

Barrott, Jared J.; Hughes, Philip F.; Osada, Takuya; Yang, Xiao-Yi; Hartman, Zachary C.; Loiselle, David R.; Spector, Neil L.; Neckers, Len; Rajaram, Narasimhan; Hu, Fangyao; Ramanujam, Nimmi; Vaidyanathan, Ganesan; Zalutsky, Michael R.; Lyerly, H. Kim; Haystead, Timothy A.

2013-01-01

Summary Hsp90 inhibitors have demonstrated unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained but could be influenced by ectopically expressed Hsp90 in tumors. We have synthesized novel Hsp90 inhibitors that can carry optical or radioiodinated probes via a PEG tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein is occurring at the plasma membrane. In mice, we show exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors targeting ectopic Hsp90 can be used to detect breast cancer malignancies through non-invasive imaging. PMID:24035283

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.

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

Nanosizing a Metal-Organic Framework Enzyme Carrier for Accelerating Nerve Agent Hydrolysis

DTIC Science & Technology

2016-10-05

Previously, biodegradable liposome nano- carriers have been shown to be effective at providing functionally significant amounts of highly purified enzymes in...AlexaFluor-647 dye was purchased from Life Technologies (Thermo Fisher Scientific). Methyl 6-(pinacolboryl)-2-naphthoate was synthesized using a published...Hitachi) and PXRD (Smartlab, Rigaku). Labeling OPAA with Fluorescent Dye . AlexaFluor-647-labeled OPAA (OPAA647) was prepared by reacting OPAA (0.5

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.

Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models

NASA Astrophysics Data System (ADS)

Maawy, Ali A.; Hiroshima, Yukihiko; Kaushal, Sharmeela; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

2013-12-01

The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.

Late magmatic controls on the origin of schorlitic and foititic tourmalines from late-Variscan peraluminous granites of the Arbus pluton (SW Sardinia, Italy): Crystal-chemical study and petrological constraints

NASA Astrophysics Data System (ADS)

Bosi, Ferdinando; Naitza, Stefano; Skogby, Henrik; Secchi, Francesco; Conte, Aida M.; Cuccuru, Stefano; Hålenius, Ulf; De La Rosa, Nathaly; Kristiansson, Per; Charlotta Nilsson, E. J.; Ros, Linus; Andreozzi, Giovanni B.

2018-05-01

Tourmalines from the late-Variscan Arbus pluton (SW Sardinia) and its metamorphic aureole were structurally and chemically characterized by single-crystal X-ray diffraction, electron and nuclear microprobe analysis, Mössbauer, infrared and optical absorption spectroscopy, to elucidate their origin and relationships with the magmatic evolution during the pluton cooling stages. The Arbus pluton represents a peculiar shallow magmatic system, characterized by sekaninaite (Fe-cordierite)-bearing peraluminous granitoids, linked via AFC processes to gabbroic mantle-derived magmas. The Fe2+-Al-dominant tourmalines occur in: a) pegmatitic layers and pods, as prismatic crystals; b) greisenized rocks and spotted granophyric dikes, as clots or nests of fine-grained crystals in small miaroles locally forming orbicules; c) pegmatitic veins and pods close to the contacts within the metamorphic aureole. Structural formulae indicate that tourmaline in pegmatitic layers is schorl, whereas in greisenized rocks it ranges from schorl to fluor-schorl. Tourmalines in thermometamorphosed contact aureole are schorl, foitite and Mg-rich oxy-schorl. The main substitution is Na + Fe2+ ↔ □ + Al, which relates schorl to foitite. The homovalent substitution (OH) ↔ F at the O1 crystallographic site relates schorl to fluor-schorl, while the heterovalent substitution Fe2+ + (OH, F) ↔ Al + O relates schorl/fluor-schorl to oxy-schorl. Tourmaline crystallization in the Arbus pluton was promoted by volatile (B, F and H2O) enrichment, low oxygen fugacity and Fe2+ activity. The mineralogical evolutive trend is driven by decreasing temperature, as follows: sekaninaite + quartz → schorl + quartz → fluor-schorl + quartz → foitite + quartz. The schorl → foitite evolution represents a distinct trend towards (Al + □) increase and unit-cell volume decrease. These trends are typical of granitic magmas and consistent with Li-poor granitic melts, as supported by the absence of elbaite and other Li-minerals in the Arbus pluton. Tourmaline-bearing rocks reflect the petrogenetic significance of contribution from a metapelitic crustal component during the evolution of magmas in the middle-upper crust.

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.

Green Tea Catechins Quench the Fluorescence of Bacteria-Conjugated Alexa Fluor Dyes

PubMed Central

Zhao, Lin; Li, Wei; Zhu, Shu; Tsai, Sheena; Li, Jianhua; Tracey, Kevin J.; Wang, Ping; Fan, Saijun; Sama, Andrew E.; Wang, Haichao

2013-01-01

Accumulating evidence suggests that Green tea polyphenolic catechins, especially the (-)-epigallocatechin gallate (EGCG), can be cross-linked to many proteins, and confer a wide range of anti-bacterial activities possibly by damaging microbial cytoplasmic lipids and proteins. At the doses that conferred protection against lethal polymicrobial infection (induced by cecal ligation and puncture), EGCG significantly reduced bacterial loads particularly in the liver and lung. To elucidate its bactericidal mechanisms, we determined whether EGCG affected the fluorescence intensities of bacteria-conjugated Alexa Fluor 488 or 594 dyes. When mixed with unconjugated Alexa Fluor 488 or 594 dyes, EGCG or analogs did not affect the fluorescence intensity of these dyes. In a sharp contrast, EGCG and some analogs (e.g., Catechin Gallate, CG), markedly reduced the fluorescence intensity of Gram-positive Staphylococcus aureus-conjugated Alexa 594 and Gram-negative Escherichia coli-conjugated Alexa 488. Interestingly, co-treatment with ethanol impaired the EGCG-mediated fluorescence quenching of the G+ S. aureus, but not of the G- E. coli-conjugated Alexa Flour dyes. In light of the notion that Alexa Fluor dyes can be quenched by aromatic amino acids, it is plausible that EGCG exerts anti-microbial activities possibly by altering microbial protein conformations and functions. This possibility can now be explored by screening other fluorescence-quenching agents for possible antimicrobial activities. PMID:24011199

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

CONTROL CHART DASHBOARDS MANAGING YOUR NUMBERS INSTEAD OF YOU NUMBER MANAGING YOU

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

PREVETTE, S.S.

2006-11-15

This paper, which documents Fluor Hanford's application of Statistical Process Control (SPC) and Dashboards to support planning and decision making, is a sequel to ''Leading with Leading Indicators'' that was presented at WM 05. This year's paper provides more detail on management's use of SPC and control charts and discusses their integration into an executive summary using the popular color-cod3ed dashboard methodology. Fluor Hanford has applied SPC in a non-traditional (that is non-manufacturing) manner. Dr. Shewhart's 75-year-old control-chart methodologies have been updated to modern data processing, but are still founded on his sound, tried and true principles. These methods aremore » playing a key role in safety and quality at what has been called the world's largest environmental cleanup project. The US Department of Energy's (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 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.« less

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.

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

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.

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

Sol-gel derived fluor-hydroxyapatite biocoatings on zirconia substrate.

PubMed

Kim, Hae-Won; Kong, Young-Min; Bae, Chang-Jun; Noh, Yoon-Jung; Kim, Hyoun-Ee

2004-07-01

Fluor-hydroxyapatite (FHA) film was coated on a zirconia (ZrO(2)) substrate by a sol-gel method. An appropriate amount of F ions was incorporated into the hydroxyapatite (HA) during the preparation of the sols. The apatite phase began to crystallize after heat treatment at 400 degrees C, and increased in intensity above 500 degrees C. No decomposition was detected by X-ray diffraction analyses up to 800 degrees C, which illustrates the high thermal stability of the FHA films. The films showed a uniform and dense morphology with a thickness of approximately 1 microm after a precisely controlled heat treatment process. These FHA films adhered firmly to the zirconia substrate, representing notable adhesion strengths of approximately 70 MPa after heat treatment above 500 degrees C. The dissolution rate of the FHA coating layer varied according to the heat treatment temperature, which was closely related to the film crystallinity. The dissolution rate of the FHA film was lower than that of the HA film, suggesting the possibility of a functional gradient coating of HA and FHA. The MG63 cells seeded onto the FHA films proliferated in a similar manner to those seeded onto pure HA ceramic and a plastic control.

Photoluminescence properties of polystyrene-hosted fluorophore thin films

NASA Astrophysics Data System (ADS)

Chakraborty, Subha; Harris, Katherine; Huang, Mengbing

2016-12-01

We report on a photo-luminescence study of four different fluorophores: Coumarin 6, 2,5-Diphenyloxazole (PPO), 1,4-Bis(5-phenyl-2-oxazolyl)benzene (POPOP) and Para-terpehnyl (PTP), doped in a polystyrene-based thin film. All of the samples are prepared by spin coating from a non-polar polymer solution at various concentrations. Their emission spectra and transient properties are characterized by photoluminescence measurements. Red-shifts in the emission spectra are observed for all four types of fluorophores as their concentration increases. We explain this phenomenon based on concentration dependence of solvatochromic effects and the results show good agreement with existing literature. We also show that the singlet-singlet annihilation processes are possibly a prevalent mechanism in the high concentration regime that affects the steady state and transient emission characteristics of the fluors. With the exception of PTP, photoluminescence quenching occurs as the fluorophore concentration in the polymer is increased. Rate equations for excited state decay mechanisms are analysed by considering different radiative and non-radiative energy transfer mechanisms. The results show consistency with our experimental observations. PTP shows the best photoluminescence results as an efficient fluor in the thin film, whereas PPO shows the strongest concentration dependent quenching and an anomalous lifetime distribution.

Allocations for HANDI 2000 business management system

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

Wilson, D.

The Data Integration 2000 Project will result in an integrated and comprehensive set of functional applications containing core information necessary to support the Project Hanford Management Contract. It is based on the Commercial-Off-The-Shelf product solution with commercially proven business processes. The COTS product solution set, of PassPort and People Soft software, supports finance, supply and chemical management/Material Safety Data Sheet, human resources. Allocations at Fluor Daniel Hanford are burdens added to base costs using a predetermined rate.

Site planning and integration fiscal year 1999 multi-year work plan (MYWP) update for WBS 1.8.2.1

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

SCHULTZ, E.A.

The primary mission of the Site Planning and Integration (SP and I) project is to assist Fluor Daniel Project Direction to ensure that all work performed under the Project Hanford Management Contract (PHMC) is adequately planned, executed, controlled, and that performance is measured and reported in an integrated fashion. Furthermore, SP and I is responsible for the development, implementation, and management of systems and processes that integrate technical, schedule, and cost baselines for PHMC work.

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

The highly successful safe remediation of the Fernald waste pits undertaken under the privatization model

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

Cherry, Mark; Lojek, Dave; Murphy, Con

2003-02-23

Remediation of eight waste pits at the Department of Energy (DOE) Fernald site, located northwest of Cincinnati, Ohio, involves excavating approximately one million tonnes in-situ of low-level waste which were placed in pits during Fernald's production era. This unique project, one of the largest in the history of CERCLA/Superfund, includes uranium and thorium contaminated waste, soils and sludges. These wet soils and sludges are thermally dried in a processing facility to meet Department of Transportation (DOT) transportation and disposal facility waste acceptance criteria, loaded into railcars and shipped to the Envirocare waste disposal facility at Clive, Utah. This project ismore » now approximately 60% complete with more than 415,000 tonnes (460,000 tons) of waste material safely shipped in 74 unit trains to Envirocare. Work is scheduled to be completed in early 2005. Success to date demonstrates that a major DOE site remediation project can be safely and successfully executed in partnership with private industry, utilizing proven commercial best practices, existing site labor resources and support of local stakeholders. In 1997 under the DOE's privatization initiative, Fluor Fernald, Inc. (Fluor Fernald) solicited the services of the remediation industry to design, engineer, procure, construct, own and operate a facility that would undertake the remediation of the waste pits. The resulting procurement was awarded to IT Corporation, currently Shaw Environmental and Infrastructure, Inc. (Shaw). The contractor was required to finance the procurement and construction of its facilities and infrastructure. The contract was performance-based and payment would be made on the successful loadout of the waste from the facility on a per-ton basis meeting the Envirocare waste acceptance criteria. This paper details the performance to date, the challenges encountered, and the seamless partnering between DOE, the Environmental Protection Agency (EPA), Fluor Fernald, Shaw, labor un ions, and the local community in creating and executing a successful project.« less

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.

The effects of an anchovy (stolephorus insularis) substrate application on the level of fluor intrusion on Sprague Dawley rat teeth (in vivo)

NASA Astrophysics Data System (ADS)

Sakinah, N. R.; Gunawan, H. A.; Puspitawati, R.

2017-08-01

Fluoride intrusion is one of the efficacy parameters of fluoridation. Anchovy (Stolephorus insularis), which contains a high fluoride concentration in the CaF2compound, can be used as a fluoridative agent which is affordable and easily obtained. The aim of this study is to prove the effectiveness of the application of an anchovy substrate (Stolephorus insularis), either by a feeding method or a topical method, for tooth fluoridation based on the depth of fluoride intrusion on the enamel. An in vivo experimental laboratory method was used. The subjects were 14 Sprague Dawley rats divided into five groups. The groups included a baseline control, a feeding negative control, a topical negative control, an anchovy feeding method, and a topical solution anchovy method. After 15 days of treatment, the teeth were cut transversely with a 0.5 mm thickness then processed to test for fluoride intrusion using fluorescence microscopy. There was increased fluor intrusion on the enamel of the experimental groups compared to the negative control groups (p<0.05).Fluoride intrusion using the topical fluoride method is higher than with the feeding method (p <0.05). Thus, the application of an anchovy substrate, either by chewing or smearing, increases fluoride intrusion on the enamel.

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

Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

PubMed

Lai, Zengzu; Schreiber, John R

2009-05-21

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

Composition-thermal expandability relations and oxidation processes in tourmaline studied by in situ Raman spectroscopy

NASA Astrophysics Data System (ADS)

Watenphul, Anke; Malcherek, Thomas; Wilke, Franziska D. H.; Schlüter, Jochen; Mihailova, Boriana

2017-11-01

The crystal chemistry of tourmaline, XY3Z6(T6O18)(BO3)3V3W, has a strong influence on the structure and physical properties. Since tourmalines occur in a wide range of geological settings and have large temperature and pressure stability fields, the understanding of the relation between the tourmaline chemistry and thermal expansion allows for better thermodynamic modeling of geological processes. Here, we report dynamic and static thermal expansions as well as mode Grüneisen parameters studied by Raman spectroscopy and single-crystal X-ray diffraction data on several tourmaline species. In addition, oxidation processes in fluor-schorl and Fe2+-bearing elbaite were followed by Raman spectroscopy. Our results emphasize the role of Y-/Z-site occupancy disorder to reduce the local strains and demonstrate that small-size octahedrally coordinated cations perturb the topology of the SiO4 rings, which in turn seems to enhance the anisotropic thermal-expansion response. In addition, it is shown that the temperature-dependent behavior of the VOH modes primarily depends on the occupancy of the Y site, whereas that of the WOH modes depends on the occupancy of the X site. High-temperature Raman experiments in air allowed to follow the oxidation of Fe2+ to Fe3+ in fluor-schorl by analyzing both the framework and OH-stretching phonon modes. It is further demonstrated that under the same conditions, no oxidation of iron is observed for Fe2+-bearing elbaite, which implies that at high oxygen fugacity, iron is only oxidized in tourmaline species with prevalent divalent cations at the Y site.

CONTRIBUTION TO THE GEOCHEMISTRY OF TANTALUM AND NIOBIUM IN THE HYDROTHERMAL-PNEUMATHOLYTIC PROCESS (in Russian)

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

Beus, A.A.; Sitnin, A.A.

1961-01-01

S>Data obtained as a result of geochemical investigations show that tantalum and niobium are typical elements of high-temperature postmagmatic processes (early albitization, greysening) connected with granites. The separation of tantalum and niobium in the hydrothermal-pneumatholytic process (greysening stage), which leads to the concentration of tantalum in albitized and greysenized granites (40 to 100 times compared to the average content in granites) is connected with the different mobility and stability of their acido- complex compounds (in particular fluor- and oxyfluorcomplexes), the existence of which in greysening solutions is suggested. A natural analogy in the behavior of both elements in the processesmore » of postmagmatic metasomatose in granites and granitic pegmatites is suggested. (tr-auth)« less

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

PubMed

Blagojević Zagorac, Gordana; Mahmutefendić, Hana; Maćešić, Senka; Karleuša, Ljerka; Lučin, Pero

2017-03-01

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Near-field fluorescence thermometry using highly efficient triple-tapered near-field optical fiber probe.

PubMed

Fujii, T; Taguchi, Y; Saiki, T; Nagasaka, Y

2012-12-01

A novel local temperature measurement method using fluorescence near-field optics thermal nanoscopy (Fluor-NOTN) has been developed. Fluor-NOTN enables nanoscale temperature measurement in situ by detecting the temperature-dependent fluorescence lifetime of CdSe quantum dots (QDs). In this paper, we report a novel triple-tapered near-field optical fiber probe that can increase the temperature measurement sensitivity of Fluor-NOTN. The performance of the proposed probe was numerically evaluated by the finite difference time domain method. Due to improvements in both the throughput and collection efficiency of near-field light, the sensitivity of the proposed probe was 1.9 times greater than that of typical double-tapered probe. The proposed shape of the triple-tapered core was successfully fabricated utilizing a geometrical model. The detected signal intensity of dried layers of QDs was greater by more than two orders than that of auto-fluorescence from the fiber core. In addition, the near-field fluorescence lifetime of the QDs and its temperature dependence were successfully measured by the fabricated triple-tapered near-field optical fiber probe. These measurement results verified the capability of the proposed triple-tapered near-field optical fiber probe to improve the collection efficiency of near-field fluorescence.

Facile green synthesis of silver doped fluor-hydroxyapatite/β-cyclodextrin nanocomposite in the dual acting fluorine-containing ionic liquid medium for bone substitute applications

NASA Astrophysics Data System (ADS)

Jegatheeswaran, S.; Selvam, S.; Sri Ramkumar, V.; Sundrarajan, M.

2016-05-01

A novel green route has approached for the synthesis of silver doped fluor-hydroxyapatite/β-cyclodextrin composite by the assistance of fluorine-based ionic liquid. The selected [BMIM]BF4 ionic liquid for this work plays a dual role as fluoride source and templating agent. It helps to improve the crystalline structures and the shape of the composites. The crystallinity, surface morphology, topographical studies of the synthesized composite were validated. The XRD results of the composite show typical Ag reflection peaks at 38.1°, 44.2° and 63.4°. The ionic liquid assisted composite displayed the hexagonal shaped HA particles, which are surrounded by spherical nano-Ag particles and these particles are uniformly dispersed in the β-cyclodextrin matrix in both horizontal and cross sections from surface morphology observations. The Ionic liquid assisted silver doped fluor-hydroxyapatite/β-cyclodextrin composite exhibited very good antibacterial activities against Escherichia coli, Salmonella typhi, Klebsiella pneumonia and Serratia liquefaciens pathogens. The antibacterial proficiencies were established using Confocal Laser Scanning Microscopic developed biofilms images and bacterial growth curve analysis. The cytotoxicity results of the ionic liquid assisted composite analyzed by cell proliferation in vitro studies using human osteosarcoma cell line (MG-63) and this study has shown excellent biocompatibility.

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

Application of "FLUOR-P" device for analysis of the space flight effects on the intracellular level.

NASA Astrophysics Data System (ADS)

Grigorieva, Olga; Rudimov, Evgeny; Buravkova, Ludmila; Galchuk, Sergey

The mechanisms of cellular gravisensitivity still remain unclear despite the intensive research in the hypogravity effects on cellular function. In most cell culture experiments on unmanned vehicles "Bion" and "Photon", as well as on the ISS only allow post-flight analysis of biological material, including fixed cells is provided. The dynamic evaluation cellular parameters over a prolonged period of time is not possible. Thus, a promising direction is the development of equipment for onboard autonomous experiments. For this purpose, the SSC RF IBMP RAS has developed "FLUOR-P" device for measurement and recording of the dynamic differential fluorescent signal from nano- and microsized objects of organic and inorganic nature (human and animal cells, unicellular algae, bacteria, cellular organelles suspension) in hermetically sealed cuvettes. Besides, the device allows to record the main physical factors affecting the analyzed object (temperature and gravity loads: position in space, any vector acceleration, shock) in sync with the main measurements. The device is designed to perform long-term programmable autonomous experiments in space flight on biological satellites. The device software of allows to carry out complex experiments using cell. Permanent registration of data on built-in flash will give the opportunity to analyze the dynamics of the estimated parameters. FLUOR-P is designed as a monobloc (5.5 kg weight), 8 functional blocks are located in the inner space of the device. Each registration unit of the FLUOR-P has two channels of fluorescence intensity and excitation light source with the wavelength range from 300 nm to 700 nm. During biosatellite "Photon" flight is supposed to conduct a full analysis of the most important intracellular parameters (mitochondria activity and intracellular pH) dynamics under space flight factors and to assess the possible contribution of temperature on the effects of microgravity. Work is supported by Roskosmos and the Russian Academy of Sciences.

Study of absorption and re-emission processes in a ternary liquid scintillation system

NASA Astrophysics Data System (ADS)

Xiao, Hua-Lin; Li, Xiao-Bo; Zheng, Dong; Cao, Jun; Wen, Liang-Jian; Wang, Nai-Yan

2010-11-01

Liquid scintillators are widely used as the neutrino target in neutrino experiments. The absorption and emission of different components of a ternary liquid scintillator (Linear Alkyl Benzene (LAB) as the solvent, 2,5-diphenyloxazole (PPO) as the fluor and p-bis-(o-methylstyryl)-benzene (bis-MSB) as wavelength shifter) are studied. It is shown that the absorption of this liquid scintillator is dominant by LAB and PPO at wavelengths less than 349 nm, and the absorption by bis-MSB becomes prevalent at the wavelength larger than 349 nm. The fluorescence quantum yields, which are the key parameters to model the absorption and re-emission processes in large liquid scintillation detectors, are measured.

Evaluation of the role of CD207 on Langerhans cells in a murine model of atopic dermatitis by in situ imaging using Cr:forsterite laser-based multimodality nonlinear microscopy

NASA Astrophysics Data System (ADS)

Lee, Jyh-Hong; Tsai, Ming-Rung; Sun, Chi-Kuang; Chiang, Bor-Luen

2012-11-01

Atopic dermatitis (AD) is an allergic inflammatory disease of skin. It remains unclear that CD207 of Langerhans cells (LCs) plays a central role in the development of allergic sensitization. There is little data on LCs within the microenviroment in vivo. We used a murine model of epicutaneous (EC) ovalbumin (OVA) sensitization inducing an inflammatory skin resembling AD to explore the role of CD207 in the pathogenesis of AD. Cr:forsterite laser-based multimodality nonlinear microscopy was applied for in situ imaging. Peritoneal injections of Alexa Fluor 647-rat anti-mouse CD207 into mice were performed to specifically trace the LCs. Peritoneal injections of OVA-Alexa Fluor 647 conjugate into mice were performed to specifically trace the OVA. We found that combining Alexa Fluor fluorescent probes with multimodality nonlinear microscopy permitted the unequivocal in situ imaging of CD207-expressing LCs. The relevant time-course, expressional, and functional studies reveal that CD207 of LCs plays an essential role during the induction of EC sensitization. We establish and validate that Cr:forsterite laser-based multimodality nonlinear microscopy is applicable for the specific detection of labeled mAb-bound LCs and labeled antigen. We suggest that CD207-expressing LCs initiate the allergic response through the CD207 mediated epicutaneous sensitization associated with the development of AD.

Effect of Fluoride Varnish on Streptococcus mutans Count in Saliva of Caries Free Children Using Dentocult SM Strip Mutans Test: A Randomized Controlled Triple Blind Study.

PubMed

A, Deepti; Jeevarathan, J; Muthu, Ms; Prabhu V, Rathna; Chamundeswari

2008-09-01

The aim of this study was to estimate the count of Streptococcus mutans in saliva of caries free children using Dentocult SM strip mutans and to evaluate the effect of fluoride varnish on the Streptococcus mutans count in saliva of these caries free children. Thirty caries free children were selected for the study based on the information obtained from a questionnaire prepared. They were randomly assigned into the control group and the study group consisting of ten and twenty children respectively. Samples of saliva were collected using the saliva strips from the Dentocult SM kit and after incubation the presence of the Streptococcus mutans was evaluated using the manufacturers' chart. The study group was subjected to Fluor Protector fluoride varnish application after 24 hours following which the samples were collected again. The average Streptococcus mutans count in primary dentition of caries free children was in the range of 10(4) to 10(5) colony forming units/ml. The average Streptococcus mutans count in primary dentition of caries free children after Fluor Protector fluoride varnish application was below 10(4) colony forming units/ml. Fluor Protector fluoride varnish application showed a statistically significant reduction in the Streptococcus mutans count in saliva of the caries free children in the study group.

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis.

PubMed

Kooy, Floor K; Ma, Muyuan; Beeftink, Hendrik H; Eggink, Gerrit; Tramper, Johannes; Boeriu, Carmen G

2009-01-15

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

Pacific Northwest National Laboratory Apatite Investigation at the 100-NR-2 Quality Assurance Project Plan

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

Fix, N. J.

This Quality Assurance Project Plan provides the quality assurance requirements and processes that will be followed by staff working on the 100-NR-2 Apatite Project. The U.S. Department of Energy, Fluor Hanford, Inc., Pacific Northwest National Laboratory, and the Washington Department of Ecology agreed that the long-term strategy for groundwater remediation at 100-N would include apatite sequestration as the primary treatment, followed by a secondary treatment. The scope of this project covers the technical support needed before, during, and after treatment of the targeted subsurface environment using a new high-concentration formulation.

Training survey -- educational profile for Hanford HANDI 2000 project

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

Wilson, D.

Fluor Daniel Hanford, Inc. (FDH) is currently adopting streamlined business processes through integrated software solutions. Replacing the legacy software (current/replacement systems, attached) also avoids significant maintenance required to resolve Year 2000 issues. This initiative is being referred to as `HANDI 2000`. The software being implemented in the first phase of this project includes Indus International`s PASSPORT Software, Peoplesoft and Primavera P3 Software. The project, which encompasses all the system replacements that will occur, has been named `HANDI 2000.` The PASSPORT applications being implemented are Inventory Management, Purchasing, Contract Management, Accounts Payable, and MSDS (Material Safety Data Sheets).

Water-Lean Solvents for Post-Combustion CO 2 Capture: Fundamentals, Uncertainties, Opportunities, and Outlook

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

Heldebrant, David J.; Koech, Phillip K.; Glezakou, Vassiliki-Alexandra

Capturing CO2 from the exhaust of coal-fired power plants is a daunting task, requiring selective removal from a dilute gas stream of millions of pounds per hour of a molecule that is considered thermodynamically and kinetically stable. There are commercial solvent technologies containing proprietary blends of aqueous amines such as Econamine FG+, KS-1, Oase® Blue, and Cansolv that may achieve this task, though only one of them has been deployed at scale, albeit in the natural gas industry.1 The Achilles’ heel of amine blends is the energy loss involved with regenerating the solvent, i.e., boiling and condensing millions of poundsmore » of water per hour. This energy loss translates to a sizeable parasitic load on a coal-fired plant, requiring the plant to burn more coal to get back to its nameplate capacity.2 Unsurprisingly, a considerable amount of research has focused on the design of more efficient technologies to lessen this parasitic load. Liquid systems are the lowest hanging fruit from a time and cost perspective, as they have the potential to use aqueous amine infrastructure, with potential for more rapid ascent up the development ladder than porous solids or membranes.« less

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

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.

35. MISCELLANEOUS ARCHITECTURAL AND STRUCTURAL DETAILS. INEEL DRAWING NUMBER 200063300287106359. ...

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

35. MISCELLANEOUS ARCHITECTURAL AND STRUCTURAL DETAILS. INEEL DRAWING NUMBER 200-0633-00-287-106359. FLUOR NUMBER 5775-CPP-633-A-9. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

Fluor-ferro-leakeite, NaNa2(FC2+2Fe3+2Li)Si8O22F2, a new alkali amphibole from the Canada Pinabete pluton, Questa, New Mexico, U.S.A.

USGS Publications Warehouse

Hawthorne, F.C.; Oberti, R.; Ungaretti, L.; Ottolini, L.; Grice, Joel D.; Czamanske, G.K.

1996-01-01

Fluor-ferro-leakeite is a new amphibole species from the Canada Pinabete pluton, Questa, New Mexico, U.S.A.; it occurs in association with quartz, alkali feldspar, acmite, ilmenite, and zircon. It forms as anhedral bluish black crystals elongated along c and up to 1 mm long. It is brittle, H = 6, Dmeas = 3.37 g/cm3, Dcalc = 3.34 g/cm3. In plane-polarized light, it is strongly pleochroic, X = very dark indigo blue, Y = gray blue, Z = yellow green; X ??? c = 10?? (in ??obtuse), Y = b, Z ??? a = 4?? (in ?? obtuse), with absorption X > Y > Z. Fluor-ferro-leakeite is biaxial positive, ?? = 1.675(2), ??= 1.683(2), ?? = 1.694(1); 2V = 87(2)??; dispersion is not visible because of the strong absorption. Fluor-ferro-leakeite is monoclinic, space group C2/m, a = 9.792(1), b = 17.938(1), c = 5.3133(4) A??, ??= 103.87(7)??, V = 906.0(1) A??3, Z = 2. The ten strongest X-ray diffraction lines in the powder pattern are [d(I,hkl)]: 2.710(100,151), 2.536(92,202), 3.404(57,131), 4.481(54,040), 8.426(45,110), 2.985(38,241), 2.585(38,061), 3.122(29,310), 2.165(26,261), and 1.586(25,403). Analysis by a combination of electron microprobe, ion microprobe, and crystal-structure refinement (Hawthorne et al. 1993) gives SiO2 51.12, Al2O3 1.13, TiO2 0.68, Fe2O3 16.73, FeO 8.87, MgO 2.02, MnO 4.51, ZnO 0.57, CaO 0.15, Na2O 9.22, K2O 1.19, Li2O 0.99, F 2.87, H2Ocalc 0.60, sum 99.44 wt%. The formula unit, calculated on the basis of 23 O atoms, is (K0.23Na0.76)(Na1.97Ca0.03)(Mg 0.46Fe2+1.4Mn2+0.59Zn0.07Fe3+1.93-Ti 0.08Al0.02Li0.61])(Si7.81Al 0.19)O22(F1.39OH0.61). A previous crystal-structure refinement (Hawthorne et al. 1993) shows Li to be completely ordered at the M3 site. Fluor-ferro-leakeite, ideally NaNa2(Fe2+2Fe3+2Li)Si8O22F2, is related to leakeite, NaNa2(Mg2Fe3+3Li)Si 8O22(OH)2, by the substitutions Fe2+ ??? Mg and F ??? OH.

27. ELEVATIONS OF EAST AND WEST SIDES. INEEL DRAWING NUMBER ...

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

27. ELEVATIONS OF EAST AND WEST SIDES. INEEL DRAWING NUMBER 200-0633-00-287-106355. FLUOR NUMBER 5775-CPP-633-A-5. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

28. NORTH AND SOUTH ELEVATIONS AND TWO SECTIONS. INEEL DRAWING ...

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

28. NORTH AND SOUTH ELEVATIONS AND TWO SECTIONS. INEEL DRAWING NUMBER 200-0633-00-287-106356. FLUOR NUMBER 5775-CPP-633-A-6. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

77 FR 61749 - Application for Final Commitment for a Long-term Loan or Financial Guarantee in Excess of $100...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-11

... Principal Supplier: Fluor Enterprises, Inc. Obligor: Reliance Industries Limited. Guarantor(s): N/A. Description of Items Being Exported Gas turbines, compressors, centrifuges, technology licenses, engineering...

L-325 Sagebrush Habitat Mitigation Project: FY2009 Compensation Area Monitoring Report

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

Durham, Robin E.; Sackschewsky, Michael R.

2009-09-29

Annual monitoring in support of the Fluor Daniel Hanford Company (Fluor) Mitigation Action Plan (MAP) for Project L-325, Electrical Utility Upgrades was conducted in June 2009. MAP guidelines defined mitigation success for this project as 3000 established sagebrush transplants on a 4.5 ha mitigation site after five monitoring years. Annual monitoring results suggest that an estimated 2130 sagebrush transplants currently grow on the site. Additional activities in support of this project included gathering sagebrush seed and securing a local grower to produce between 2250 and 2500 10-in3 tublings for outplanting during the early winter months of FY2010. If the minimummore » number of seedlings grown for this planting meets quality specifications, and planting conditions are favorable, conservative survival estimates indicate the habitat mitigation goals outlined in the MAP will be met in FY2014.« less

Polysiloxane scintillator composition

DOEpatents

Walker, J.K.

1992-05-05

A plastic scintillator useful for detecting ionizing radiation comprising a matrix which comprises an optically transparent polysiloxane having incorporated therein at least one ionizing radiation-hard fluor capable of converting electromagnetic energy produced in the polysiloxane upon absorption of ionizing radiation to detectable light.

Polysiloxane scintillator composition

DOEpatents

Walker, James K.

1992-01-01

A plastic scintillator useful for detecting ionizing radiation comprising a matrix which comprises an optically transparent polysiloxane having incorporated therein at least one ionizing radiation-hard fluor capable of converting electromagnetic energy produced in the polysiloxane upon absorption of ionizing radiation to detectable light.

41. OPERATING CORRIDOR PLAN AND SECTIONS, INCLUDING SOME ISOMETRIC DETAILS. ...

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

41. OPERATING CORRIDOR PLAN AND SECTIONS, INCLUDING SOME ISOMETRIC DETAILS. INEEL DRAWING NUMBER 200-0633-00-287-106455. FLUOR NUMBER 5775-CPP-633-P-60 - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

Fluor Hanford (FH) River Corridor Transition Plan

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

MCBRIDE, D.J.

2002-08-28

This Transition Plan defines the scope and schedule for actions that are critical for a smooth transition of the River Corridor scope of work and to ensure the achievement of transition as planned, with minimal or no impact to ongoing baseline activities.

29. FLOOR PLAN OF WASTE CALCINATION FACILITY SHOWING MAIN ABOVEGRADE ...

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

29. FLOOR PLAN OF WASTE CALCINATION FACILITY SHOWING MAIN ABOVE-GRADE FLOOR LEVEL. INEEL DRAWING NUMBER 200-0633-00-287-106354. FLUOR NUMBER 5775-CPP-633-A-4. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

38. SECTIONS OF ACCESS CORRIDOR, INCLUDES SECTION SHOWING ARRANGEMENT OF ...

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

38. SECTIONS OF ACCESS CORRIDOR, INCLUDES SECTION SHOWING ARRANGEMENT OF NAVY GUN BARRELS. INEEL DRAWING NUMBER 200-0633-00-287-106454. FLUOR NUMBER 5775-CPP-633-P-59. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

Development of a thyroperoxidase inhibition assay for high-throughput screening

EPA Science Inventory

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluores...

31. FLOOR PLANS OF WASTE CALCINATION FACILITY. SHOWS ACCESS CORRIDOR ...

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

31. FLOOR PLANS OF WASTE CALCINATION FACILITY. SHOWS ACCESS CORRIDOR AT MEZZANINE AND LOWER LEVELS. INEEL DRAWING NUMBER 200-0633-00-287-106352. FLUOR NUMBER 5775-CPP-633-A-2. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

LESSONS LEARNED IN OPERATING THE HOSE-IN-HOSE SYSTEM FOR TRANSFSERRING SLUDGE AT HANFORDS K-BASINS

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

PERES MW

In May 2007, the Department of Energy and the Fluor Hanford K Basin Closure Project completed transferring sludge from the K East Basin to new containers in the K West Basin using a Hose-in-Hose system. This project presented a number of complex and unique technical, operational, and management challenges that had to be resolved to complete the required transfers and satisfy project milestones. The project team (including DOE; regulators; and Fluor management, operations, maintenance, engineering and all other support organizations) found innovative solutions to each challenge. This paper records lessons learned during the operational phase of the sludge transfer viamore » the Hose-In-Hose system. The subject is limited to the operational phase and does not cover design, development, testing or turnover. A discussion of the situation or problem encountered is provided, along with the lesson learned as applicable to a future program or project.« less

39. CALCINER CELL PLANS. TOGETHER WITH HAER ID33C37 ILLUSTRATES COMPLEXITY ...

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

39. CALCINER CELL PLANS. TOGETHER WITH HAER ID-33-C-37 ILLUSTRATES COMPLEXITY OF PIPING. INEEL DRAWING NUMBER 200-0633-00-287-106445. FLUOR NUMBER 5775-CPP-633-P-50 - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

40. CALCINER CELL SECTIONS. TOGETHER WITH HAER ID33C37 ILLUSTRATES COMPLEXITY ...

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

40. CALCINER CELL SECTIONS. TOGETHER WITH HAER ID-33-C-37 ILLUSTRATES COMPLEXITY OF PIPING. INEEL DRAWING NUMBER 200-0633-00-287-106446. FLUOR NUMBER 5775-CPP-P-51. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

30. FLOOR PLANS OF WASTE CALCINATION FACILITY. SHOWS LEVELS ABOVE ...

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

30. FLOOR PLANS OF WASTE CALCINATION FACILITY. SHOWS LEVELS ABOVE GRADE AND AT LEVEL OF OPERATING CORRIDOR. INEEL DRAWING NUMBER 200-0633-00-287-106351. FLUOR NUMBER 5775-CPP-633-A-1. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

37. PLAN OF ACCESS CORRIDOR PIPING INCLUDES WASTE HOLD TANK ...

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

37. PLAN OF ACCESS CORRIDOR PIPING INCLUDES WASTE HOLD TANK CELL, OFFGAS CELL, ADSORBER CELL, AND OFFGAS FILTER CELL. INEEL DRAWING NUMBER 200-0633-00-287-106453. FLUOR NUMBER 5775-CPP-P-58. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

PNNL Apatite Investigation at 100-NR-2 Quality Assurance Project Plan

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

Fix, N. J.

In 2004, the U.S. Department of Energy, Fluor Hanford, Inc., Pacific Northwest National Laboratory (PNNL), and the Washington Department of Ecology agreed that the long-term strategy for groundwater remediation at the 100-N Area would include apatite sequestration as the primary treatment, followed by a secondary treatment if necessary. Since then, the agencies have worked together to agree on which apatite sequestration technology has the greatest chance of reducing strontium-90 flux to the Columbia River. This Quality Assurance Project Plan provides the quality assurance requirements and processes that will be followed by staff working on the PNNL Apatite Investigation at 100-NR-2more » Project. The plan is designed to be used exclusively by project staff.« less

Disaster recovery plan for HANDI 2000 business management system

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

Adams, D.E.

The BMS production implementation will be complete by October 1, 1998 and the server environment will be comprised of two types of platforms. The PassPort Supply and the PeopleSoft Financials will reside on LNIX servers and the PeopleSoft Human Resources and Payroll will reside on Microsoft NT servers. Because of the wide scope and the requirements of the COTS products to run in various environments backup and recovery responsibilities are divided between two groups in Technical Operations. The Central Computer Systems Management group provides support for the LTNIX/NT Backup Data Center, and the Network Infrastructure Systems group provides support formore » the NT Application Server Backup outside the Data Center. The disaster recovery process is dependent on a good backup and recovery process. Information and integrated system data for determining the disaster recovery process is identified from the Fluor Daniel Hanford (FDH) Risk Assessment Plan, Contingency Plan, and Backup and Recovery Plan, and Backup Form for HANDI 2000 BMS.« less

Fluorination of thiocarbonyl compounds with Bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-fluor reagent): A facile synthesis of gem-difluorides

PubMed

Lal; Lobach; Evans

2000-08-11

A variety of thiocarbonyl derivatives (thioketone, thioester, thioamide, dithioester, and dithiocarbamate) were converted to the corresponding gem-difluorides in excellent yields on reaction with the fluorinating agent, bis(2-methoxyethyl)aminosulfur trifluoride, in the presence of SbCl(3).

Decay Of Bacterial Pathogen, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure Amended Soils

EPA Science Inventory

This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria, and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manre-amended agricultural soils. Known concentrations of transformed green fluore...

Transition and closeout of the Fernald Closure Project

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

Bilson, H.E.; Terry, T.; Reising, J.

The U.S. Department of Energy (DOE) and Fluor Fernald have completed the majority of the cleanup of the Fernald Site. The over 1,000 acre complex for processing uranium has been demolished and soil contamination has been remediated. With acres of wetlands and prairies replacing the buildings and waste pits. At the end of the project the focus shifted to developing demonstrating the completion of the project and the contract, as well as ensuring a smooth transition of the facility from the DOE's Environmental Management (EM) Program to the DOE's Legacy Management (LM) Program. Working with the DOE, each portion ofmore » the closure contract was examined for specific closure definition. From this negotiation effort the Comprehensive Exit and Transition Plan (CE/T Plan) was written. The CE/T Plan is intended to assist DOE in the analysis that the site is ready for transfer into long-term stewardship (LTS) (also referred to as legacy management) and that Fluor Fernald, Inc. has satisfactorily completed the closure contract statement of work elements. Following the Lessons Learned from the closure of the Rocky Flats Site, the DOE's Legacy Management Program created a matrix of Transition Elements required to ensure adequate information was in place to allow the new prime contractor to perform the Legacy Management scope of work. The transition plan included over 1,000 elements broken down into functional areas and relied on specific Fernald Responsibility Transition Packages (RTPs) for detailed transition actions. The template for Closure and Transition Planning used at the Fernald Site was developed using the best Lessons Learned from across the DOE Complex. The template could be used for other sites, and lessons learned from this closure and transition will be appropriate for all closure projects. (authors)« less

EFFECTS OF SEEDING PROCEDURES AND WATER QUALITY ON RECOVERY OF CRYPTOSPORIDIUM OOCYSTS FROM STREAM WATER BY USING U.S. ENVIRONMENTAL PROTECTION AGENCY METHOD 1623

EPA Science Inventory

U.S.EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluor...

32. SECTIONS AA, BB, CC, DD, AND EE WASTE CALCINATION ...

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

32. SECTIONS A-A, B-B, C-C, D-D, AND E-E WASTE CALCINATION FACILITY SHOWING RELATIONSHIPS OF DIFFERENT FLOOR LEVELS TO ONE ANOTHER. INEEL DRAWING NUMBER 200-0633-00-287-106353. FLUOR NUMBER 5775-CPP-633-A-3. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

36. ARCHITECTURAL AND STRUCTURAL DETAILS OF ELEVATOR HOUSING, NaK HEATER ...

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

36. ARCHITECTURAL AND STRUCTURAL DETAILS OF ELEVATOR HOUSING, NaK HEATER STACK ROOF FLASHING, HOOD ELEVATION DETAIL. INCLUDES PARTIAL 'BILL OF MATERIAL.' INEEL DRAWING NUMBER 200-0633-00-287-106361. FLUOR NUMBER 5775-CPP-633-A-11. - Idaho National Engineering Laboratory, Old Waste Calcining Facility, Scoville, Butte County, ID

77 FR 50102 - Application for Final Commitment for a Long-Term Loan or Financial Guarantee in Excess of $100...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-20

... Incorporated; Jacobs Engineering Group Incorporated; Foster Wheeler AG; Fluor Corporation. Obligor: The obligor is a special purpose vehicle anticipated to be named ``Sadara Chemical Company.'' Guarantor(s): The Dow Chemical Company, Dow Europe Holding B.V., and Saudi Arabian Oil Company. Description of Items...

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

Quantitative estimation of the fluorescent parameters for crop leaves with the Bayesian inversion

USDA-ARS?s Scientific Manuscript database

In this study, the fluorescent parameters of crop leaves were retrieved from the leaf hyperspectral measurements by inverting the FluorMODleaf model, which is a leaf-level fluorescence model that is based on the widely used and validated PROSPECT (leaf optical properties) model and can simulate the ...

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

PubMed Central

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 intact virions after in vitro stimulation. Overall, Alexa Fluor dye labeled -DENV are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. PMID:25497702

High-Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor.

PubMed

Foote, Peter K; Krist, David T; Statsyuk, Alexander V

2017-09-14

HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases. The release of fluorophore upon transthiolation allows fluorescence polarization detection of HECT E3 activity. In the presence of inhibitors, HECT E3 activity is ablated, and thus no reaction and no change in FP are observed. This assay has been adapted for high-throughput screening of small molecules against HECT E3 ligases, and its utility has been proven in the discovery of HECT E3 ligase inhibitors. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Advanced Plant Habitat Test Harvest

NASA Image and Video Library

2017-08-24

John "JC" Carver, a payload integration engineer with NASA Kennedy Space Center's Test and Operations Support Contract, uses a FluorPen to measure the chlorophyll fluorescence of Arabidopsis thaliana plants inside the growth chamber of the Advanced Plant Habitat (APH) Flight Unit No. 1. Half the plants were then harvested. The harvest is part of an ongoing verification test of the APH unit, which is located inside the International Space Station Environmental Simulator in Kennedy's Space Station Processing Facility. The APH undergoing testing at Kennedy is identical to one on the station and uses red, green and broad-spectrum white LED lights to grow plants in an environmentally controlled chamber. The seeds grown during the verification test will be grown on the station to help scientists understand how these plants adapt to spaceflight.

Li- and F-bearing alkali amphibole from granitic pegmatite at Hurricane Mountain, Carroll County, New Hampshire

USGS Publications Warehouse

Foord, E.E.; Erd, Richard C.; Robie, S.B.; Lichte, F.E.; King, V.T.

1996-01-01

At Hurricane Mountain, Carroll County, New Hampshire, bodies of granitic pegmatite in riebeckite granite contain large (up to 10 cm long and 2 cm across) primary crystals of Li-bearing fluor-arfvedsonite in miarolitic cavities, grading to euhedral Li- and F-poor arfvedsonite. Fine-grained, fibrous, light blue-gray riebeckite occurs as a late-stage hydrothermal filling in the miarolitic cavities. The early, Li-rich, fluor-arfvedsonite has: a 9.836(5), b 17.997(7), c 5.316(4) A??, ?? 103.735(4)??, V 914.20(6) A??3; Z = 2, Dmeas. 3.34 g/cm3, Dcalc. 3.353 g/cm3; biaxial (-), 2Vmeas. 44(1)??, 2Vcalc. 46??; ?? 1.681(2), ?? 1.692(2), ?? 1.694(2), inclined dispersion, r > v; X ??? c -7??, Y = b, Z ??? a +7??; X dark blue, Y lavender gray, Z pale yellowish brown; X > Y > Z; X is opaque at 0.03 mm thickness. A structural formula, on the basis of 24 (O,OH,F) atoms is: (Na0.86K0.25)Na2(Fe2+2.54Fe3+1.485Mn0.10Zn 0.02Li0.49Ti0.07)(Si7.71Al 0.07)O22(F1.34OH0.63). Arfvedsonite within the miarolitic cavities contains less Li and F than that of the earlier generation, and the still later riebeckite contains only 0.09 wt.% Li2O and 0.3 wt.% F. The Fe3+:Fe2+ ratio of the early Li-bearing fluor-arfvedsonite and that of the euhedral arfvedsonite crystals within miarolitic cavities is 0.58. The late, fibrous, cavity-filling riebeckite has an Fe3+:Fe2+ ratio of 0.99. The total iron content of the three amphiboles increases with continued crystallization. These amphiboles are products of peralkaline pegmatites locally derived from peralkaline granite.

CSER 99-002: CSER for unrestricted moderation of sludge material with two-boat operations in gloveboxes HC-21A and HC21-C

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

LAN, J.S.

1999-04-29

This Criticality Safety Evaluation Report was prepared by Fluor Daniel Northwest under contract to BWHC. This document establishes the criticality safety parameters for unrestricted moderation of Sludge material with two-boat operations in gloveboxes HC-21A and HC-21C.

Bone Marrow-derived Mesenchymal Stem Cells (MSCs) as a Selective Delivery Vehicle for a PSA-Activated Protoxin for Advanced Prostate Cancer

DTIC Science & Technology

2013-03-01

tissue have been optimized • Optimization of flow cytometry-based analyses to characterize co- incident expression of multiple MSC-related markers on a...CA). Mouse anti-human vimentin (clone LN-6) was purchased from Sigma-Aldrich (St. Louis, MO). Goat anti-mouse Alexa Fluor 488, Roswell Park

Experimental study of the influence of the counter and scintillator on the universal curves in the cross-efficiency method in LSC.

PubMed

Cassette, P; Tartès, I

2014-05-01

The cross-efficiency method in LSC is one of the approaches proposed for the extension of the Système International de Référence (SIR) to radionuclides emitting no gamma radiation. This method is based on a so-called "universal cross-efficiency curve", establishing a relationship between the detection efficiency of the radionuclide to be measured and the detection efficiency of a suitable tracer. This paper reports a study at LNHB on the influence of the scintillator and of the LS counter on the cross-efficiency curves. This was done by measuring the cross-efficiency curves obtained for (63)Ni and (55)Fe vs. (3)H, using three different commercial LS counters (Guardian 1414, Tricarb 3170 and Quantulus 1220), three different liquid scintillator cocktails (Ultima Gold, Hionic Fluor and PicoFluor 15 from Perkin Elmer(®)), and for chemical and colour-quenched sources. This study shows that these cross-efficiency curves are dependent on the scintillator, on the counter used and on the nature of the quenching phenomenon, and thus cannot definitively be considered as "universal". © 2013 Published by Elsevier Ltd.

Oligoethylene Glycol-substituted Aza-BODIPY Dyes As Red Emitting ER-Probes

PubMed Central

Kamkaew, Anyanee; Thavornpradit, Sopida; Puangsamlee, Thamon; Xin, Dongyue; Wanichacheva, Nantanit; Burgess, Kevin

2015-01-01

This study features aza-BODIPY (BF2-chelated azadipyrromethene) dyes with two aromatic substituents linked by oligoethylene glycol fragments to increase hydrophilicity of aza-BODIPY for applications in intracellular imaging. To prepare these, two chalcones were attached α,ω onto oligoethylene glycol fragments, then reacted with nitromethane anion. Conjugate addition products from this reaction were then subjected to typical conditions for synthesis of aza-BODIPY dyes (NH4OAc, nBuOH, 120 °C); formation of boracycles in this reaction was concomitant with creation of macrocycles containing the oligoethylene glycol fragments. Similar dyes with acyclic oligoelythene glycol substituents in the same position were used to compare the efficiencies of the intra- and inter-molecular aza-BODIPY forming reactions, and the characteristics of the products. All the fluors with oligoethylene glycol fragments, ie cyclic or acyclic, localized in the endoplasmic reticulum of a fibroblast cell line (WEHI-13VAR), the human pancreatic cancer cell line (PANC-1, rough ER predominates) and human liver cancer cell line (HepG2, smooth ER prevalent). These fluors are potentially useful for near IR (λmax emis at 730 nm) ER staining probes. PMID:26138325

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.

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.

Bone marrow cells stained by azide-conjugated Alexa fluors in the absence of an alkyne label.

PubMed

Lin, Guiting; Ning, Hongxiu; Banie, Lia; Qiu, Xuefeng; Zhang, Haiyang; Lue, Tom F; Lin, Ching-Shwun

2012-09-01

Thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) has recently been introduced as an alternative to 5-bromo-2-deoxyuridine (BrdU) for cell labeling and tracking. Incorporation of EdU into replicating DNA can be detected by azide-conjugated fluors (eg, Alexa-azide) through a Cu(i)-catalyzed click reaction between EdU's alkyne moiety and azide. While this cell labeling method has proven to be valuable for tracking transplanted stem cells in various tissues, we have found that some bone marrow cells could be stained by Alexa-azide in the absence of EdU label. In intact rat femoral bone marrow, ~3% of nucleated cells were false-positively stained, and in isolated bone marrow cells, ~13%. In contrast to true-positive stains, which localize in the nucleus, the false-positive stains were cytoplasmic. Furthermore, while true-positive staining requires Cu(i), false-positive staining does not. Reducing the click reaction time or reducing the Alexa-azide concentration failed to improve the distinction between true- and false-positive staining. Hematopoietic and mesenchymal stem cell markers CD34 and Stro-1 did not co-localize with the false-positively stained cells, and these cells' identity remains unknown.

High effective atomic number polymer scintillators for gamma ray spectroscopy

DOEpatents

Cherepy, Nerine Jane; Sanner, Robert Dean; Payne, Stephen Anthony; Rupert, Benjamin Lee; Sturm, Benjamin Walter

2014-04-15

A scintillator material according to one embodiment includes a bismuth-loaded aromatic polymer having an energy resolution at 662 keV of less than about 10%. A scintillator material according to another embodiment includes a bismuth-loaded aromatic polymer having a fluor incorporated therewith and an energy resolution at 662 keV of less than about 10%. Additional systems and methods are also presented.

Bi-layered zirconia/fluor-apatite bridges supported by ceramic dental implants: a prospective case series after thirty months of observation.

PubMed

Spies, Benedikt Christopher; Witkowski, Siegbert; Butz, Frank; Vach, Kirstin; Kohal, Ralf-Joachim

2016-10-01

The aim of this study was to determine the success and survival rate of all-ceramic bi-layered implant-supported three-unit fixed dental prostheses (IS-FDPs) 3 years after implant placement. Thirteen patients (seven males, six females; age: 41-78 years) received two one-piece ceramic implants (alumina-toughened zirconia) each in the region of the premolars or the first molar and were finally restored with adhesively cemented bi-layered zirconia-based IS-FDPs (3 in the maxilla, 10 in the mandible) composed of CAD/CAM-fabricated zirconia frameworks pressed-over with fluor-apatite glass-ceramic ingots. At prosthetic delivery and the follow-ups after 1, 2 and 3 years, the restorations were evaluated using modified United States Public Health Service (USPHS) criteria. Restorations with minor veneer chippings, a small-area occlusal roughness, slightly soundable restoration margins, minimal contour deficiencies and tolerable color deviations were regarded as success. In case of more distinct defects that could, however, be repaired to a clinically acceptable level, IS-FDPs were regarded as surviving. Kaplan-Meier plots were used for the success/survival analyses. To verify an impact on subjective patients' perceptions, satisfaction was evaluated by visual analog scales (VAS). All patients were seen 3 years after implant installation. No IS-FDP had to be replaced, resulting in 100% survival after a mean observation period of 29.5 months (median: 30.7). At the 3-year follow-up, 7/13 IS-FDPs showed a veneer chipping, 13/13 an occlusal roughness and 12/13 minimal deficiencies of contour/color. Since six restorations showed a major chipping and/or a major occlusal roughness, the Kaplan-Meier success rate was 53.8%. However, patients' significantly improved perceptions of function, esthetics, sense, and speech at prosthetic delivery remained stable over time. Bi-layered zirconia/fluor-apatite IS-FDPs entirely survived the observation period but showed a high frequency of technical complications. Nevertheless, the treatment highly satisfied patients' expectations. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Turboexpanders aid condensate recovery

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

Houghton, J.; McLay, J.D.

1973-03-05

Turboexpander plants built by Fluor are operating in 18 locations throughout the world, with the first one having been built in 1963. These plants, plus those under construction, account for a combined gas capacity or more than 4,000 MMscfd. In both gas-processing and cryogenic operations, the turboexpander is gaining wide acceptance in such areas as North and South America, the Middle East, and N. Africa. In a typical plant, feed gas is first dehydrated before being chilled by heat exchange with the gas product from the turboexpander. Condensate liquids are separated from the gas stream which is then expanded. Furthermore » condensed liquids resulting from the expansion-cooling effect are separated for fractionation. The degree of expansion can be varied to recover up to 60 to 90% of the ethane contained in the feed gas. The most efficient use of turboexpander refrigeration is dependent upon the product required.« less

Thermal expansion behavior of fluor-chlorapatite crystalline solutions

NASA Astrophysics Data System (ADS)

Hovis, G.; Harlov, D.; Gottschalk, M.; Hudacek, W.; Wildermuth, S.

2009-04-01

Apatite Ca5(PO4)3(F,Cl,OH,CO3) occurs widely as an accessory mineral in many igneous and metamorphic rocks and in nature displays a wide range of F-Cl-OH-CO3 mixtures (e.g., O'Reilly and Griffin, 2000) that have been used to interpret the role of fluids, e.g. Cl, F, and OH activities, during metamorphic and igneous processes (e.g., Harlov and Förster, 2002). It is important, therefore, to understand the thermodynamic behavior of these solid solutions, including their thermal expansion properties. Fluorapatite - chlorapatite samples were synthesized at the GFZ-Potsdam (Hovis, Harlov, Hahn and Steigert, 2007) using an adaptation of the molten flux method of Cherniak (2000). Dry CaF2 and CaCl2 (0.1 mole total) were mixed with Ca3(PO4)2 (0.03 moles), placed in a Pt crucible, equilibrated for 15 hours at 1375 °C, cooled to 1220 °C at 3 °C/hour, removed from the oven and cooled in air. Crystals were separated from the flux by boiling the quenched product in water. F:Cl fractions for each sample were determined via Rietveld refinement of X-ray powder diffraction data. Chemical homogeneity was confirmed by Rietveld refinement and high-contrast back-scattered electron imaging. Room-temperature unit-cell volumes were determined at the GFZ-Potsdam through Rietveld analysis of X-ray powder diffraction data and also at Lafayette College by standard unit-cell refinement techniques (Holland and Redfern, 1997) using NBS/NIST 640a Si as an internal standard. High-temperature unit-cell dimensions were calculated from X-ray powder diffraction data collected at Cambridge University from room temperature to 1000 °C on a Bruker D8 X-ray diffractometer. NBS Si again was utilized as an internal standard; high-temperature Si peak positions were taken from Parrish (1953). Results indicate that despite the considerable size difference between fluorine and chlorine ions, reflected by substantially different unit-cell sizes at room temperature, the coefficient of thermal expansion across the fluor-chlorapatite series is little affected by composition. This contrasts with relationships in alkali feldspars (Hovis and coworkers, 1997, 1999), which show that K-rich feldspars expand less than Na-rich feldspars. It contrasts also with the behavior of additional AlSi3 feldspars (Hovis and others, 2008), in which room-temperature chemical expansion limits the degree to which the structure can expand thermally. It also differs from expansion in kalsilite crystalline solutions (Hovis and coworkers, 2003, 2006), which depends on K:Na ratio. Among the minerals we have studied previously, only nepheline displays expansion behavior similar to that of fluor-chlorapatite crystalline solutions in that thermal expansion shows little sensitivity to composition. In AlSi3 feldspars and kalsilite one observes a single crystallographically distinct alkali site and a dominating SiO4 tetrahedral framework that limits the vibrational characteristics of the alkali-site occupant(s). Fluor-chlorapatite crystalline solutions have no such structural framework. Moreover, the anion site in the latter changes structural character in the transition from fluorapatite to chlorapatite. This flexibility apparently allows anion vibrational characteristics, coupled with those of Ca polyhedral components, to change continuously and in a compensating manner across the series. The thermal expansion data also imply that volumes of F-Cl mixing in fluor-chlorapatite are constant from room temperature to 1000 °C. References: Cherniak, D.J. (2000) Rare earth element diffusion in apatite. Geochimica et Cosmochimica Acta 64, 3871-3885. Harlov, D.E. and Förster, H-J. (2002) High grade fluid metasomatism on both a local and regional Scale: the Seward Peninsula, Alaska and the Ivrea-Verbano Zone, Northern Italy Part II: phosphate mineral chemistry. Journal of Petrology 43, 801-824. Holland, T.J.B. and Redfern, S.A.T. (1997) Unit-cell refinement: Changing the dependent variable, and use of regression diagnostics. Mineralogical Magazine 61, 65-77. Hovis, G.L., Brennan, S., Keohane, M., Crelling, J. (1999) High-temperature X-ray investigation of sanidine - analbite crystalline solutions: Thermal expansion, phase transitions, and volumes of mixing. The Canadian Mineralogist 37, 701-709. Hovis, G.L., Crelling, J., Wattles, D., Dreibelbis, B., Dennison, A., Keohane, M., and Brennan, S. (2003) Thermal expansion of nepheline - kalsilite crystalline solutions. Mineralogical Magazine 67, 535-546. Hovis, G.L. and Graeme-Barber, A. (1997) Volumes of K-Na mixing for low albite - microcline crystalline solutions at elevated temperature: A test of regular solution thermodynamic models. American Mineralogist 82, 158-164. Hovis, G.L., Harlov, D.E., Hahn, A., and Steigert, H. (2007) Enthalpies and volumes of F-Cl mixing in fluorapatite - chlorapatite crystalline solutions. Geophysical Research Abstracts 9, abstract 01748. Hovis, G.L., Morabito, J.R. Spooner, R., Mott, A. Person, E.L., Henderson, C. Michael B., Roux, J., and Harlov, D. (2008) A simple predictive model for the thermal expansion of AlSi3 feldspars. American Mineralogist 98, 1568-1573. Hovis, G.L., Person, E., Spooner, A., and Roux, J. (2006) Thermal expansion of highly silicic nepheline - kalsilite crystalline solutions. Mineralogical Magazine 70, 383-396. O'Reilly, S.Y. and Griffin, W.L. (2000) Apatite in the mantle: implications for metasomatic processes and high heat production in Phanerozoic mantle. Lithos 53, 217-232. Parrish, W. (1953) X-Ray reflection angle tables for several standards. Technical Report No. 68, Philips Laboratories Incorporated, Irvington on Hudson, New York.

Proposed Test of Relative Phase as Hidden Variable in Quantum Mechanics

DTIC Science & Technology

2012-01-01

implicitly due to its ubiquity in quantum theory , but searches for dependence of measurement outcome on other parameters have been lacking. For a two -state...implemen- tation for the specific case of an atomic two -state system with laser-induced fluores- cence for measurement. Keywords Quantum measurement...Measurement postulate · Born rule 1 Introduction 1.1 Problems with Quantum Measurement Quantum theory prescribes probabilities for outcomes of measurements

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

Effects of Sterilizing Agents on Microorganisms

DTIC Science & Technology

1963-03-01

light increases with the incubation of Escherichia coli at 15, but the rate of in- 85. ESTUDIO DE LOS EFECTOS QUE PRODUCE duction of mutations...between the deoxyribonucleic acid content of a cell and its response to irradiation: 5-fluor- 86. ESTUDIO DE LOS EFECTOS QUE PRODUCE ouracil treated...EFFECTS OF ULTRAVIOLET LIGHT ON 84. ESTUDIO DE LOS EFECTOS QUE PRODUCE BACTERIA. III. ALTERATIONS IN THE LA LUZ ULTRAVIOLETA SOBRE LAS PHYSIOLOGICAL

["Mixed" and "miscellaneous" vulvovaginitis: diagnostics and therapy of vaginal administration of nystatin and nifuratel].

PubMed

Cepický, P; Malina, J; Líbalová, Z; Kuzelová, M

2005-05-01

The evaluation of combined and miscellaneous vulvovaginal infections incidence and their treatment with combined vaginal products containing nifuratel and nystatin. Prospective study. Gynecologic outpatient department LEVRET, Prague; Laboratories of Microbiology AescuLab, Prague. 70 consecutive patients were examined with complaint of vaginal fluor and/or pruritus. We established macroscopic features of fluor, pH, amine test and mounts stained with Giemsa and Gram. We qualified the cases with more diagnostic criteria (mycosis, lactobacillosis, anaerobic vaginosis, aerobic vaginitis) as combined infection, those with no diagnostic criteria as miscellaneous. We treated all patients with vaginal tablets nystatin + nifuratel (Macmiror complex). We prescribed clotrimazol cream, if pruritus was present. We evaluated withdrawals of symptoms and relapses during 3 months after treatment. Combined infection was found in 21 patients from 70 (30%). The most frequent combination was that of mycosis and aerobic vaginitis (13/70, 18.6%) or mycosis and anaerobic vaginosis (4/70, 5.7%); 11 patients fulfilled criteria of no diagnosis. We concluded them as "miscelaneous". The treatment was successful in all cases, 10 women relapsed in 3 months. Combined vaginal infection findings are present very often (30%), likewise miscellaneous ones (15%) occur. The treatment of these women in successful with vaginal tablets with nystatin + nifuratel.

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

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

Effect of aging temperature on formation of sol-gel derived fluor-hydroxyapatite nanoparticles.

PubMed

Joughehdoust, S; Behnamghader, A; Jahandideh, R; Manafi, S

2010-04-01

Synthetic hydroxyapatite (HA) has been recognized as one of the most important bone substitute materials in orthopaedics and dentistry over past few decades because of its chemical and biological similarity to the mineral phase of human bone. One solution for reduction the solubility of HA in biological environments is replacing F- by OH in HA structure and forming fluor-hydroxyapatite (FHA) solid solution. In this paper, FHA nanoparticles were successfully synthesized by a sol-gel method. Also, the influence of aging temperature on formation of FHA powder was studied. Equimolar solutions of calcium nitrate tetrahydrate, triethyl phosphite and ammonium fluoride in ethanol were used as Ca, P and F precursors. After aging at different temperatures, the synthesized powders were heat treated at 550 degrees C. The powders were investigated with X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), selected area electron diffraction pattern (SAED), energy dispersive analysis of X-ray (EDAX) and zetasizer measurement. The results of XRD proved the presence of fluorapatite (FA) and HA in all samples. In addition, the formation of FHA was confirmed by FT-IR results. XRD studies also showed that the crystallites were in nanometric scale. At the same time, this result was in good agreement with the result of zetasizer analysis.

A fusion-spliced near-field optical fiber probe using photonic crystal fiber for nanoscale thermometry based on fluorescence-lifetime measurement of quantum dots.

PubMed

Fujii, Takuro; Taguchi, Yoshihiro; Saiki, Toshiharu; Nagasaka, Yuji

2011-01-01

We have developed a novel nanoscale temperature-measurement method using fluorescence in the near-field called fluorescence near-field optics thermal nanoscopy (Fluor-NOTN). Fluor-NOTN enables the temperature distributions of nanoscale materials to be measured in vivo/in situ. The proposed method measures temperature by detecting the temperature dependent fluorescence lifetimes of Cd/Se quantum dots (QDs). For a high-sensitivity temperature measurement, the auto-fluorescence generated from a fiber probe should be reduced. In order to decrease the noise, we have fabricated a novel near-field optical-fiber probe by fusion-splicing a photonic crystal fiber (PCF) and a conventional single-mode fiber (SMF). The validity of the novel fiber probe was assessed experimentally by evaluating the auto-fluorescence spectra of the PCF. Due to the decrease of auto-fluorescence, a six- to ten-fold increase of S/N in the near-field fluorescence lifetime detection was achieved with the newly fabricated fusion-spliced near-field optical fiber probe. Additionally, the near-field fluorescence lifetime of the quantum dots was successfully measured by the fabricated fusion-spliced near-field optical fiber probe at room temperature, and was estimated to be 10.0 ns.

Regulation of Tumor Progression by Mgat5-Dependent Glycosylation

DTIC Science & Technology

2002-07-01

PfA, P. found in mammals are also conserved in this nematode (9-13). vudgaris leucoaggiutinin EST, expressed sequence tag; FACS, fluores- cence...this paper, we establish that the pCSYK-L116RtogeneratepEGFP-L116R. The introduced segment was nematode orthologue is functionally equivalent to that...into the between 30 and 100 pl. Enzyme sources were nematode microsomal EcoRV site of pZErO-2 (Invitrogen). Independent recombinants were membranes

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

Experimental Analysis and Computational Modeling of Network States and Drug Responses in the PI3K/Akt/mTOR Network

DTIC Science & Technology

2010-09-01

Fixation was in 2% formaldehyde, followed by permeabilization in 100% methanol at -20 C. Blocking and incubation with primary/secondary antibodies ...was performed in Odyssey blocking buffer (LICOR). Primary antibodies were obtained from Cell Signaling, BD Biosciences, and Santa Cruz...Biotechnology, and Alexa fluor 488-, 555-, or 647-conjugated secondary antibodies were obtained from Invitrogen. Data were collected using an Applied Precision

Ternary liquid scintillator for optical fiber applications

DOEpatents

Franks, Larry A.; Lutz, Stephen S.

1982-01-01

A multicomponent liquid scintillator solution for use as a radiation-to-light converter in conjunction with a fiber optic transmission system. The scintillator includes a quantity of 5-amino-9-diethylaminobenz (a) phenoxazonium nitrate (Nile Blue Nitrate) as a solute in a fluor solvent such as benzyl alcohol. The use of PPD as an additional solute is also disclosed. The system is controllable by addition of a suitable quenching agent, such as phenol.

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

DTIC Science & Technology

2016-12-01

Aldefluor reagent. (B) A549 control lung cancer cells were incubated with Alde- fluor regent and DEAB, an inhibitor of aldehyde dehydro- genase. (C...increase in liquid colony formation or in cell proliferation compared to SHH- cells. Therefore, we turned to identify aldehyde dehydrogenase (ALDH...in which a green fluorescent BODIPY moiety is linked to aminoacetaldehyde, an aldehyde dehydrogenase substrate, and thus, cells expressing ALDH

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.

Liquid scintillators for optical fiber applications

DOEpatents

Franks, Larry A.; Lutz, Stephen S.

1982-01-01

A multicomponent liquid scintillator solution for use as a radiation-to-light converter in conjunction with a fiber optic transmission system. The scintillator includes a quantity of 1, 2, 4, 5, 3H, 6H, 1 OH, tetrahydro-8-trifluoromethyl (1) benzopyrano (9, 9a, 1-gh) quinolizin-10-one (Coumarin) as a solute in a fluor solvent such as benzyl alcohol or pseudo-cumene. The use of BIBUQ as an additional or primary solute is also disclosed.

Cancer Localization in the Prostate with F-18 Fluorocholine Positron Emission Tomography. Addendum

DTIC Science & Technology

2010-01-01

of malignancy in anatomical sextants of the prostate gland. The rationale for evaluating fluorocholine as an oncologic tracer applicable to...interest in radiolabeled choline deriv- atives as oncologic tracers for positron emission tomogra- phy (PET) [6, 7]. This approach has shown feasibility in...prostate cancer using the tracer fluorine-18 fluor- omethylcholine (18F-choline) [8–11]. As a preliminary step in evaluating 18F-choline PET/CT as a

QUEST Hanford Site Computer Users - What do they do?

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

WITHERSPOON, T.T.

2000-03-02

The Fluor Hanford Chief Information Office requested that a computer-user survey be conducted to determine the user's dependence on the computer and its importance to their ability to accomplish their work. Daily use trends and future needs of Hanford Site personal computer (PC) users was also to be defined. A primary objective was to use the data to determine how budgets should be focused toward providing those services that are truly needed by the users.

Hydroxyapatite, fluor-hydroxyapatite and fluorapatite produced via the sol-gel method: dissolution behaviour and biological properties after crystallisation.

PubMed

Tredwin, Christopher J; Young, Anne M; Abou Neel, Ensanya A; Georgiou, George; Knowles, Jonathan C

2014-01-01

Hydroxyapatite (HA), fluor-hydroxyapatite (FHA) with varying levels of fluoride ion substitution and fluorapatite (FA) were synthesised by the sol-gel method as possible implant coating or bone-grafting materials. Calcium nitrate and triethyl phosphite were used as precursors under an ethanol-water based solution. Different amounts of ammonium fluoride were incorporated for the preparation of the FHA and FA sol-gels. After heating and powdering the sol-gels, dissolution behaviour was assessed using ion chromatography to measure Ca(2+) and PO4 (3-) ion release. Biological behaviour was assessed using cellular proliferation with human osteosarcoma cells and alamarBlue™ assay. Statistical analysis was performed with a two way analysis of variance and post hoc testing with a Bonferroni correction. Increasing fluoride substitution into an apatite structure decreased the dissolution rate. Increasing the firing temperature of the HA, FHA and FA sol-gels up to 1,000 °C decreased the dissolution rate. There was significantly higher cellular proliferation on highly substituted FHA and FA than on HA or Titanium. The properties of an implant coating or bone grafting material can be tailored to meet specific requirements by altering the amount of fluoride that is incorporated into the original apatite structure. The dissolution behaviour can further be altered by the temperature at which the sol-gel is fired.

A survey on polycyclic aromatic hydrocarbon concentrations in soil in Chiang-Mai, Thailand

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

Amagai, Takashi; Takahashi, Yukari; Matsushita, Hidetsuru

Soil samples were collected at 30 sampling sites along roadsides in the city of Chiang-Mai, Thailand, in February 1996, and concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) were determined. The distribution of PAH concentration in the soil samples was almost log-normal for all PAHs. Concentrations of pyrene (Py) and fluoranthene (Fluor) were the highest, followed by those of benzo[ghi]perylene and coronene (Cor). Since PAH concentrations were highest on the roadside where the traffic density was high, vehicles were the main determinants of PAH concentration in soil in Chiang-Mai. Significant correlations among PAH concentrations were found for almost all PAHs. PAHmore » profiles in the air were different from those in the soil. For example, relative benzo[a]pyrene (BaP) concentration in the soil was significantly lower than that in the air. Relative concentrations of Fluor, Py, chrysene, and Cor in the soil were considerably higher than those in the air, due presumably to their difference in photochemical reactivities and in sources. The sampling of soil has advantages relative to that of air: (1) collection of soil is easy; (2) it needs no special equipment and electricity; (3) it takes little time; and (4) it can be collected anywhere. Therefore PAH analysis in soil was useful as a proxy-screening tool for air pollution levels with consideration of compositional differences between soil and air samples.« less

Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis.

PubMed

Hilliard, Mark; Alley, William R; McManus, Ciara A; Yu, Ying Qing; Hallinan, Sinead; Gebler, John; Rudd, Pauline M

Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.

The Vibrational Deactivation of HF(v = 3) and HF(v = 2) by H Atoms

DTIC Science & Technology

1977-08-18

experiments. A silicon flat in front of the photomultiplier restricted the monitored fluores- cence to wavelengths > I 4tm. An RCA C-31034 (GaAs... stent with the present results f’ir HF(v =1) and HI-F(v = 2’ remioval rates but can not explain the fast HF(v = 3) rate. Wilk-ins performed trajectory...Sciences Laboratory: Development of new materials; metal matrix composites and new forms of carbon; test and evaluation of graphite and ceramics in

Center for the Evaluation of Biomarkers for the Early Detection of Breast Cancer

DTIC Science & Technology

2009-10-01

Table S1, including mammography (BI-RADS) score and breast density at the last mammogram before diagnosis, lymph node positivity, tumor size, number of...Bibliography 1.& Fletcher RH. Should all people over the age of 50 have regular fecal occult-blood tests? If it works, why not do it? New England Journal...blocked for 15 minutes at room temperature in fluores- cence-activated cell sorting (FACS) buffer (PBS, 0.1% bovine serum albumin (BSA), 0.02% sodium

Extension of Storage Stability in Energy-Dense Encapsulated Systems by Minimization of Lipid Oxidation

DTIC Science & Technology

1988-01-01

of compounds like gallic 8 and chlorogenic acids . There are, of course, modifying circumstances. BHA and BHT are quite volatile and may be partially...GROUP FLUORE-SCEI-NCEI AIITox I l)ATI ON ASCORBIC ACID MA LLARI) REIA’IION ANT lOX I DANTs SOLID SAMPLE FLUORESCENCE ICALI.ATEI) IIYDROP1I 11 1. A...By contrast, ascorbic acid produced little to no effect in the compressed system. ....DG, a lipophile, was mcre than twic.Pas effective as propyl

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

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

Optimization of Antibody-Conjugated Magnetic Nanoparticles for Target Preconcentration and Immunoassays

DTIC Science & Technology

2010-01-01

protein, AlexaFluor647– chicken IgG (Alexa647–chick IgG). Antibody-labeled MNPs (Alexa647– chick–MNPs) were used to preconcentrate the target via magnetic...separation and as the tracer to dem- onstrate binding to slides modified with anti- chicken IgG as a capture agent. A full optimization study of the...magnetically assisted transport evanescent field fluoroimmunoassay; Alexa647–chick–MNPs, MNPs functionalized with fluorescently labeled target chicken IgG

Application of Time-Resolved Spectroscopies to the Study of Energetic Materials - 1982

DTIC Science & Technology

1983-05-24

fluores- cence intensity as a function of UV pulse energy, for individual laser shots. The lower curve shows the UV + probe induced fluorescence... intensity as a function of UV pulse energy, for individual laser shots. The lower curve shows the UV + probe Induced fluorescence, at 1 ns delay...locked Nd:YAG Laser Pulse ", Appl. Phys. Lett 26, 501-503 (1975). 97 43. A. J. Campillo, V. H. Kollman and S. L. Shapiro, " Intensity Dependence of

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.

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

External Performance Evaluation Program Participation at Fluor Hanford (FH) 222S Lab

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

CLARK, G.A.

2002-06-01

Fluor Hanford operates the U. S. Department of Energy's (DOE) 2224 Laboratory on the Hanford Site in Southeastern Washington State. 222-S Laboratory recently celebrated its 50th anniversary of providing laboratory services to DOE and DOE contractors on the Hanford Site. The laboratory operated for many years as a production support analytical laboratory, but in the last two decades has supported the Hanford Site cleanup mission. The laboratory performs radioanalytical, inorganic, and organic characterization analyses on highly radioactive liquid and solid tank waste that will eventually be vitrified for long-term storage and or disposal. It is essential that the laboratory reportmore » defensible, highly credible data in its role as a service provider to DOE and DOE contractors. Among other things, the participation in a number of performance evaluation (PE) programs helps to ensure the credibility of the laboratory. The laboratory currently participates in Environmental Resource Associates' Water Pollution (WP) Studies and the DOE Environmental Management Laboratory (EML) Quality Assessment Program (QAP). DOE has mandated participation of the laboratory in the EML QAP. This EML program evaluates the competence of laboratories performing environmental radioanalytical measurements for DOE, and is the most comprehensive and well-established PE program in the DOE community for radiochemical laboratories. Samples are received and analyzed for radionuclides in air filter, soil, vegetation, and water matrices on a semiannual basis. The 222-S Laboratory has performed well in this program over the years as evidenced by the scores in the chart below.« less

Intracellular zinc distribution in mitochondria, ER and the Golgi apparatus

PubMed Central

Lu, Qiping; Haragopal, Hariprakash; Slepchenko, Kira G; Stork, Christian; Li, Yang V

2016-01-01

Zinc (Zn2+) is required for numerous cellular functions. As such, the homeostasis and distribution of intracellular zinc can influence cellular metabolism and signaling. However, the exact distribution of free zinc within live cells remains elusive. Previously we showed the release of zinc from thapsigargin/IP3-sensitive endoplasmic reticulum (ER) storage in cortical neurons. In the present study, we investigated if other cellular organelles also contain free chelatable zinc and function as organelle storage for zinc. To identify free zinc within the organelles, live cells were co-stained with Zinpyr-1, a zinc fluorescent dye, and organelle-specific fluorescent dyes (MitoFluor Red 589: mitochondria; ER Tracker Red: endoplasmic reticulum; BODIPY TR ceramide: Golgi apparatus; Syto Red 64: nucleus). We examined organelles that represent potential storing sites for intracellular zinc. We showed that zinc fluorescence staining was co-localized with MitoFluor Red 589, ER Tracker Red, and BODIPY TR ceramide respectively, suggesting the presence of free zinc in mitochondria, endoplasmic reticulum, and the Golgi apparatus. On the other hand, cytosol and nucleus had nearly no detectable zinc fluorescence. It is known that nucleus contains high amount of zinc binding proteins that have high zinc binding affinity. The absence of zinc fluorescence suggests that there is little free zinc in these two regions. It also indicates that the zinc fluorescence detected in mitochondria, ER and Golgi apparatus represents free chelatable zinc. Taken together, our results support that these organelles are potential zinc storing organelles during cellular zinc homeostasis. PMID:27186321

Diffusion of flexible random-coil dextran polymers measured in anisotropic brain extracellular space by integrative optical imaging.

PubMed

Xiao, Fanrong; Nicholson, Charles; Hrabe, Jan; Hrabetová, Sabina

2008-08-01

There are a limited number of methods available to quantify the extracellular diffusion of macromolecules in an anisotropic brain region, e.g., an area containing numerous aligned fibers where diffusion is faster along the fibers than across. We applied the integrative optical imaging method to measure diffusion of the fluorophore Alexa Fluor 488 (molecular weight (MW) 547) and fluorophore-labeled flexible random-coil dextran polymers (dex3, MW 3000; dex75, MW 75,000; dex282, MW 282,000; dex525, MW 525,000) in the extracellular space (ECS) of the anisotropic molecular layer of the isolated turtle cerebellum. For all molecules, two-dimensional images acquired an elliptical shape with major and minor axes oriented along and across, respectively, the unmyelinated parallel fibers. The effective diffusion coefficients, D*(major) and D*(minor), decreased with molecular size. The diffusion anisotropy ratio (DAR = D*(major)/D*(minor)) increased for Alexa Fluor 488 through dex75 but then unexpectedly reached a plateau. We argue that dex282 and dex525 approach the ECS width and deform to diffuse. In support of this concept, scaling theory shows the diffusion behavior of dex282 and dex525 to be consistent with transition to a reptation regime, and estimates the average ECS width at approximately 31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover.

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.

ATR Spent Fuel Options Study

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

Connolly, Michael James; Bean, Thomas E.; Brower, Jeffrey O.

The Advanced Test Reactor (ATR) is a materials and fuels test nuclear reactor that performs irradiation services for the U.S. Department of Energy (DOE) Office of Nuclear Energy (NE), Naval Reactors, the National Nuclear Security Administration (NNSA), and other research programs. ATR achieved initial criticality in 1967 and is expected to operate in support of needed missions until the year 2050 or beyond. It is anticipated that ATR will generate approximately 105 spent nuclear fuel (SNF) elements per year through the year 2050. Idaho National Laboratory (INL) currently stores 2,008 ATR SNF elements in dry storage, 976 in wet storage,more » and expects to have 1,000 elements in wet storage before January 2017. A capability gap exists at INL for long-term (greater than the year 2050) management, in compliance with the Idaho Settlement Agreement (ISA), of ATR SNF until a monitored retrievable geological repository is open. INL has significant wet and dry storage capabilities that are owned by the DOE Office of Environmental Management (EM) and operated and managed by Fluor Idaho, which include the Idaho Nuclear Technology and Engineering Center’s (INTEC’s) CPP-666, CPP-749, and CPP-603. In addition, INL has other capabilities owned by DOE-NE and operated and managed by Battelle Energy Alliance, LLC (BEA), which are located at the Materials and Fuel Complex (MFC). Additional storage capabilities are located on the INL Site at the Naval Reactors Facility (NRF). Current INL SNF management planning, as defined in the Fluor Idaho contract, shows INTEC dry fuel storage, which is currently used for ATR SNF, will be nearly full after transfer of an additional 1,000 ATR SNF from wet storage. DOE-NE tasked BEA with identifying and analyzing options that have the potential to fulfill this capability gap. BEA assembled a team comprised of SNF management experts from Fluor Idaho, Savannah River Site (SRS), INL/BEA, and the MITRE Corp with an objective of developing and analyzing options for fulfilling the capability gap. This management options analysis is not an alternatives analysis as defined by DOE Order 413.3B; rather, it is an evaluation of near-term, mid term and long-term actions needed to fulfill the capability gap. The actions are described in sufficient detail to inform stakeholders and DOE decision makers regarding a potential path forward. The recommended path forward will inform Fiscal Year 2019 budget formulation, support potential National Environmental Policy Act (NEPA) analyses, and may or may not include capital asset projects.« less

User Manual for the AZ-101 Data Acquisition System (AS-101 DAS)

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

BRAYTON, D.D.

2000-02-17

User manual for the TK AZ-101 Waste Retrieval System Data Acquisition System. The purpose of this document is to describe use of the AZ-101 Data Acquisition System (AZ-101 DAS). The AZ-101 DAS is provided to fulfill the requirements for data collection and monitoring as defined in Letters of Instruction (LOI) from Numatec Hanford Corporation (NHC) to Fluor Federal Services (FFS). For a complete description of the system, including design, please refer to the AZ-101 DAS System Description document, RPP-5572.

PPO-ethanol system as wavelength shifter for the Cherenkov counting technique using a liquid scintillation counter

NASA Astrophysics Data System (ADS)

Takiue, Makoto; Fujii, Haruo; Ishikawa, Hiroaki

1984-12-01

2, 5-diphenyloxazole (PPO) has been proposed as a wavelength shifter for Cherenkov counting. Since PPO is not incorporated with water, we have introduced the fluor into water in the form of micelle using a PPO-ethanol system. This technique makes it possible to obtain a high Cherenkov counting efficiency under stable sample conditions, attributed to the proper spectrometric features of the PPO. The 32P Cherenkov counting efficiency (68.4%) obtained from this technique is large as that measured with a conventional Cherenkov technique.

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

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.

PROGRESS WITH K BASINS SLUDGE RETRIEVAL STABILIZATION & PACKAGING AT THE HANFORD NUCLEAR SITE

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

KNOLLMEYER, P.M.; PHILLIPS, C; TOWNSON, P.S.

This paper shows how Fluor Hanford and BNG America have combined nuclear plant skills from the U.S. and the U.K. to devise methods to retrieve and treat the sludge that has accumulated in K Basins at the Hanford Site over many years. Retrieving the sludge is the final stage in removing fuel and sludge from the basins to allow them to be decontaminated and decommissioned, so as to remove the threat of contamination of the Columbia River. A description is given of sludge retrieval using vacuum lances and specially developed nozzles and pumps into Consolidation Containers within the basins. Themore » special attention that had to be paid to the heat generation and potential criticality issues with the irradiated uranium-containing sludge is described. The processes developed to re-mobilize the sludge from the Consolidation Containers and pump it through flexible and transportable hose-in-hose piping to the treatment facility are explained with particular note made of dealing with the abrasive nature of the sludge. The treatment facility, housed in an existing Hanford building, is described, and the uranium-corrosion and grout packaging processes explained. The uranium corrosion process is a robust, tempered process very suitable for dealing with a range of differing sludge compositions. Optimization and simplification of the original sludge corrosion process design is described and the use of transportable and reusable equipment is indicated. The processes and techniques described in the paper are shown to have wide applicability to nuclear cleanup.« less

A near-infrared interferometric survey of debris-disc stars. VI. Extending the exozodiacal light survey with CHARA/JouFLU

NASA Astrophysics Data System (ADS)

Nuñez, P. D.; Scott, N. J.; Mennesson, B.; Absil, O.; Augereau, J.-C.; Bryden, G.; ten Brummelaar, T.; Ertel, S.; Coudé du Foresto, V.; Ridgway, S. T.; Sturmann, J.; Sturmann, L.; Turner, N. J.; Turner, N. H.

2017-12-01

We report the results of high-angular-resolution observations that search for exozodiacal light in a sample of main sequence stars and sub-giants. Using the "jouvence" of the fiber linked unit for optical recombination (JouFLU) at the center for high angular resolution astronomy (CHARA) telescope array, we have observed a total of 44 stars. Out of the 44 stars, 33 are new stars added to the initial, previously published survey of 42 stars performed at CHARA with the fiber linked unit for optical recombination (FLUOR). Since the start of the survey extension, we have detected a K-band circumstellar excess for six new stars at the 1% level or higher, four of which are known or candidate binaries, and two for which the excess could be attributed to exozodiacal dust. We have also performed follow-up observations of 11 of the stars observed in the previously published survey and found generally consistent results. We do however detect a significantly larger excess on three of these follow-up targets: Altair, υ And and κ CrB. Interestingly, the last two are known exoplanet host stars. We perform a statistical analysis of the JouFLU and FLUOR samples combined, which yields an overall exozodi detection rate of . We also find that the K-band excess in FGK-type stars correlates with the existence of an outer reservoir of cold (≲100 K) dust at the 99% confidence level, while the same cannot be said for A-type stars.

HPSEC reveals ubiquitous components in fluorescent dissolved organic matter across aquatic ecosystems

NASA Astrophysics Data System (ADS)

Wünsch, Urban; Murphy, Kathleen; Stedmon, Colin

2017-04-01

Absorbance and fluorescence spectroscopy are efficient tools for tracing the supply, turnover and fate of dissolved organic matter (DOM). The fluorescent fraction of DOM (FDOM) can be characterized by measuring excitation-emission matrices and decomposing the combined fluorescence signal into independent underlying fraction using Parallel Factor Analysis (PARAFAC). Comparisons between studies, facilitated by the OpenFluor database, reveal highly similar components across different aquatic systems and between studies. To obtain PARAFAC models in sufficient quality, scientists traditionally rely on analyzing dozens to hundreds of samples spanning environmental gradients. A cross-validation of this approach using different analytical tools has not yet been accomplished. In this study, we applied high-performance size-exclusion chromatography (HPSEC) to characterize the size-dependent optical properties of dissolved organic matter of samples from contrasting aquatic environments with online absorbance and fluorescence detectors. Each sample produced hundreds of absorbance spectra of colored DOM (CDOM) and hundreds of matrices of FDOM intensities. This approach facilitated the detailed study of CDOM spectral slopes and further allowed the reliable implementation of PARAFAC on individual samples. This revealed a high degree of overlap in the spectral properties of components identified from different sites. Moreover, many of the model components showed significant spectral congruence with spectra in the OpenFluor database. Our results provide evidence of the presence of ubiquitous FDOM components and additionally provide further evidence for the supramolecular assembly hypothesis. They demonstrate the potential for HPSEC to provide a wealth of new insights into the relationship between optical and chemical properties of DOM.

IMPLEMENTATION OF THE SEISMIC DESIGN CRITERIA OF DOE-STD-1189-2008 APPENDIX A [FULL PAPER

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

OMBERG SK

2008-05-14

This paper describes the approach taken by two Fluor Hanford projects for implementing of the seismic design criteria from DOE-STD-1189-2008, Appendix A. The existing seismic design criteria and the new seismic design criteria is described, and an assessment of the primary differences provided. The gaps within the new system of seismic design criteria, which necessitate conduct of portions of work to the existing technical standards pending availability of applicable industry standards, is discussed. Two Hanford Site projects currently in the Control Decision (CD)-1 phase of design have developed an approach to implementation of the new criteria. Calculations have been performedmore » to determine the seismic design category for one project, based on information available in early CD-1. The potential effects of DOE-STD-1189-2008, Appendix A seismic design criteria on the process of project alternatives analysis is discussed. Present of this work is expected to benefit others in the DOE Complex that may be implementing DOE-STD-1189-2008.« less

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.

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

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.

Growth of Third-Harmonic Signal in Optical Glass Fibre

DTIC Science & Technology

1990-01-01

harmonic signal In optical glass fibres , illuminated vith 10kV peak pover pulses from A NdYAG lasers has been observed. Broadband fluores.enc from the third...J’T. Al-0002 GROWTH OF THIFRO-HARMVONIC SIGNAL IN OPTICAL GLASS FIORE Irdexim tems 0,rkl f. N motwvij p~ .G For mo i fibres the Sit signal strtd g0r...Amorphous nature of glass ) but with time, as the fibre is illuminated with inltense laser light at 4 w1O6pm, the S1t signal 3rows. What is believed to

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.

Fast Electron Deposition in Laser Shock Compressed Plastic Targets

NASA Astrophysics Data System (ADS)

Hall, T. A.; Ellwi, S.; Batani, D.; Bernardinello, A.; Masella, V.; Koenig, M.; Benuzzi, A.; Krishnan, J.; Pisani, F.; Djaoui, A.; Norreys, P.; Neely, D.; Rose, S.; Key, M. H.; Fews, P.

1998-08-01

We present the first results of fast electron deposition in a laser shock compressed plasma. The interaction of a 3 ps, 15 J laser pulse with solid polyethylene targets is used to produce fast electrons on one side of foil targets and a 2 ns duration laser pulse is used to drive a shock wave into the target from the opposite side. Kα emission from chlorine fluor buried layers is used to measure the electron transport. The hot electron range in the shock compressed plastic is found to be approximately twice as large as the range in the solid density plastic.

Plutonium Finishing Plant (PFP) Final Safety Analysis Report (FSAR) [SEC 1 THRU 11

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

ULLAH, M K

2001-02-26

The Plutonium Finishing Plant (PFP) is located on the US Department of Energy (DOE) Hanford Site in south central Washington State. The DOE Richland Operations (DOE-RL) Project Hanford Management Contract (PHMC) is with Fluor Hanford Inc. (FH). Westinghouse Safety Management Systems (WSMS) provides management support to the PFP facility. Since 1991, the mission of the PFP has changed from plutonium material processing to preparation for decontamination and decommissioning (D and D). The PFP is in transition between its previous mission and the proposed D and D mission. The objective of the transition is to place the facility into a stablemore » state for long-term storage of plutonium materials before final disposition of the facility. Accordingly, this update of the Final Safety Analysis Report (FSAR) reflects the current status of the buildings, equipment, and operations during this transition. The primary product of the PFP was plutonium metal in the form of 2.2-kg, cylindrical ingots called buttoms. Plutonium nitrate was one of several chemical compounds containing plutonium that were produced as an intermediate processing product. Plutonium recovery was performed at the Plutonium Reclamation Facility (PRF) and plutonium conversion (from a nitrate form to a metal form) was performed at the Remote Mechanical C (RMC) Line as the primary processes. Plutonium oxide was also produced at the Remote Mechanical A (RMA) Line. Plutonium processed at the PFP contained both weapons-grade and fuels-grade plutonium materials. The capability existed to process both weapons-grade and fuels-grade material through the PRF and only weapons-grade material through the RMC Line although fuels-grade material was processed through the line before 1984. Amounts of these materials exist in storage throughout the facility in various residual forms left from previous years of operations.« less

Hydrothermal Testing of K Basin Sludge and N Reactor Fuel at Sludge Treatment Project Operating Conditions

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

Delegard, Calvin H.; Schmidt, Andrew J.; Thornton, Brenda M.

The Sludge Treatment Project (STP), managed for the U. S. DOE by Fluor Hanford (FH), was created to design and operate a process to eliminate uranium metal from K Basin sludge prior to packaging for Waste Isolation Pilot Plant (WIPP). The STP process uses high temperature liquid water to accelerate the reaction, produce uranium dioxide from the uranium metal, and safely discharge the hydrogen. Under nominal process conditions, the sludge will be heated in pressurized water at 185°C for as long as 72 hours to assure the complete reaction (corrosion) of up to 0.25-inch diameter uranium metal pieces. Under contractmore » to FH, the Pacific Northwest National Laboratory (PNNL) conducted bench-scale testing of the STP hydrothermal process in November and December 2006. Five tests (~50 ml each) were conducted in sealed, un-agitated reaction vessels under the hydrothermal conditions (e.g., 7 to 72 h at 185°C) of the STP corrosion process using radioactive sludge samples collected from the K East Basin and particles/coupons of N Reactor fuel also taken from the K Basins. The tests were designed to evaluate and understand the chemical changes that may be occurring and the effects that any changes would have on sludge rheological properties. The tests were not designed to evaluate engineering aspects of the process. The hydrothermal treatment affected the chemical and physical properties of the sludge. In each test, significant uranium compound phase changes were identified, resulting from dehydration and chemical reduction reactions. Physical properties of the sludge were significantly altered from their initial, as-settled sludge values, including, shear strength, settled density, weight percent water, and gas retention.« less

Hydroxyapatite, fluor-hydroxyapatite and fluorapatite produced via the sol-gel method: bonding to titanium and scanning electron microscopy.

PubMed

Tredwin, Christopher J; Georgiou, George; Kim, Hae-Won; Knowles, Jonathan C

2013-05-01

Hydroxyapatite (HA), fluor-hydroxyapatite (FHA) with varying levels of fluoride ion substitution and fluorapatite (FA) production has been characterised and optimised by the sol-gel method and the dissolution and biological properties of these materials were investigated. It was the objective of this study to investigate the potential bond strength and interaction of these materials with titanium. HA, FHA and FA were synthesised by a sol-gel method. Calcium nitrate and triethyl phosphite were used as precursors under an ethanol-water based solution. Different amounts of ammonium fluoride (NH4F) were incorporated for the preparation of the FHA and FA sol-gels. Using a spin coating technique the sol-gels were coated onto commercially pure titanium disks and crystallised at various temperatures. Using scanning electron microscopy (SEM) and elemental analysis, the surface characteristics, coating thickness and interaction of the Ti substrate and coating were investigated. The bond strengths of the coating to the Ti were investigated using an Instron Universal Load Testing Machine. Statistical analysis was performed with a two-way analysis of variance and post hoc testing with a Bonferroni correction. (1) Coating speed inversely influenced the coating thickness. (2) Increasing fluoride ion substitution and heating temperature significantly increased bond strength and (3) increasing fluoride ion substitution increased the coating thickness. FHA and FA synthesised using the sol-gel technique may offer a superior alternative to coating titanium implants with HA and plasma spraying. HA, FHA and FA materials synthesised by the sol-gel method may also have a use as bone grafting materials. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

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

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.

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.

Absorbed radiation dosimetry of the D3-specific PET radioligand [18F]FluorTriopride estimated using rodent and nonhuman primate.

PubMed

Laforest, Richard; Karimi, Morvarid; Moerlein, Stephen M; Xu, Jinbin; Flores, Hubert P; Bognar, Christopher; Li, Aixiao; Mach, Robert H; Perlmutter, Joel S; Tu, Zhude

2016-01-01

[ 18 F]FluorTriopride ([ 18 F]FTP) is a dopamine D 3 -receptor preferring radioligand with potential for investigation of neuropsychiatric disorders including Parkinson disease, dystonia and schizophrenia. Here we estimate human radiation dosimetry for [ 18 F]FTP based on the ex-vivo biodistribution in rodents and in vivo distribution in nonhuman primates. Biodistribution data were generated using male and female Sprague-Dawley rats injected with ~370 KBq of [ 18 F]FTP and euthanized at 5, 30, 60, 120, and 240 min. Organs of interest were dissected, weighed and assayed for radioactivity content. PET imaging studies were performed in two male and one female macaque fascicularis administered 143-190 MBq of [ 18 F]FTP and scanned whole-body in sequential sections. Organ residence times were calculated based on organ time activity curves (TAC) created from regions of Interest. OLINDA/EXM 1.1 was used to estimate human radiation dosimetry based on scaled organ residence times. In the rodent, the highest absorbed radiation dose was the upper large intestines (0.32-0.49 mGy/MBq), with an effective dose of 0.07 mSv/MBq in males and 0.1 mSv/MBq in females. For the nonhuman primate, however, the gallbladder wall was the critical organ (1.81 mGy/MBq), and the effective dose was 0.02 mSv/MBq. The species discrepancy in dosimetry estimates for [ 18 F]FTP based on rat and primate data can be attributed to the slower transit of tracer through the hepatobiliary track of the primate compared to the rat, which lacks a gallbladder. Out findings demonstrate that the nonhuman primate model is more appropriate model for estimating human absorbed radiation dosimetry when hepatobiliary excretion plays a major role in radiotracer elimination.

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.

Development of drug delivery systems based on nanostructured porous silicon loaded with the anti-tumoral drug emodin adsorbed on silver nanoparticles

NASA Astrophysics Data System (ADS)

Hernández, Margarita; Recio, Gonzalo; Sevilla, Paz; Torres-Costa, Vicente; García-Ramos, José V.; Domingo, Concepción; Martín-Palma, Raúl J. J.

2012-10-01

A study of the fluorescence and Raman spectra of a new and complex drug delivery system formed by emodin adsorbed on silver nanoparticles embedded into a matrix of porous silicon is here reported. Several experimental methods of inclusion of the drug-silver set inside the pores, without previous functionalization of porous silicon, have been tested in order to optimize the conditions for the fluorescence detection of emodin. In this sense, we have also added bovine serum albumin to the system, finding that the presence of the protein enhances the fluores-cence signal from emodin.

Plume Delineation in the BC Cribs and Trenches Area

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

Rucker, Dale F.; Sweeney, Mark D.

2004-11-30

HydroGEOPHYSICS, Inc. and Pacific Northwest National Laboratory (PNNL) were contracted by Fluor Hanford Group, Inc. to conduct a geophysical investigation in the area of the BC Cribs and Trenches (subject site) at the Hanford Site in Richland, Washington. The BC Cribs and Trenches are located south of the 200 East Area. This document provides the details of the investigation to identify existing infrastructure from legacy disposal activities and to delineate the edges of a groundwater plume that contains radiological and heavy metal constituents beneath the 216-B-26 and 216-B-52 Trenches, and the 216-B-14 through 216-B-19 Cribs.

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.

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.

Oxygen quenching in a LAB based liquid scintillator and the nitrogen bubbling model

NASA Astrophysics Data System (ADS)

Xiao, Hua-Lin; Deng, Jing-Shan; Wang, Nai-Yan

2010-05-01

The oxygen quenching effect in a Linear Alkl Benzene (LAB) based liquid scintillator (LAB as the solvent, 3 g/L 2, 5 diphe-nyloxazole (PPO) as the fluor and 15 mg/L p-bis-(o-methylstyryl)-benzene (bis-MSB) as the λ-shifter) is studied by measuring the light yield as a function of the nitrogen bubbling time. It is shown that the light yield of the fully purged liquid scintillator is increased by 11% at room temperature and the room atmospheric pressure. A simple nitrogen bubbling model is proposed to describe the relationship between the relative light yield (oxygen quenching factor) and the bubbling time.

Badgastein spa—Austrian central Alps

NASA Astrophysics Data System (ADS)

Zötl, J. G.

1995-12-01

The Badgastein spa shows almost all the special geological and hydrological features existing in the northeastern central Alps. Situated within the Tauern window, the open joint system enables the infiltration of surface water to great depths. There it becomes heated and loaded with radon and trace elements such as fluor. The water discharges as thermal water springs at the steep right flank of the cascade of the Gastein-Ache. This hydraulic process occurs during a span of 3600 3800 years. The springs are captured in galleries, and the largest discharge is in gallery IX (2518 m3/d-1). The spa became known worldwide in the 19th century by visiting aristocrats from Central and Eastern Europe. The most important event in the 20th century was the discovery of the heat joint in the Böckstein gallery. In 1940 an effort to reactivate gold mining in the Radhausberg near Böckstein south of Badgastein failed. However, the miners had the unexpected experience of being cured of rheumatism. A connection between the great depth and high temperature in the gallery was made by the discovery of the joint set with heat and radon emanation. In the medical stations of the Böckstein gallery treatment of polyarthritis has become especially successful.

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

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.

Executive roundtable on coal-fired generation

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

NONE

2009-09-15

Power Engineering magazine invited six industry executives from the coal-fired sector to discuss issues affecting current and future prospects of coal-fired generation. The executives are Tim Curran, head of Alstom Power for the USA and Senior Vice President and General Manager of Boilers North America; Ray Kowalik, President and General Manager of Burns and McDonnell Energy Group; Jeff Holmstead, head of Environmental Strategies for the Bracewell Giuliani law firm; Jim Mackey, Vice President, Fluor Power Group's Solid Fuel business line; Tom Shelby, President Kiewit Power Inc., and David Wilks, President of Energy Supply for Excel Energy Group. Steve Blankinship, themore » magazine's Associate Editor, was the moderator. 6 photos.« less

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

IR and SiO Maser Observations of Miras

NASA Astrophysics Data System (ADS)

Cotton, W. D.; Mennesson, B.; Diamond, P. J.; Perrin, G.; Coudé du Foresto, V.; Chagnon, G.; van Langevelde, H. J.; Ridgway, S.; Waters, R.; Vlemmings, W.; Morel, S.; Traub, W.; Carleton, N.; Lacasse, M.

2005-12-01

Preliminary results of a coordinated program of near IR and SiO maser interferometric observations of Mira variables are reported. The 2.2 and 3.6 micron results are from the FLUOR/TISIS beam combiners on the IOTA interferometer and the SiO maser observations from the VLBA. The ratio of the SiO ring diameter to the apparent diameter at 2.2 microns for stars in our sample cluster around 2, whereas the 3.6 micron diameters range from slightly larger than the 2.2 micron diameter to approximately the SiO ring diameter. This may be due to differences in the opacity of the molecular envelope at 3.6 microns.

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.

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.

Autotrophic denitrification supported by biotite dissolution in crystalline aquifers: (2) transient mixing and denitrification dynamic during long-term pumping.

PubMed

Roques, Clément; Aquilina, Luc; Boisson, Alexandre; Vergnaud-Ayraud, Virginie; Labasque, Thierry; Longuevergne, Laurent; Laurencelle, Marc; Dufresne, Alexis; de Dreuzy, Jean-Raynald; Pauwels, Hélène; Bour, Olivier

2018-04-01

We investigated the mixing and dynamic of denitrification processes induced by long-term pumping in the crystalline aquifer of Ploemeur (Brittany, France). Hydrological and geochemical parameters have been continuously recorded over 15 boreholes in 5km 2 on a 25-year period. This extensive spatial and temporal monitoring of conservative as well as reactive compounds is a key opportunity to identify aquifer-scale transport and reactive processes in crystalline aquifers. Time series analysis of the conservative elements recorded at the pumped well were used to determine mixing fractions from different compartments of the aquifer on the basis of a Principal Component Analysis approach coupled with an end-member mixing analysis. We could reveal that pumping thus induces a thorough reorganization of fluxes known as capture, favoring infiltration and vertical fluxes in the recharge zone, and upwelling of deep and distant water at long-term time scales. These mixing fractions were then used to quantify the extent of denitrification linked to pumping. Based on the results from batch experiments described in a companion paper, our computations revealed that i) autotrophic denitrification processes are dominant in this context where carbon sources are limited, that ii) nitrate reduction does not only come from the oxidation of pyrite as classically described in previous studies analyzing denitrification processes in similar contexts, and that iii) biotite plays a critical role in sustaining the nitrate reduction process. Both nitrate reduction, sulfate production as well as fluor release ratios support the hypothesis that biotite plays a key role of electron donor in this context. The batch-to-site similarities support biotite availability and the role by bacterial communities as key controls of nitrate removal in such crystalline aquifers. However, the long term data monitoring also indicates that mixing and reactive processes evolve extremely slowly at the scale of the decade. Copyright © 2017 Elsevier B.V. All rights reserved.

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).

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.

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

CLOSING IN ON CLOSURE PERSPECTIVES FROM HANFORD & FERNALD AN UPDATE

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

CONNELL, J.D.

2004-12-22

In World War II, the arms dramatically changed from machine guns and incendiary bombs to nuclear weapons. Hanford and Fernald, two government-run sites, were part of the infrastructure established for producing the fissile material for making these weapons, as well as building a nuclear arsenal to deter future aggression by other nations. This paper compares and contrasts, from a communications point of view, these two Department of Energy (DOE) closure sites, each with Fluor as a prime contractor. The major differences between the two sites--Hanford in Washington state and Fernald in Ohio--includes the following: size of the site and themore » workforce, timing of closure, definition of end state, DOE oversight, proximity to population centers, readiness of local population for closure, and dependence of the local economy on the site's budget. All of these elements affect how the sites' communication professionals provide information even though the objectives are the same: build public acceptance and support for DOE's mission to accelerate cleanup, interface with stakeholders to help ensure that issues are addressed and goals are met, help workers literally work themselves out of jobs--faster, and prepare the ''host'' communities to deal with the void left when the sites are closed and the government contractors are gone. The 12-months between January 04 and January 05 have seen dramatic transformations at both sites, as Fernald is now just about a year away from closure and FLuor's work at Hanford has made the transition from operations to deactivation and demolition. While Fernald continues to clean out silos of waste and ship it off site, Hanford is dealing with recent state legislation that has the potential to significantly impact the progress of cleanup. These changes have even further accentuated the differences in the content, distribution, and impact of communications.« less

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.

Absorbed radiation dosimetry of the D3-specific PET radioligand [18F]FluorTriopride estimated using rodent and nonhuman primate

PubMed Central

Laforest, Richard; Karimi, Morvarid; Moerlein, Stephen M; Xu, Jinbin; Flores, Hubert P; Bognar, Christopher; Li, Aixiao; Mach, Robert H; Perlmutter, Joel S; Tu, Zhude

2016-01-01

[18F]FluorTriopride ([18F]FTP) is a dopamine D3-receptor preferring radioligand with potential for investigation of neuropsychiatric disorders including Parkinson disease, dystonia and schizophrenia. Here we estimate human radiation dosimetry for [18F]FTP based on the ex-vivo biodistribution in rodents and in vivo distribution in nonhuman primates. Biodistribution data were generated using male and female Sprague-Dawley rats injected with ~370 KBq of [18F]FTP and euthanized at 5, 30, 60, 120, and 240 min. Organs of interest were dissected, weighed and assayed for radioactivity content. PET imaging studies were performed in two male and one female macaque fascicularis administered 143-190 MBq of [18F]FTP and scanned whole-body in sequential sections. Organ residence times were calculated based on organ time activity curves (TAC) created from regions of Interest. OLINDA/EXM 1.1 was used to estimate human radiation dosimetry based on scaled organ residence times. In the rodent, the highest absorbed radiation dose was the upper large intestines (0.32-0.49 mGy/MBq), with an effective dose of 0.07 mSv/MBq in males and 0.1 mSv/MBq in females. For the nonhuman primate, however, the gallbladder wall was the critical organ (1.81 mGy/MBq), and the effective dose was 0.02 mSv/MBq. The species discrepancy in dosimetry estimates for [18F]FTP based on rat and primate data can be attributed to the slower transit of tracer through the hepatobiliary track of the primate compared to the rat, which lacks a gallbladder. Out findings demonstrate that the nonhuman primate model is more appropriate model for estimating human absorbed radiation dosimetry when hepatobiliary excretion plays a major role in radiotracer elimination. PMID:28078183

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

Durham, Robin E.; Becker, James M.

This document provides a review and status of activities conducted in support of the Fluor Daniel Hanford Company (Fluor), now Mission Support Alliance (MSA), Mitigation Action Plan (MAP) for Project L-325, Electrical Utility Upgrades (2007). Three plantings have been installed on a 4.5-hectare mitigation area to date. This review provides a description and chronology of events, monitoring results, and mitigative actions through fiscal year (FY) 2012. Also provided is a review of the monitoring methods, transect layout, and FY 2012 monitoring activities and results for all planting years. Planting densities and performance criteria stipulated in the MAP were aimed atmore » a desired future condition (DFC) of 10 percent mature sagebrush (Artemisia tridentata ssp wyomingensis) cover. Current recommendations for yielding this DFC are based upon a conceptual model planting of 1000 plants/ha (400/ac) exhibiting a 60-percent survival rate after 5 monitoring years (DOE 2003). Accordingly, a DFC after 5 monitoring years would not be less than 600 plants/ha (240/ac). To date, about 8700 sagebrush plants have been grown and transplanted onto the mitigation site. Harsh site conditions and low seedling survival have resulted in an estimated 489 transplants/ha on the mitigation site, which is 111 plants/ha short of the target DFC. Despite this apparent shortcoming, 71, 91, and 24 percent of the surviving seedlings planted in FY 2007 and FY 2008 and FY 2010, respectively, showed signs of blooming in FY 2012. Blooming status may be a positive indication of future sagebrush recruitment, and is therefore a potential source for reaching the target DFC of 600 plants/ha on this mitigation site over time. Because of the difficulty establishing small transplants on this site, we propose that no additional plantings be considered for this mitigation area and to rely upon the potential recruitment by established seedlings to achieve the mitigation commitment set forth in the MAP of 600 plants/ha.« less

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.

Lake Charles CCS Project

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

Leib, Thomas; Cole, Dan

In late September 2014 development of the Lake Charles Clean Energy (LCCE) Plant was abandoned resulting in termination of Lake Charles Carbon Capture and Sequestration (CCS) Project which was a subset the LCCE Plant. As a result, the project was only funded through Phase 2A (Design) and did not enter Phase 2B (Construction) or Phase 2C (Operations). This report was prepared relying on information prepared and provided by engineering companies which were engaged by Leucadia Energy, LLC to prepare or review Front End Engineering and Design (FEED) for the Lake Charles Clean Energy Project, which includes the Carbon Capture andmore » Sequestration (CCS) Project in Lake Charles, Louisiana. The Lake Charles Carbon Capture and Sequestration (CCS) Project was to be a large-scale industrial CCS project intended to demonstrate advanced technologies that capture and sequester carbon dioxide (CO 2) emissions from industrial sources into underground formations. The Scope of work was divided into two discrete sections; 1) Capture and Compression prepared by the Recipient Leucadia Energy, LLC, and 2) Transport and Sequestration prepared by sub-Recipient Denbury Onshore, LLC. Capture and Compression-The Lake Charles CCS Project Final Technical Report describes the systems and equipment that would be necessary to capture CO 2 generated in a large industrial gasification process and sequester the CO 2 into underground formations. The purpose of each system is defined along with a description of its equipment and operation. Criteria for selection of major equipment are provided and ancillary utilities necessary for safe and reliable operation in compliance with environmental regulations are described. Construction considerations are described including a general arrangement of the CCS process units within the overall gasification project. A cost estimate is provided, delineated by system area with cost breakdown showing equipment, piping and materials, construction labor, engineering, and other costs. The CCS Project Final Technical Report is based on a Front End Engineering and Design (FEED) study prepared by SK E&C, completed in [June] 2014. Subsequently, Fluor Enterprises completed a FEED validation study in mid-September 2014. The design analyses indicated that the FEED package was sufficient and as expected. However, Fluor considered the construction risk based on a stick-build approach to be unacceptable, but construction risk would be substantially mitigated through utilization of modular construction where site labor and schedule uncertainty is minimized. Fluor’s estimate of the overall EPC project cost utilizing the revised construction plan was comparable to SKE&C’s value after reflecting Fluor’s assessment of project scope and risk characteristic. Development was halted upon conclusion of Phase 2A FEED and the project was not constructed.Transport and Sequestration – The overall objective of the pipeline project was to construct a pipeline to transport captured CO 2 from the Lake Charles Clean Energy project to the existing Denbury Green Line and then to the Hastings Field in Southeast Texas to demonstrate effective geologic sequestration of captured CO 2 through commercial EOR operations. The overall objective of the MVA portion of the project was to demonstrate effective geologic sequestration of captured CO 2 through commercial Enhanced Oil Recovery (EOR) operations in order to evaluate costs, operational processes and technical performance. The DOE target for the project was to capture and implement a research MVA program to demonstrate the sequestration through EOR of approximately one million tons of CO 2 per year as an integral component of commercial operations.« less

INTEGRATED GASIFICATION COMBINED CYCLE PROJECT 2 MW FUEL CELL DEMONSTRATION

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

FuelCell Energy

2005-05-16

With about 50% of power generation in the United States derived from coal and projections indicating that coal will continue to be the primary fuel for power generation in the next two decades, the Department of Energy (DOE) Clean Coal Technology Demonstration Program (CCTDP) has been conducted since 1985 to develop innovative, environmentally friendly processes for the world energy market place. The 2 MW Fuel Cell Demonstration was part of the Kentucky Pioneer Energy (KPE) Integrated Gasification Combined Cycle (IGCC) project selected by DOE under Round Five of the Clean Coal Technology Demonstration Program. The participant in the CCTDP Vmore » Project was Kentucky Pioneer Energy for the IGCC plant. FuelCell Energy, Inc. (FCE), under subcontract to KPE, was responsible for the design, construction and operation of the 2 MW fuel cell power plant. Duke Fluor Daniel provided engineering design and procurement support for the balance-of-plant skids. Colt Engineering Corporation provided engineering design, fabrication and procurement of the syngas processing skids. Jacobs Applied Technology provided the fabrication of the fuel cell module vessels. Wabash River Energy Ltd (WREL) provided the test site. The 2 MW fuel cell power plant utilizes FuelCell Energy's Direct Fuel Cell (DFC) technology, which is based on the internally reforming carbonate fuel cell. This plant is capable of operating on coal-derived syngas as well as natural gas. Prior testing (1992) of a subscale 20 kW carbonate fuel cell stack at the Louisiana Gasification Technology Inc. (LGTI) site using the Dow/Destec gasification plant indicated that operation on coal derived gas provided normal performance and stable operation. Duke Fluor Daniel and FuelCell Energy developed a commercial plant design for the 2 MW fuel cell. The plant was designed to be modular, factory assembled and truck shippable to the site. Five balance-of-plant skids incorporating fuel processing, anode gas oxidation, heat recovery, water treatment/instrument air, and power conditioning/controls were built and shipped to the site. The two fuel cell modules, each rated at 1 MW on natural gas, were fabricated by FuelCell Energy in its Torrington, CT manufacturing facility. The fuel cell modules were conditioned and tested at FuelCell Energy in Danbury and shipped to the site. Installation of the power plant and connection to all required utilities and syngas was completed. Pre-operation checkout of the entire power plant was conducted and the plant was ready to operate in July 2004. However, fuel gas (natural gas or syngas) was not available at the WREL site due to technical difficulties with the gasifier and other issues. The fuel cell power plant was therefore not operated, and subsequently removed by October of 2005. The WREL fuel cell site was restored to the satisfaction of WREL. FuelCell Energy continues to market carbonate fuel cells for natural gas and digester gas applications. A fuel cell/turbine hybrid is being developed and tested that provides higher efficiency with potential to reach the DOE goal of 60% HHV on coal gas. A system study was conducted for a 40 MW direct fuel cell/turbine hybrid (DFC/T) with potential for future coal gas applications. In addition, FCE is developing Solid Oxide Fuel Cell (SOFC) power plants with Versa Power Systems (VPS) as part of the Solid State Energy Conversion Alliance (SECA) program and has an on-going program for co-production of hydrogen. Future development in these technologies can lead to future coal gas fuel cell applications.« less

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.

A pH-sensitive fluor, CypHer 5, used to monitor agonist-induced G protein-coupled receptor internalization in live cells.

PubMed

Adie, E J; Kalinka, S; Smith, L; Francis, M J; Marenghi, A; Cooper, M E; Briggs, M; Michael, N P; Milligan, G; Game, S

2002-11-01

G protein-coupled receptors (GPCRs) are the largest family of proteins involved in transmembrane signal transduction and are actively studied because of their suitability as therapeutic small-molecule drug targets. Agonist activation of GPCRs almost invariably results in the receptor being desensitized. One of the key events in receptor desensitization is the sequestration of the receptor from the cell surface into acidic intracellular endosomes. Therefore, a convenient, generic, and noninvasive monitor of this process is desirable. A novel, pH-sensitive, red-excited fluorescent dye, CypHer 5, was synthesized. This dye is non-fluorescent at neutral pH and is fluorescent at acidic pH. Anti-epitope antibodies labeled with this dye were internalized in an agonist concentration- and time-dependent manner, following binding on live cells to a range of GPCRs that had been modified to incorporate the epitope tags in their extracellular N-terminal domain. This resulted in a large signal increase over background. When protonated, the red fluorescence of CypHer 5 provides a generic reagent suitable for monitoring the internalization of GPCRs into acidic vesicles. This approach should be amenable to the study of many other classes of cell surface receptors that also internalize following stimulation.

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

Cherenkov and scintillation light separation in organic liquid scintillators

NASA Astrophysics Data System (ADS)

Caravaca, J.; Descamps, F. B.; Land, B. J.; Yeh, M.; Orebi Gann, G. D.

2017-12-01

The CHErenkov/Scintillation Separation experiment (CHESS) has been used to demonstrate the separation of Cherenkov and scintillation light in both linear alkylbenzene (LAB) and LAB with 2 g/L of PPO as a fluor (LAB/PPO). This is the first successful demonstration of Cherenkov light detection from the more challenging LAB/PPO cocktail and improves on previous results for LAB. A time resolution of 338± 12 ps FWHM results in an efficiency for identifying Cherenkov photons in LAB/PPO of 70 ± 3 % and 63± 8% for time- and charge-based separation, respectively, with scintillation contamination of 36± 5% and 38± 4%. LAB/PPO data is consistent with a rise time of τ _r=0.72± 0.33 ns.

Unraveling the Mystery of Exozodiacal Dust

NASA Astrophysics Data System (ADS)

Ertel, S.; Augereau, J.-C.; Thébault, P.; Absil, O.; Bonsor, A.; Defrère, D.; Kral, Q.; Le Bouquin, J.-B.; Lebreton, J.; Coudé du Foresto, V.

2014-01-01

Exozodiacal dust clouds are thought to be the extrasolar analogs of the Solar System's zodiacal dust. Studying these systems provides insights in the architecture of the innermost regions of planetary systems, including the Habitable Zone. Furthermore, the mere presence of the dust may result in major obstacles for direct imaging of earth-like planets. Our EXOZODI project aims to detect and study exozodiacal dust and to explain its origin. We are carrying out the first large, near-infrared interferometric survey in the northern (CHARA/FLUOR) and southern (VLTI/PIONIER) hemispheres. Preliminary results suggest a detection rate of up to 30% around A to K type stars and interesting trends with spectral type and age. We focus here on presenting the observational work carried out by our team.

Systems and methods for imaging using radiation from laser produced plasmas

DOEpatents

Renard-Le Galloudec, Nathalie; Cowan, Thomas E.; Sentoku, Yasuhiko; Rassuchine, Jennifer

2009-06-30

In particular embodiments, the present disclosure provides systems and methods for imaging a subject using radiation emitted from a laser produced plasma generating by irradiating a target with a laser. In particular examples, the target includes at least one radiation enhancing component, such as a fluor, cap, or wire. In further examples, the target has a metal layer and an internal surface defining an internal apex, the internal apex of less than about 15 .mu.m, such as less than about 1 .mu.m. The targets may take a variety of shapes, including cones, pyramids, and hemispheres. Certain aspects of the present disclosure provide improved imaging of a subject, such as improved medical images of a radiation dose than typical conventional methods and systems.

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

Hanford Waste Vitrification Plant technical manual

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

Larson, D.E.; Watrous, R.A.; Kruger, O.L.

1996-03-01

A key element of the Hanford waste management strategy is the construction of a new facility, the Hanford Waste Vitrification Plant (HWVP), to vitrify existing and future liquid high-level waste produced by defense activities at the Hanford Site. The HWVP mission is to vitrify pretreated waste in borosilicate glass, cast the glass into stainless steel canisters, and store the canisters at the Hanford Site until they are shipped to a federal geological repository. The HWVP Technical Manual (Manual) documents the technical bases of the current HWVP process and provides a physical description of the related equipment and the plant. Themore » immediate purpose of the document is to provide the technical bases for preparation of project baseline documents that will be used to direct the Title 1 and Title 2 design by the A/E, Fluor. The content of the Manual is organized in the following manner. Chapter 1.0 contains the background and context within which the HWVP was designed. Chapter 2.0 describes the site, plant, equipment and supporting services and provides the context for application of the process information in the Manual. Chapter 3.0 provides plant feed and product requirements, which are primary process bases for plant operation. Chapter 4.0 summarizes the technology for each plant process. Chapter 5.0 describes the engineering principles for designing major types of HWVP equipment. Chapter 6.0 describes the general safety aspects of the plant and process to assist in safe and prudent facility operation. Chapter 7.0 includes a description of the waste form qualification program and data. Chapter 8.0 indicates the current status of quality assurance requirements for the Manual. The Appendices provide data that are too extensive to be placed in the main text, such as extensive tables and sets of figures. The Manual is a revision of the 1987 version.« less

Research in Materials Science

DTIC Science & Technology

1975-05-31

COMMERCE - - ■ t^m^m^mmi mmt m i ««■■OTiHiuiiii. ii pi tv^i^^m^mmm^^m wntm ^•1 I I I I I I I i I I I I I I...5 Er: Iio/p» Ho: I7 * Er: IIR/?’ HO: U w’’tfl t^ e Ho: ^5 decaying rapidly to the fluor- 5 escing level Ho: Ig by multiphonon emission. At higher...8217-■ — ■ IWI^P ■ -13- 15 e 10 Ü IO Q < ÜJ S3/2 ^ 9/2 9/2 4i 11/2 4I 13/2 15/2 Er 3F, 3F. ’H, 3K ’H, Tm 5Fe 5I, 5L »I, 2.06/i

An image engineering system for the inspection of transparent construction materials

NASA Astrophysics Data System (ADS)

Hinz, S.; Stephani, M.; Schiemann, L.; Zeller, K.

This article presents a modular photogrammetric recording and image analysis system for inspecting the material characteristics of transparent foils, in particular Ethylen-TetraFluorEthylen-Copolymer (ETFE) foils. The foils are put under increasing air pressure and are observed by a stereo camera system. Determining the time-variable 3D shape of transparent material imposes a number of challenges: especially the automatic point transfer between stereo images and, in temporal domain, from one image pair to the next. We developed an automatic approach that accommodates for these particular circumstances and allows reconstruction of the 3D shape for each epoch as well as determining 3D translation vectors between epochs by feature tracking. Examples including numerical results and accuracy measures prove the applicability of the system.

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

Cherepy, Nerine J.; Sanner, Robert D.; Beck, Patrick R.

In this paper, transparent plastic scintillators based on polyvinyltoluene (PVT) have been fabricated with high loading of bismuth carboxylates for gamma spectroscopy, and with lithium carboxylates for neutron detection. When activated with a combination of standard fluors, 2,5-diphenyloxazole (PPO) and tetraphenylbutadiene (TPB), gamma light yields with 15 wt% bismuth tripivalate of 5000 Ph/MeV are measured. A PVT plastic formulation including 30 wt% lithium pivalate and 30 wt% PPO offers both pulse shape discrimination, and a neutron capture peak at ~400 keVee. Finally, in another configuration, a bismuth-loaded PVT plastic is coated with ZnS( 6Li) paint, permitting simultaneous gamma and neutronmore » detection via pulse shape discrimination with a figure-of-merit of 3.8, while offering gamma spectroscopy with energy resolution of R(662 keV)=15%.« less

Bismuth- and lithium-loaded plastic scintillators for gamma and neutron detection

NASA Astrophysics Data System (ADS)

Cherepy, Nerine J.; Sanner, Robert D.; Beck, Patrick R.; Swanberg, Erik L.; Tillotson, Thomas M.; Payne, Stephen A.; Hurlbut, Charles R.

2015-04-01

Transparent plastic scintillators based on polyvinyltoluene (PVT) have been fabricated with high loading of bismuth carboxylates for gamma spectroscopy, and with lithium carboxylates for neutron detection. When activated with a combination of standard fluors, 2,5-diphenyloxazole (PPO) and tetraphenylbutadiene (TPB), gamma light yields with 15 wt% bismuth tripivalate of 5000 Ph/MeV are measured. A PVT plastic formulation including 30 wt% lithium pivalate and 30 wt% PPO offers both pulse shape discrimination, and a neutron capture peak at 400 keVee. In another configuration, a bismuth-loaded PVT plastic is coated with ZnS(6Li) paint, permitting simultaneous gamma and neutron detection via pulse shape discrimination with a figure-of-merit of 3.8, while offering gamma spectroscopy with energy resolution of R(662 keV)=15%.

Cherenkov and scintillation light separation in organic liquid scintillators

DOE PAGES

Caravaca, J.; Descamps, F. B.; Land, B. J.; ...

2017-11-29

The CHErenkov/Scintillation Separation experiment (CHESS) has been used to demonstrate the separation of Cherenkov and scintillation light in both linear alkylbenzene (LAB) and LAB with 2 g/L of PPO as a fluor (LAB/PPO). This is the first successful demonstration of Cherenkov light detection from the more challenging LAB/PPO cocktail and improves on previous results for LAB. A time resolution of 338 ± 12 ps FWHM results in an efficiency for identifying Cherenkov photons in LAB/PPO of 70 ± 3 % and 63 ± 8 % for time- and charge-based separation, respectively, with scintillation contamination of 36 ± 5 % andmore » 38 ± 4 %. LAB/PPO data is consistent with a rise time of τ r = 0.72 ± 0.33 ns.« less

Cherenkov and scintillation light separation in organic liquid scintillators

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

Caravaca, J.; Descamps, F. B.; Land, B. J.

The CHErenkov/Scintillation Separation experiment (CHESS) has been used to demonstrate the separation of Cherenkov and scintillation light in both linear alkylbenzene (LAB) and LAB with 2 g/L of PPO as a fluor (LAB/PPO). This is the first successful demonstration of Cherenkov light detection from the more challenging LAB/PPO cocktail and improves on previous results for LAB. A time resolution of 338 ± 12 ps FWHM results in an efficiency for identifying Cherenkov photons in LAB/PPO of 70 ± 3 % and 63 ± 8 % for time- and charge-based separation, respectively, with scintillation contamination of 36 ± 5 % andmore » 38 ± 4 %. LAB/PPO data is consistent with a rise time of τ r = 0.72 ± 0.33 ns.« less

WASTE TREATMENT PLANT (WTP) LIQUID EFFLUENT TREATABILITY EVALUATION

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

LUECK, K.J.

2004-10-18

A forecast of the radioactive, dangerous liquid effluents expected to be produced by the Waste Treatment Plant (WTP) was provided by Bechtel National, Inc. (BNI 2004). The forecast represents the liquid effluents generated from the processing of Tank Farm waste through the end-of-mission for the WTP. The WTP forecast is provided in the Appendices. The WTP liquid effluents will be stored, treated, and disposed of in the Liquid Effluent Retention Facility (LERF) and the Effluent Treatment Facility (ETF). Both facilities are located in the 200 East Area and are operated by Fluor Hanford, Inc. (FH) for the US. Department ofmore » Energy (DOE). The treatability of the WTP liquid effluents in the LERF/ETF was evaluated. The evaluation was conducted by comparing the forecast to the LERF/ETF treatability envelope (Aromi 1997), which provides information on the items which determine if a liquid effluent is acceptable for receipt and treatment at the LERF/ETF. The format of the evaluation corresponds directly to the outline of the treatability envelope document. Except where noted, the maximum annual average concentrations over the range of the 27 year forecast was evaluated against the treatability envelope. This is an acceptable approach because the volume capacity in the LERF Basin will equalize the minimum and maximum peaks. Background information on the LERF/ETF design basis is provided in the treatability envelope document.« less

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.

Status Report for Remediation Decision Support Project, Task 1, Activity 1.B – Physical and Hydraulic Properties Database and Interpretation

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

Rockhold, Mark L.

2008-09-26

The objective of Activity 1.B of the Remediation Decision Support (RDS) Project is to compile all available physical and hydraulic property data for sediments from the Hanford Site, to port these data into the Hanford Environmental Information System (HEIS), and to make the data web-accessible to anyone on the Hanford Local Area Network via the so-called Virtual Library. In past years efforts were made by RDS project staff to compile all available physical and hydraulic property data for Hanford sediments and to transfer these data into SoilVision{reg_sign}, a commercial geotechnical software package designed for storing, analyzing, and manipulating soils data.more » Although SoilVision{reg_sign} has proven to be useful, its access and use restrictions have been recognized as a limitation to the effective use of the physical and hydraulic property databases by the broader group of potential users involved in Hanford waste site issues. In order to make these data more widely available and useable, a decision was made to port them to HEIS and to make them web-accessible via a Virtual Library module. In FY08 the objectives of Activity 1.B of the RDS Project were to: (1) ensure traceability and defensibility of all physical and hydraulic property data currently residing in the SoilVision{reg_sign} database maintained by PNNL, (2) transfer the physical and hydraulic property data from the Microsoft Access database files used by SoilVision{reg_sign} into HEIS, which has most recently been maintained by Fluor-Hanford, Inc., (3) develop a Virtual Library module for accessing these data from HEIS, and (4) write a User's Manual for the Virtual Library module. The development of the Virtual Library module was to be performed by a third party under subcontract to Fluor. The intent of these activities is to make the available physical and hydraulic property data more readily accessible and useable by technical staff and operable unit managers involved in waste site assessments and remedial action decisions for Hanford. This status report describes the history of this development effort and progress to date.« less

[Influence of the fluoride releasing dental materials on the bacterial flora of dental plaque].

PubMed

Płuciennik, Małgorzata; Sakowska, Danuta; Krzemiński, Zbigniew; Piatowska, Danuta

2008-01-01

The assessment of influence of silver-free, fluor releasing dental materials on dental plaque bacteria quantity. 17 patients were included into the study. 51 restorations were placed following manufacturers recommendations. Following materials were used: conventional glassionomer Ketac-Molar ESPE, resin modified glassionomer Fuji II LC GC and fluor containing composite Charisma Heraeus Kulzer Class V restorations were placed in following teeth of upper and lower jaw: canines, first bicuspids, second bicuspids. Sound enamel was a control. After 10 weeks the 72 hours old dental plaque was collected from surface of restorations and control using sterile probe. Total amount of 68 dental plaques were investigated. Each plaque was placed on scaled and sterile aluminum foil. The moist weight of dental plaque was scaled. Dental plaque was moved into 7 ml 0.85% NaCl solution reduced by cystein chlorine hydrogen and disintegrated by ultrasounds (power:100 Watt, wave amplitude: 5 micorm). The suspension of dental plaque was serially diluted from 10(-4) to 10(-5) in sterile 0,85% NaCl solution, and seeded with amount of 0.1 ml on appropriate base. In dental plaque trials the amount of cariogenic bacteria was calculated--Streptococcus mutans, Streptococcus, Lactobacillus, Veillonella and Neisseria, and also total amount of aerobic and anaerobic bacteria was measured. Microbiologic studies were performed in Institute of Microbiology, Medical University, Łódź. Statistical analysis of collected data was accomplished. In 72 hours old dental plaques collected from the surfaces of Ketac -Molar, Fuji II LC, Charisma after 10 weeks since being placed into the class V cavity, results show no statistically significant differences in the amount of Streptococcus mutans, Streptococcus spp., Lactobacillus spp., Veillonella spp., Neisseria spp, in total amount of aerobic and anaerobic bacteria and in the quantity proportion of Streptococcus mutans versus Streptococcus spp. in comparison with control trail. Results show no statistically significant differences in the amount of listed above bacteria and in the proportion of Streptococcus mutans versus Streptococcus spp. in 72 hours old dental plaques collected from surfaces of investigated restorative materials.

Fluor Daniel Hanford implementation plan for DOE Order 5480.28, Natural phenomena hazards mitigation

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

Conrads, T.J.

1997-09-12

Natural phenomena hazards (NPH) are unexpected acts of nature that pose a threat or danger to workers, the public, or the environment. Earthquakes, extreme winds (hurricane and tornado), snow, flooding, volcanic ashfall, and lightning strikes are examples of NPH that could occur at the Hanford Site. U.S. Department of Energy (DOE) policy requires facilities to be designed, constructed, and operated in a manner that protects workers, the public, and the environment from hazards caused by natural phenomena. DOE Order 5480.28, Natural Phenomena Hazards Mitigation, includes rigorous new natural phenomena criteria for the design of new DOE facilities, as well asmore » for the evaluation and, if necessary, upgrade of existing DOE facilities. The Order was transmitted to Westinghouse Hanford Company in 1993 for compliance and is also identified in the Project Hanford Management Contract, Section J, Appendix C. Criteria and requirements of DOE Order 5480.28 are included in five standards, the last of which, DOE-STD-1023, was released in fiscal year 1996. Because the Order was released before all of its required standards were released, enforcement of the Order was waived pending release of the last standard and determination of an in-force date by DOE Richland Operations Office (DOE-RL). Agreement also was reached between the Management and Operations Contractor and DOE-RL that the Order would become enforceable for new structures, systems, and components (SSCS) 60 days following issue of a new order-based design criteria in HNF-PRO-97, Engineering Design and Evaluation. The order also requires that commitments addressing existing SSCs be included in an implementation plan that is to be issued 1 year following the release of the last standard. Subsequently, WHC-SP-1175, Westinghouse Hanford Company Implementation Plan for DOE Order 5480.28, Natural Phenomena Hazards Mitigation, Rev. 0, was issued in November 1996, and this document, HNF-SP-1175, Fluor Daniel Hanford Implementation Plan for DOE Order 5480.28, Natural Phenomena Hazards Mitigation, is Rev. 1 of that plan.« less

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.

Feasibility study for hydrocarbon complex in southern seaboard. Petroleum Authority of Thailand

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

NONE

This study, conducted by Fluor Daniel, was funded by the U.S. Trade and Development Agency, on behalf of the Petroleum Authority of Thailand. The primary objective of the study was to investigate the economic viability of the related facilities and determine how each could help to industrialize and build up the Southern Seaboard area of Thailand. The focus of the report is in three areas including; Crude Oil Transportation System, Refinery, and Petrochemical Complex. Another objective of the study was to offer an alternative for large crude carrier traffic by proposing the completion of a crude oil pipeline. The reportmore » is divided into the following sections: (1) Executive Summary; (2) Introduction; (3) Crude Oil Transportation System; (4) Refinery Project; (5) Petrochemical Complex; (6) Key Issues & Considerations; (7) Financial Evaluations; (8) Summary & Conclusions.« less

Bismuth- and lithium-loaded plastic scintillators for gamma and neutron detection

DOE PAGES

Cherepy, Nerine J.; Sanner, Robert D.; Beck, Patrick R.; ...

2015-01-09

In this paper, transparent plastic scintillators based on polyvinyltoluene (PVT) have been fabricated with high loading of bismuth carboxylates for gamma spectroscopy, and with lithium carboxylates for neutron detection. When activated with a combination of standard fluors, 2,5-diphenyloxazole (PPO) and tetraphenylbutadiene (TPB), gamma light yields with 15 wt% bismuth tripivalate of 5000 Ph/MeV are measured. A PVT plastic formulation including 30 wt% lithium pivalate and 30 wt% PPO offers both pulse shape discrimination, and a neutron capture peak at ~400 keVee. Finally, in another configuration, a bismuth-loaded PVT plastic is coated with ZnS( 6Li) paint, permitting simultaneous gamma and neutronmore » detection via pulse shape discrimination with a figure-of-merit of 3.8, while offering gamma spectroscopy with energy resolution of R(662 keV)=15%.« less

Spectral distribution of UV range diffuse reflectivity for Si+ ion implanted polymers

NASA Astrophysics Data System (ADS)

Balabanov, S.; Tsvetkova, T.; Borisova, E.; Avramov, L.; Bischoff, L.

2008-05-01

The analysis of the UV range spectral characteristics can supply additional information on the formed sub-surface buried layer with implanted dopants. The near-surface layer (50÷150 nm) of bulk polymer samples have been implanted with silicon (Si+) ions at low energies (E = 30 keV) and a wide range of ion doses (D = 1.1013 ÷ 1, 2.1017 cm-2). The studied polymer materials were: ultra-high-molecular-weight polyethylene (UHMWPE), poly-methyl-metacrylate (PMMA) and poly-tetra-fluor-ethylene (PTFE). The diffuse optical reflectivity spectra Rd = f(λ) of the ion implanted samples have been measured in the UV range (λ = 220÷350 nm). In this paper the dose dependences of the size and sign of the diffuse optical reflectivity changes λRd = f(D) have been analysed.

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.

XMM-Newton Proposal 03039101

NASA Astrophysics Data System (ADS)

Hernanz, Margarita

2004-10-01

Two recent galactic novae, V2487 Oph 1998 and V4633 Sgr 1998, have been detec- ted in both soft and hard X-rays with XMM. V2487 Oph showed an iron fluores- cent line only 2.7 yrs after its explosion, a clear signature of the very fast recovery of accretion, and V4633 Sgr displayed hard X-ray emission not clearly attributable to shocked ejecta or accretion. Longer observations are needed to do accurate timing and high resolution spectroscopy, essential to determine the main properties of the underlying cataclysmic variable in V2487 Oph (first nova seen in X-rays prior and after exploding) and to disentangle the origin of the hard X-rays from V4633 Sgr, through an analysis of the chemical compo- sition of the emitting thermal plasma, solar if accretion, non solar if ejecta.

Multi-color interferometric observations of Mira stars

NASA Technical Reports Server (NTRS)

Mennesson, B.; Creech-Eakman, M.; Thompson, B. B.; Foresto, V. Coude du; Merand, A.; Ridgway, S.; Perrin, G.

2002-01-01

Interferometric observations in the atmospheric windows of the near infrared (1 to 4 microns) can be efficiently used to probe the chemical composition of Miras atmosphere and provide direct measurements of extended gas layers around these stars. This is illustrated by recent Miras observations carried out with the FLUOR instrument of the IOTA interferometer (Mennesson et al. 2002, and Perrin et al. proceedings of this conference) and with the PTI test-bed (Thompson 2002, Thompson et al. 2002). These visibility measurements show evidence for continuum emission from very extended ( 2-3 stellar radii) semi-transparent gaseous atmospheric layers, and large apparent diameter changes with pulsation phase. Interestingly these observations are consistent with the extended molecular gas layers (H20, CO ...) already inferred around some of these objects from IS0 and high resolution ground-based FTS infrared spectra.

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

Hutzel, Margaret; Siegrist, Lindsey; Wilson, Natalie

The report that follows is a summary of the U.S. Department of Energy Portsmouth Annual Site Environmental Report for 2012 (ASER), regarding PORTS located near Piketon, Ohio. The summary has been compiled by the 2015 WHS Environmental Science class, made up of juniors and seniors at WHS during the 2014-2015 school year. Even with most of the class having lived in this region for their entire lives, it became apparent how little of the workings of the plant were known by the members of the class. In the process of putting this summary together, we were able to gain amore » better understanding of the history, function, and possible future of the site. The presentations provided by Ohio University, Fluor-B&W Portsmouth LLC (FBP), U.S. DOE, and Rio Grande University were greatly appreciated and provided invaluable understanding of the materials which we were asked to summarize. Not only did we learn from the presentations, but we greatly enjoyed the opportunities to participate in the field studies that gave us a glimpse into what is being done at the plant site to ensure the environmental safety of people and wildlife of this region. Our goal from this summary has been to make the information concerning the monitoring and cleanup of the PORTS facility better understood by the people who it most affects. We hope that this summary makes the information useful to you and that you can gain a better understanding of the cleanup processes that are going on around the site to ensure your safety. Though it has been hard work, we appreciate the opportunity that we have been presented with to learn and share with the people of our community.« less

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.

NiTi shape memory alloys treated by plasma-polymerized tetrafluoroethylene. A physicochemical and electrochemical characterization.

PubMed

Yahia, L H; Lombardi, S; Piron, D; Klemberg-Sapieha, J E; Wertheimer, M R

NiTi alloy specimens were plasma cleaned and then coated with a thin film of plasma-polymerized tetrafluoroethylene (TFE) in a Radio-Frequency reactor. The corrosion protection provided by these films was studied by potentiodynamic tests performed in Hank's physiological solution. Surface properties which determine biocompatibility were characterized by X-ray photoelectron spectroscopy (XPS). The results showed that the surface of untreated NiTi was mostly composed by oxygen, carbon, titanium oxide (TiO2) with traces of nickel oxides (NiO and Ni2O3) and metallic Ni. The passivity of untreated NiTi was found to be unstable in the simulated human body media. After plasma treatment, the NiTi surface contained only carbon and fluor. The plasma-polymerized thin film was found to stabilize the NiTi passivity and to increase its pitting potential. This treatment provides a good protection against dissolution of nickel from NiTi alloys.

Characterization of an anthraquinone fluor from the bioluminescent, pelagic polychaete Tomopteris

PubMed Central

Francis, Warren R; Powers, Meghan L; Haddock, Steven H D

2014-01-01

Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report the isolation of the fluorescent yellow–orange pigment found in the luminous exudate and in the body of the animals. Liquid chromatography-mass spectrometry revealed the mass to be 270 m/z with a molecular formula of C15H10O5, which ultimately was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence. PMID:24760626

Multi-Phenotypic subtyping of circulating tumor cells using sequential fluorescent quenching and restaining

NASA Astrophysics Data System (ADS)

Adams, Daniel L.; Alpaugh, R. Katherine; Tsai, Susan; Tang, Cha-Mei; Stefansson, Steingrimur

2016-09-01

In tissue biopsies formalin fixed paraffin embedded cancer blocks are micro-sectioned producing multiple semi-identical specimens which are analyzed and subtyped proteomically, and genomically, with numerous biomarkers. In blood based biopsies (BBBs), blood is purified for circulating tumor cells (CTCs) and clinical utility is typically limited to cell enumeration, as only 2-3 positive fluorescent markers and 1 negative marker can be used. As such, increasing the number of subtyping biomarkers on each individual CTC could dramatically enhance the clinical utility of BBBs, allowing in depth interrogation of clinically relevant CTCs. We describe a simple and inexpensive method for quenching the specific fluors of fluorescently stained CTCs followed by sequential restaining with additional biomarkers. As proof of principle a CTC panel, immunosuppression panel and stem cell panel were used to sequentially subtype individual fluorescently stained patient CTCs, suggesting a simple and universal technique to analyze multiple clinically applicable immunomarkers from BBBs.

Surface Functionalization of Exosomes Using Click Chemistry

PubMed Central

2015-01-01

A method for conjugation of ligands to the surface of exosomes was developed using click chemistry. Copper-catalyzed azide alkyne cycloaddition (click chemistry) is ideal for biocojugation of small molecules and macromolecules to the surface of exosomes, due to fast reaction times, high specificity, and compatibility in aqueous buffers. Exosomes cross-linked with alkyne groups using carbodiimide chemistry were conjugated to a model azide, azide-fluor 545. Conjugation had no effect on the size of exosomes, nor was there any change in the extent of exosome adherence/internalization with recipient cells, suggesting the reaction conditions were mild on exosome structure and function. We further investigated the extent of exosomal protein modification with alkyne groups. Using liposomes with surface alkyne groups of a similar size and concentration to exosomes, we estimated that approximately 1.5 alkyne groups were present for every 150 kDa of exosomal protein. PMID:25220352

The Identification of Aluminum in Human Brain Tissue Using Lumogallion and Fluorescence Microscopy

PubMed Central

Mirza, Ambreen; King, Andrew; Troakes, Claire; Exley, Christopher

2016-01-01

Aluminum in human brain tissue is implicated in the etiologies of neurodegenerative diseases including Alzheimer’s disease. While methods for the accurate and precise measurement of aluminum in human brain tissue are widely acknowledged, the same cannot be said for the visualization of aluminum. Herein we have used transversely-heated graphite furnace atomic absorption spectrometry to measure aluminum in the brain of a donor with Alzheimer’s disease, and we have developed and validated fluorescence microscopy and the fluor lumogallion to show the presence of aluminum in the same tissue. Aluminum is observed as characteristic orange fluorescence that is neither reproduced by other metals nor explained by autofluorescence. This new and relatively simple method to visualize aluminum in human brain tissue should enable more rigorous testing of the aluminum hypothesis of Alzheimer’s disease (and other neurological conditions) in the future. PMID:27472886

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.

Users speak out on technology deployment

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

Peters, Mark; Prochaska, Marty; Cromer, Paul

2001-02-25

This report summarizes user feedback data collected during a recent Accelerated Site Technology Deployment (ASTD) project: the Fluor Fernald ASTD Technology Deployment Project from May, 1999 through September, 2000. The main goal of the ASTD project was to use the ''Fernald approach'' to expedite the deployment of new or innovative technologies with superior safety, cost, and/or productivity benefits to Department of Energy (DOE) facilities. The Fernald approach targets technology end-users and their managers and directly involves them with hands-on demonstrations of new or innovative technologies during technology transfer sessions. The two technologies deployed through this project were the Personal Icemore » Cooling System (PICS) and the oxy-gasoline torch. Participants of technology transfer sessions were requested to complete feedback surveys. Surveys evaluated the effectiveness of the Fernald approach to technology deployment and assessed the responsiveness of employees to new technologies. This report presents the results of those surveys.« less

AVIRIS foreoptics, fiber optics and on-board calibrator

NASA Technical Reports Server (NTRS)

Chrisp, Michael P.; Chrien, Thomas G.; Steimle, L.

1987-01-01

The foreoptics, fiber optic system and calibration source of the Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) are described. The foreoptics, based on a modified Kennedy scanner, is coupled by optical fibers to the four spectrometers. The optical fibers allow convenient positioning of the spectrometers in the limited space and enable simple compensation of the scanner's thermal defocus (at the -23 C operating temp) by active control of the fiber focal plane position. A challenging requirement for the fiber optic system was the transmission to the spectral range 1.85 to 2.45 microns at .45 numerical aperture. This was solved with custom fluoride glass fibers from Verre Fluore. The onboard calibration source is also coupled to the spectrometers by the fibers and provides two radiometric levels and a reference spectrum to check the spectrometers' alignment. Results of the performance of the assembled subsystems are presented.

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.

Preservation of rodent bones from El Harhoura 2 cave (Morocco, Neolithic - Middle Palaeolithic): Microstructure, mineralogy, crystallinity and composition

NASA Astrophysics Data System (ADS)

Farre, Bastien; Massard, Pierre; Nouet, Julius; Dauphin, Yannicke

2014-04-01

Thin sections, scanning electron microscopy (SEM), diffraction X (DRX) and infrared spectrometry (FTIR) have been used to study the structure, mineralogy, crystallinity and bulk composition of fossil rodent long bones extracted from a succession of sedimentary layers in a cave from Morocco (Neolithic - Middle Palaeolithic, El Harhoura 2). The microstructure of fossil bones is well-preserved at this scale of observation, and encrusted deposits are rare. All bones are preserved in apatite, but the crystallinity is modified, as well as the crystallite shape, the organic content and the organic-mineral ratio. No fluor enrichment has been observed. Alone or together, the studied parameters do not show a regular trend from the upper to the lower layers of the cave. The preservation of the fossil bones does not confirm the sequence of arid and humid periods inferred from taphonomic analyses.

100-NR-2 Apatite Treatability Test: High-Concentration Calcium-Citrate-Phosphate Solution Injection for In Situ Strontium-90 Immobilization

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

Vermeul, Vincent R.; Fritz, Brad G.; Fruchter, Jonathan S.

2010-09-01

Following an evaluation of potential strontium-90 (90Sr) treatment technologies and their applicability under 100-NR-2 hydrogeologic conditions, the U.S. Department of Energy (DOE), Fluor Hanford, Inc. (now CH2M Hill Plateau Remediation Company [CHPRC]), Pacific Northwest National Laboratory, and the Washington State Department of Ecology agreed that the long-term strategy for groundwater remediation at the 100-N Area should include apatite as the primary treatment technology. This agreement was based on results from an evaluation of remedial alternatives that identified the apatite permeable reactive barrier (PRB) technology as the approach showing the greatest promise for reducing 90Sr flux to the Columbia River atmore » a reasonable cost. This letter report documents work completed to date on development of a high-concentration amendment formulation and initial field-scale testing of this amendment solution.« less

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

Effect of a chlorhexidine/thymol and a fluoride varnish on caries development in erupting permanent molars: a comparative study.

PubMed

Flamee, S; Gizani, S; Caroni, C; Papagiannoulis, L; Twetman, S

2015-12-01

To compare the caries preventive effect of a chlorhexidine/thymol-containing antibacterial varnish with a fluoride varnish when topically applied during the eruption of permanent molars. The study group consisted of 189 patients, 5-14 years of age, with one 1st or 2nd permanent molar in the process of eruption. After stratification for type of molar and stage of eruption, the patients were randomised to either quarterly topical applications with an antibacterial varnish (Cervitec(®) Plus; CV group) or biannual applications with a fluoride varnish plus biannual treatments with placebo varnish (Fluor Protector; FV group). The duration of the study was 2 years. The primary endpoint was caries incidence (initial and cavitated) in the erupting molars and the secondary outcome was salivary mutans streptococci (MS) counts. The groups were balanced with respect to socio-economy, oral hygiene, dietary habits and caries experience at baseline. The dropout rate was 11.6 %. The caries incidence was low (< 10 %) in both groups and there was no significant difference between the CV and FV groups with respect to occlusal caries development in the erupting molars (relative risk 1.08, 95 % CI 0.94-1.25). Significantly lower levels of salivary MS were disclosed in the CV group at the end of the study (p < 0.05). No difference in occlusal caries development in young permanent molars was displayed after topical applications of either a chlorhexidine/thymol varnish or a fluoride varnish during tooth eruption.

High affinity binding of amyloid β-peptide to calmodulin: Structural and functional implications.

PubMed

Corbacho, Isaac; Berrocal, María; Török, Katalin; Mata, Ana M; Gutierrez-Merino, Carlos

2017-05-13

Amyloid β-peptides (Aβ) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid β precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid β(1-42) HiLyte™-Fluor555, we show in this work that Aβ binds with high affinity to CaM through the neurotoxic Aβ25-35 domain. In addition, the affinity of Aβ for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of K d of 0.98 ± 0.11 nM obtained for Aβ1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aβ peptides. A major functional consequence of Aβ-CaM interaction is that it slowdowns Aβ fibrillation. The novel and high affinity interaction between calmodulin and Aβ shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aβ in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Strategic plan for infrastructure optimization

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

Donley, C.D.

This document represents Fluor Daniel Hanford`s and DynCorp`s Tri-Cities Strategic Plan for Fiscal Years 1998--2002, the road map that will guide them into the next century and their sixth year of providing safe and cost effective infrastructure services and support to the Department of Energy (DOE) and the Hanford Site. The Plan responds directly to the issues raised in the FDH/DOE Critical Self Assessment specifically: (1) a strategy in place to give DOE the management (systems) and physical infrastructure for the future; (2) dealing with the barriers that exist to making change; and (3) a plan to right-size the infrastructuremore » and services, and reduce the cost of providing services. The Plan incorporates initiatives from several studies conducted in Fiscal Year 1997 to include: the Systems Functional Analysis, 200 Area Water Commercial Practices Plan, $ million Originated Cost Budget Achievement Plan, the 1OO Area Vacate Plan, the Railroad Shutdown Plan, as well as recommendations from the recently completed Review of Hanford Electrical Utility. These and other initiatives identified over the next five years will result in significant improvements in efficiency, allowing a greater portion of the infrastructure budget to be applied to Site cleanup. The Plan outlines a planning and management process that defines infrastructure services and structure by linking site technical base line data and customer requirements to work scope and resources. The Plan also provides a vision of where Site infrastructure is going and specific initiatives to get there.« less

Biocatalyzed approach for the surface functionalization of poly(L-lactic acid) films using hydrolytic enzymes.

PubMed

Pellis, Alessandro; Acero, Enrique Herrero; Weber, Hansjoerg; Obersriebnig, Michael; Breinbauer, Rolf; Srebotnik, Ewald; Guebitz, Georg M

2015-09-01

Poly(lactic acid) as a biodegradable thermoplastic polyester has received increasing attention. This renewable polyester has found applications in a wide range of products such as food packaging, textiles and biomedical devices. Its major drawbacks are poor toughness, slow degradation rate and lack of reactive side-chain groups. An enzymatic process for the grafting of carboxylic acids onto the surface of poly(L-lactic acid) (PLLA) films was developed using Candida antarctica lipase B as a catalyst. Enzymatic hydrolysis of the PLLA film using Humicola insolens cutinase in order to increase the number of hydroxyl and carboxylic groups on the outer polymer chains for grafting was also assessed and showed a change of water contact angle from 74.6 to 33.1° while the roughness and waviness were an order of magnitude higher in comparison to the blank. Surface functionalization was demonstrated using two different techniques, (14) C-radiochemical analysis and X-ray photoelectron spectroscopy (XPS) using (14) C-butyric acid sodium salt and 4,4,4-trifluorobutyric acid as model molecules, respectively. XPS analysis showed that 4,4,4-trifluorobutyric acid was enzymatically coupled based on an increase of the fluor content from 0.19 to 0.40%. The presented (14) C-radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

MONA, LISA and VINCI Soon Ready to Travel to Paranal

NASA Astrophysics Data System (ADS)

2000-11-01

First Instruments for the VLT Interferometer Summary A few months from now, light from celestial objects will be directed for the first time towards ESO's Very Large Telescope Interferometer (VLTI) at the Paranal Observatory (Chile). During this "First Light" event and the subsequent test phase, the light will be recorded with a special test instrument, VINCI (VLT INterferometer Commissioning Instrument). The main components of this high-tech instrument are aptly named MONA (a system that combines the light beams from several telescopes by means of optical fibers) and LISA (the infrared camera). VINCI was designed and constructed within a fruitful collaboration between ESO and several research institutes and industrial companies in France and Germany . It is now being assembled at the ESO Headquarters in Garching (Germany) and will soon be ready for installation at the telescope on Paranal. With the VLTI and VINCI, Europe's astronomers are now entering the first, crucial phase of an exciting scientific and technology venture that will ultimately put the world's most powerful optical/IR interferometric facility in their hands . PR Photo 31/00 : VINCI during tests at the ESO Headquarters in Garching. The VLT Interferometer (VLTI) ESO Press Photo 31/00 ESO Press Photo 31/00 [Preview; JPEG: 400 x 301; 43k] [Normal; JPEG: 800 x 602;208xk] [Full-Res; JPEG: 1923 x 1448; 2.2Mb] PR Photo 31/00 shows the various components of the complex VINCI instrument for the VLT Interferometer , during the current tests at the Optical Laboratory at the ESO Headquarters in Garching (Germany). It will later be installed in "clean-room" conditions within the Interferometric Laboratory at the Paranal Observatory. This electronic photo was obtained for documentary purposes. VINCI (VLT INterferometer Commissioning Instrument) is the "First Light" instrument for the Very Large Telescope Interferometer (VLTI) at the Paranal Observatory (Chile). Early in 2001, it will be used for the first tests of this very complex system. Subsequently, it will serve to tune this key research facility to the highest possible performance. The VLTI is based on the combination of light (beams) from the telescopes at Paranal. Of these, the four 8.2-m Unit Telescopes are already in operation - they will soon be joined by three 1.8-m telescopes that can be relocated on rails, cf. PR Photo 43b/99. By means of a system of mirrors, the light from two or more of these telescopes will be guided to the central Interferometric Laboratory , at the center of the observing platform on Paranal. Information about the heart of this complex system, the Delay Lines that are located in the underground Interferometric Tunnel, is available with the recent ESO PR Photos 26a-e/00. The VLTI will later receive other front-line instruments, e.g. AMBER , MIDI and PRIMA. When fully ready some years from now, the VLTI will produce extremely sharp images. This will have a major impact on different types of exciting astronomical observations, e.g.: * the direct discovery and imaging of extra-solar planets comparable to Jupiter, * the discovery and imaging of low-mass stars such as brown dwarfs, * observations of star-forming regions and to better understand the physical processes that give birth to stars, * spectral analysis of the atmospheres of nearby stars, and * imaging the objects of the very core of our Galaxy and the detection of black holes in active nuclei of galaxies. The VINCI test instrument The new instrument, VINCI , will soon be delivered to Paranal by the Département de Recherche Spatiale (Department for Space Research), a joint unit of the Centre Nationale de la Recherche Scientifique (French National Centre for Scientific Research) and the Paris Observatory. VINCI is a functional copy of the FLUOR instrument - now at the IOTA (Infrared Optical Telescope Array) interferometer - that has been upgraded and adapted to the needs of the VLTI. FLUOR was developed by the Département de Recherche Spatiale (DESPA) of the Paris Observatory. It was used in 1991 at the Kitt Peak National Observatory (Arizona, USA), for the first (coherent) combination of the light beams from two independent telescopes by means of optical fibers of fluoride glass. It has since been in operation for five years as a focal instrument at the IOTA Interferometer (Mount Hopkins, Arizona, USA) within a collaboration with the Harvard Smithsonian Center for Astrophysics), producing a rich harvest of scientific data. The VINCI partners The VINCI instrument is constructed in a collaboration between ESO (that also finances it) and the following laboratories and institutes: * DESPA (Paris Observatory) provides the expertise, the general concept, the development and integration of the optomechanics (with the exception of the camera) and the electronics, * Observatoire Midi-Pyrénées that produces the control software * The LISA infrared camera is developed by the Max-Planck-Institut für Extraterrestrische Physik (Garching, Germany), and * ESO provides the IR camera electronics and the overall observational software and is also responsible for the final integration. DESPA delivered VINCI to ESO in Garching on September 27, 2000, and is now assembling the instrument in the ESO optical workshop. It will stay here during three months, until it has been fully integrated and thoroughly tested. It will then be shipped to Paranal at the beginning of next year. After set-up and further tests, the first observations on the sky are expected in late March 2001. Fluoride fibers guide the light The heart of VINCI - named MONA - is a fiber optics beam combine unit. It is the outcome of a fertile, 10-year research partnership between Science (DESPA) and Industry ("Le Verre Fluoré" [2]). Optical fibers will be used to combine the light from two telescopes inside VINCI . Since the instrument will be working in the near-infrared region of the spectrum (wavelength 2-2.5 µm), it is necessary to use optical fibers made of a special type of glass that is transparent at these wavelengths. By far the best best material for this is fluoride glass . It was invented by one of the co-founders of the company "Le Verre Fluoré", the only manufacturer of this kind of highly specialized material in the world. Optical fibers of fluoride glass from this company are therefore used in VINCI . They are of a special type ("monomode") with a very narrow core measuring only 6.5 µm (0.065 mm) across. Light that is collected by one of the telescopes in the VLTI array (e.g., by the 50 m 2 mirror of a VLT Unit Telescope) is guided through the VLTI system of optics and finally enters this core. The fibers guide the light and at the same time "clean" the light beam by eliminating the errors introduced by the atmospheric turbulence, hereby improving the accuracy of the measurements by a factor of 10. DESPA has shown that this is indeed possible by means of real astronomical observations with the FLUOR experiment. Following this positive demonstration, it has been decided to equip the instrumentation of all interferometers currently under construction with fibers or equivalent systems.

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

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.

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.

Waste Preparation and Transport Chemistry: Results of the FY 2001 Studies

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

Hunt, R.D.

2002-03-25

During FY 2001, tank farm operations at Hanford and the Savannah River Site (SRS) continued to be negatively impacted by the unintended formation of solids. At Hanford, the primary solids formation problem involves a series of plugged pipes and pumps during the saltwell pumping activities of the interim stabilization program. For example, transfers of tank S-102 waste were suspended due to a plugged pipeline or a mechanical problem with the transfer pump. The replacement pump then failed within 2 weeks. In contrast, since full-scale waste remediation activities such as vitrification were initiated, the SRS has encountered a wider range ofmore » problems due to unwanted solids. The 2H evaporator system was shut down because of the formation of aluminosilicate deposits with enriched uranium in the evaporator pot. While high concentrations of aluminum are expected in the tank waste due to previous canyon operations, the primary source of silicon is the recycle stream from the vitrifier. While solids formation can be expected when waste streams are combined, the formation of the aluminosilicate deposits required an elevated temperature within the evaporator. The shutdown of the 2H evaporator led to a severe shortage of tank space. Therefore, the SRS tank farm was forced to transfer highly concentrated waste, which led to a plugged transfer pump in tank 32. For each of the proposed cesium removal technologies for the SRS, unwanted solids formation occurred during the large laboratory-scale tests prior to the final selection of the solvent extraction process. It can be expected that further problems will be encountered as more unit operations of the remediation effort are deployed and as more waste streams are combined. Since these problems have already led to costly schedule delays, the tank farm operators at both sites have identified the prevention of solids formation as a high-priority need. In response to this need, the Tank Focus Area has assembled a team of researchers of researchers from AEA Technology, Florida International University (FIU), Fluor Hanford, Mississippi State University (MSU), Oak Ridge National Laboratory (ORNL), and Savannah River Technology Center (SRTC) to evaluate various aspects of the waste preparation and transport chemistry. The majority of this effort was focused on saltcake dissolution and saltwell pumping. The results of the AEA Technology, FIU, and MSU studies of saltcake dissolution and slurry transfers for Hanford are discussed in detail in a companion report prepared by T. D. Welch in 2001 (ORNIJTM-2001097). Staff members at Fluor Hanford have continued to conduct saltcake dissolution tests on actual tank waste (documented in reports prepared by D. L. Herting in 2000 and 2001). It should be noted that full-scale saltcake dissolution at Hanford is scheduled to begin in FY 2002. While the Hanford effort is focused on the transfer of waste from one tank to another, the objective of the SRTC study is the formation of aluminosilicates at elevated temperatures, which are present in the waste evaporator.« less

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.

Pathogenesis of Persistent Hyperplastic Primary Vitreous in Mice Lacking the Arf Tumor Suppressor Gene

PubMed Central

Martin, Amy C.; Thornton, J. Derek; Liu, Jiewiu; Wang, XiaoFei; Zuo, Jian; Jablonski, Monica M.; Chaum, Edward; Zindy, Frederique; Skapek, Stephen X.

2006-01-01

Purpose Persistent hyperplastic primary vitreous (PHPV) is an idiopathic developmental eye disease associated with failed involution of the hyaloid vasculature. The present work addressed the pathogenesis of PHPV in a mouse model that replicates many aspects of the human disease. Methods Ophthalmoscopic and histologic analyses documented pathologic processes in eyes of mice lacking the Arf gene compared with Ink4a-deficient and wild-type control animals. Immunohistochemical staining, in situ hybridization, and RT-PCR demonstrated the expression of relevant gene products. Arf gene expression was determined by in situ hybridization using wholemounts of wild-type mouse eyes and by immunofluorescence staining for green fluores-cent protein (GFP) in Arf+/GFP heterozygous knock-in mouse eyes. Results Abnormalities in Arf−/− mice mimicked those found in patients with severe PHPV. The mice had microphthalmia; fibrovascular, retrolental tissue containing retinal pigment epithelial cells and remnants of the hyaloid vascular system; posterior lens capsule destruction with lens degeneration and opacity; and severe retinal dysplasia and detachment. Eyes of mice lacking the overlapping Ink4a gene were normal. Arf was selectively expressed in perivascular cells within the vitreous of the postnatal eye. Cells composing the retrolental mass in Arf−/− mice expressed the Arf promoter. The remnant hyaloid vessels expressed Flk-1. Its ligand, vascular endothelial growth factor (Vegf), was expressed in the retrolental tissue and the adjacent dysplastic neuroretina. Conclusions Arf−/− mice have features that accurately mimic severe PHPV. In the HVS, Arf expression in perivascular cells may block their accumulation or repress Vegf expression to promote HVS involution and prevent PHPV. PMID:15452040

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.

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.

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

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

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.

Environmental Management Performance Report June 2000

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

EDER, D.M.

2000-06-01

The purpose of this report is to provide the Department of Energy Richland Operations Office (DOE-RL) a monthly summary of the Project Hanford Management Contractor's (PHMC) Environmental Management (EM) performance by Fluor Hanford (FH) and its subcontractors. In addition to project-specific information, it includes some PHMC-level data not detailed elsewhere in the report. Section A, Executive Summary, provides an executive level summary of the cost, schedule, and technical performance described in this report. It summarizes performance for the period covered, highlights areas worthy of management attention, and provides a forward look to some of the upcoming key performance activities asmore » extracted from the PHMC baseline. The remaining sections provide detailed performance data relative to each individual Project (e.g., Waste Management, Spent Nuclear Fuels, etc.), in support of Section A of the report. Unless otherwise noted, the Safety, Conduct of Operations, and Cost/Schedule data contained herein is as of April 30, 2000. All other information is updated as of May 19, unless otherwise noted.« less

Preparation of Gd Loaded Liquid Scintillator for Daya Bay Neutrino Experiment

NASA Astrophysics Data System (ADS)

Ya-yun, Ding; Zhi-yong, Zhang

2010-05-01

Gadolinium loaded liquid scintillator (Gd-LS) is an excellent target material for reactor antineutrino experiments. Ideal Gd-LS should have long attenuation length, high light yield, long term stability, low toxicity, and should be compatible with the material used to build the detector. We have developed a new Gd-LS recipe in which carboxylic acid 3,5,5-trimethylhexanoic acid is used as the complexing ligand to gadolinium, 2,5-diphenyloxazole (PPO) and 1,4-bis[2-methylstyryl]benzene (bis-MSB) are used as primary fluor and wavelength shifter, respectively. The scintillator base is linear alkyl benzene (LAB). Eight hundred liters of Gd-LS has been synthesized and tested in a prototype detector. Results show that the Gd-LS has high quality and is suitable for underground experiments in large quantity. Large scale production facility has been built. A full batch production of 4 t Gd-LS has been produced and monitored for several months. The production of 180 t Gd-LS will be carried out in the near future.

Preparation and performance study of a novel liquid scintillator with mixed solvent as the matrix

NASA Astrophysics Data System (ADS)

Zheng, Zhanlong; Zhu, Jiayi; Luo, Xuan; Xu, Yewei; Zhang, Qianfeng; Zhang, Xing; Bi, Yutie; Zhang, Lin

2017-04-01

A novel liquid scintillator with the mixed solvent as the matrix was prepared for obtaining a good comprehensive performance. In this ternary liquid scintillator, the combination of 20% pseudocumene (PC) and 80% linear-alkyl benzene (LAB) by volume was chosen as the mixed solvent, and 2,5-diphenyloxazole (PPO) and 1,4-bis(2-Methylstyryl) benzene (bis-MSB) were as the primary fluor and wavelength shifter, respectively. The optimum prescription was obtained with regard to the light yield. Some characterizations based on the optimal formulation were conducted. The fluorescence emission spectra and wavelength-dependent optical attenuation length of the sample were measured by the fluorescence spectrophotometer and an UV-Vis spectrometer, respectively. The light yield was characterized by adopting the home-made optical platform device. The decay time was tested by adopting the time-correlated single photon counting (TCSPC) technique featured in high dynamic range of several orders of magnitude in light intensity. The experimental test results showed that the sample had a fairly good comprehensive performance.

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

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.

Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

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

Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and themore » IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.« less

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

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

OPEN AIR DEMOLITION OF FACILITIES HIGHLY CONTAMINATED WITH PLUTONIUM

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

LLOYD, E.R.

2007-05-31

The demolition of highly contaminated plutonium buildings usually is a long and expensive process that involves decontaminating the building to near free- release standards and then using conventional methods to remove the structure. It doesn't, however, have to be that way. Fluor has torn down buildings highly contaminated with plutonium without excessive decontamination. By removing the select source term and fixing the remaining contamination on the walls, ceilings, floors, and equipment surfaces; open-air demolition is not only feasible, but it can be done cheaper, better (safer), and faster. Open-air demolition techniques were used to demolish two highly contaminated buildings tomore » slab-on-grade. These facilities on the Department of Energy's Hanford Site were located in, or very near, compounds of operating nuclear facilities that housed hundreds of people working on a daily basis. To keep the facilities operating and the personnel safe, the projects had to be creative in demolishing the structures. Several key techniques were used to control contamination and keep it within the confines of the demolition area: spraying fixatives before demolition; applying fixative and misting with a fine spray of water as the buildings were being taken down; and demolishing the buildings in a controlled and methodical manner. In addition, detailed air-dispersion modeling was done to establish necessary building and meteorological conditions and to confirm the adequacy of the proposed methods. Both demolition projects were accomplished without any spread of contamination outside the modest buffer areas established for contamination control. Furthermore, personnel exposure to radiological and physical hazards was significantly reduced by using heavy equipment rather than ''hands on'' techniques.« less

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

Hydroxyapatite, fluor-hydroxyapatite and fluorapatite produced via the sol-gel method. Optimisation, characterisation and rheology.

PubMed

Tredwin, Christopher J; Young, Anne M; Georgiou, George; Shin, Song-Hee; Kim, Hae-Won; Knowles, Jonathan C

2013-02-01

Currently, most titanium implant coatings are made using hydroxyapatite and a plasma spraying technique. There are however limitations associated with plasma spraying processes including poor adherence, high porosity and cost. An alternative method utilising the sol-gel technique offers many potential advantages but is currently lacking research data for this application. It was the objective of this study to characterise and optimise the production of Hydroxyapatite (HA), fluorhydroxyapatite (FHA) and fluorapatite (FA) using a sol-gel technique and assess the rheological properties of these materials. HA, FHA and FA were synthesised by a sol-gel method. Calcium nitrate and triethylphosphite were used as precursors under an ethanol-water based solution. Different amounts of ammonium fluoride (NH4F) were incorporated for the preparation of the sol-gel derived FHA and FA. Optimisation of the chemistry and subsequent characterisation of the sol-gel derived materials was carried out using X-ray Diffraction (XRD) and Differential Thermal Analysis (DTA). Rheology of the sol-gels was investigated using a viscometer and contact angle measurement. A protocol was established that allowed synthesis of HA, FHA and FA that were at least 99% phase pure. The more fluoride incorporated into the apatite structure; the lower the crystallisation temperature, the smaller the unit cell size (changes in the a-axis), the higher the viscosity and contact angle of the sol-gel derived apatite. A technique has been developed for the production of HA, FHA and FA by the sol-gel technique. Increasing fluoride substitution in the apatite structure alters the potential coating properties. Crown Copyright © 2012. Published by Elsevier Ltd. 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.

Submittal for 2003 Project of the Year K Basins Fuel Transfer System Project

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

GERBER, M.S.

2003-01-29

Fluor Hanford, Inc. is pleased to submit the K Basins Fuel Transfer System (FTS) for consideration by the Project Management Institute as Project of the Year for 2003. The FTS involved installing a unique, unproven system in an inhospitable and deteriorating radiological and hazardous environment, under very stringent requirements and within an extremely condensed schedule, just 19 months, from authorization to full operations. The FTS, therefore, is an excellent example of effective project management, and the dynamic involvement of an integrated team representing a broad spectrum of personnel, disciplines, and services. The FTS is an integral and critical part ofmore » a larger project at Hanford -the Spent Nuclear Fuel Project (SNF). The mission of the SNF Project is to relocate used, or spent, nuclear fuel to safe interim storage, permanently dispose of radioactive debris in the K-Basins, and deactivate all related facilities and prepare them for demolition. Today, the FTS is being used to remove highly radioactive nuclear fuel from an aging, and potentially unstable storage in underground pools of water--the K-Basins--and safely transport it to a processing area to be cleaned, dried and sent to safe storage. The role the FTS plays in successfully completing the mission of the SNF Project is concrete evidence of the intrinsic value of project management and a testimonial to the innovation, ingenuity, and teamwork of many--from workers to management and subcontractors, and regulators to stakeholders. It's a true success story and one that will have a happy ending, safely eliminating the risk of potentially contaminating one of Washington state's most valuable natural resources, the Columbia River. This nomination is dedicated to that Project Team.« less

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.

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.

Preparation of Gd Loaded Liquid Scintillator for Daya Bay Neutrino Experiment

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

Ding Yayun; Zhang Zhiyong

2010-05-12

Gadolinium loaded liquid scintillator (Gd-LS) is an excellent target material for reactor antineutrino experiments. Ideal Gd-LS should have long attenuation length, high light yield, long term stability, low toxicity, and should be compatible with the material used to build the detector. We have developed a new Gd-LS recipe in which carboxylic acid 3,5,5-trimethylhexanoic acid is used as the complexing ligand to gadolinium, 2,5-diphenyloxazole (PPO) and 1,4-bis[2-methylstyryl]benzene (bis-MSB) are used as primary fluor and wavelength shifter, respectively. The scintillator base is linear alkyl benzene (LAB). Eight hundred liters of Gd-LS has been synthesized and tested in a prototype detector. Results showmore » that the Gd-LS has high quality and is suitable for underground experiments in large quantity. Large scale production facility has been built. A full batch production of 4 t Gd-LS has been produced and monitored for several months. The production of 180 t Gd-LS will be carried out in the near future.« less

New method for GC/FID and GC-C-IRMS Analysis of plasma free fatty acid concentration and isotopic enrichment

PubMed Central

Kangani, Cyrous O.; Kelley, David E.; DeLany, James P.

2008-01-01

A simple, direct and accurate method for the determination of concentration and enrichment of free fatty acids in human plasma was developed. The validation and comparison to a conventional method are reported. Three amide derivatives, dimethyl, diethyl and pyrrolidide, were investigated in order to achieve optimal resolution of the individual fatty acids. This method involves the use of dimethylamine/Deoxo-Fluor to derivatize plasma free fatty acids to their dimethylamides. This derivatization method is very mild and efficient, and is selective only towards free fatty acids so that no separation from a total lipid extract is required. The direct method gave lower concentrations for palmitic acid and stearic acid and increased concentrations for oleic acid and linoleic acid in plasma as compared to methylester derivative after thin-layer chromatography. The [13C]palmitate isotope enrichment measured using direct method was significantly higher than that observed with the BF3/MeOH-TLC method. The present method provided accurate and precise measures of concentration as well as enrichment when analyzed with gas chromatography combustion-isotope ratio-mass spectrometry. PMID:18757250

New method for GC/FID and GC-C-IRMS analysis of plasma free fatty acid concentration and isotopic enrichment.

PubMed

Kangani, Cyrous O; Kelley, David E; Delany, James P

2008-09-15

A simple, direct and accurate method for the determination of concentration and enrichment of free fatty acids (FFAs) in human plasma was developed. The validation and comparison to a conventional method are reported. Three amide derivatives, dimethyl, diethyl and pyrrolidide, were investigated in order to achieve optimal resolution of the individual fatty acids. This method involves the use of dimethylamine/Deoxo-Fluor to derivatize plasma free fatty acids to their dimethylamides. This derivatization method is very mild and efficient, and is selective only towards FFAs so that no separation from a total lipid extract is required. The direct method gave lower concentrations for palmitic acid and stearic acid and increased concentrations for oleic acid and linoleic acid in plasma as compared to methyl ester derivative after thin-layer chromatography. The [(13)C]palmitate isotope enrichment measured using direct method was significantly higher than that observed with the BF(3)/MeOH-TLC method. The present method provided accurate and precise measures of concentration as well as enrichment when analyzed with gas chromatography combustion-isotope ratio-mass spectrometry.

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.

A Rationally Designed, General Strategy for Membrane Orientation of Photoinduced Electron Transfer-Based Voltage-Sensitive Dyes.

PubMed

Kulkarni, Rishikesh U; Yin, Hang; Pourmandi, Narges; James, Feroz; Adil, Maroof M; Schaffer, David V; Wang, Yi; Miller, Evan W

2017-02-17

Voltage imaging with fluorescent dyes offers promise for interrogating the complex roles of membrane potential in coordinating the activity of neurons in the brain. Yet, low sensitivity often limits the broad applicability of optical voltage indicators. In this paper, we use molecular dynamics (MD) simulations to guide the design of new, ultrasensitive fluorescent voltage indicators that use photoinduced electron transfer (PeT) as a voltage-sensing switch. MD simulations predict an approximately 16% increase in voltage sensitivity resulting purely from improved alignment of dye with the membrane. We confirm this theoretical finding by synthesizing 9 new voltage-sensitive (VoltageFluor, or VF) dyes and establishing that all of them display the expected improvement of approximately 19%. This synergistic outworking of theory and experiment enabled computational and theoretical estimation of VF dye orientation in lipid bilayers and has yielded the most sensitive PeT-based VF dye to date. We use this new voltage indicator to monitor voltage spikes in neurons from rat hippocampus and human pluripotent-stem-cell-derived dopaminergic neurons.

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

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.

Enhancing the luminescence of Eu3+ /Eu2+ ion-doped hydroxyapatite by fluoridation and thermal annealing.

PubMed

Van, Hoang Nhu; Tam, Phuong Dinh; Kien, Nguyen Duc Trung; Huy, Pham Thanh; Pham, Vuong-Hung

2017-08-01

This paper reports a novel way for the synthesis of a europium (Eu)-doped fluor-hydroxyapatite (FHA) nanostructure to control the luminescence of hydroxyapatite nanophosphor, particularly, by applying optimum fluorine concentrations, annealed temperatures and pH value. The Eu-doped FHA was made using the co-precipitation method followed by thermal annealing in air and reducing in a H 2 atmosphere to control the visible light emission center of the nanophosphors. The intensities of the OH - group decreased with the increasing fluorine concentrations. For the specimens annealed in air, the light emission center of the nanophosphor was 615 nm, which was emission from the Eu 3 + ion. However, when they were annealed in reduced gas (Ar + 5% H 2 ), a 448 nm light emission center from the Eu 2 + ion of FHA was observed. The presence of fluorine in Eu-doped FHA resulted in a significant enhancement of nanophosphor luminescence, which has potential application in light emission and nanomedicine. Copyright © 2016 John Wiley & Sons, Ltd.

Experiment to demonstrate separation of Cherenkov and scintillation signals

DOE PAGES

Caravaca, J.; Descamps, F. B.; Land, B. J.; ...

2017-05-05

The ability to separately identify the Cherenkov and scintillation light components produced in scintillating mediums holds the potential for a major breakthrough in neutrino detection technology, allowing development of a large, low-threshold, directional detector with a broad physics program. Furthermore, the CHESS (CHErenkov/Scintillation Separation) experiment employs an innovative detector design with an array of small, fast photomultiplier tubes and state-of-the-art electronics to demonstrate the reconstruction of a Cherenkov ring in a scintillating medium based on photon hit time and detected photoelectron density. Our paper describes the physical properties and calibration of CHESS along with first results. The ability to reconstructmore » Cherenkov rings are demonstrated in a water target, and a time precision of 338 ± 12 ps FWHM is achieved. Finally, Monte Carlo–based predictions for the ring imaging sensitivity with a liquid scintillator target predict an efficiency for identifying Cherenkov hits of 94 ± 1 % and 81 ± 1 % in pure linear alkyl benzene (LAB) and LAB loaded with 2 g/L of a fluor, PPO, respectively, with a scintillation contamination of 12 ± 1 % and 26 ± 1 % .« less

Experiment to demonstrate separation of Cherenkov and scintillation signals

NASA Astrophysics Data System (ADS)

Caravaca, J.; Descamps, F. B.; Land, B. J.; Wallig, J.; Yeh, M.; Orebi Gann, G. D.

2017-05-01

The ability to separately identify the Cherenkov and scintillation light components produced in scintillating mediums holds the potential for a major breakthrough in neutrino detection technology, allowing development of a large, low-threshold, directional detector with a broad physics program. The CHESS (CHErenkov/Scintillation Separation) experiment employs an innovative detector design with an array of small, fast photomultiplier tubes and state-of-the-art electronics to demonstrate the reconstruction of a Cherenkov ring in a scintillating medium based on photon hit time and detected photoelectron density. This paper describes the physical properties and calibration of CHESS along with first results. The ability to reconstruct Cherenkov rings is demonstrated in a water target, and a time precision of 338 ±12 ps FWHM is achieved. Monte Carlo-based predictions for the ring imaging sensitivity with a liquid scintillator target predict an efficiency for identifying Cherenkov hits of 94 ±1 % and 81 ±1 % in pure linear alkyl benzene (LAB) and LAB loaded with 2 g/L of a fluor, PPO, respectively, with a scintillation contamination of 12 ±1 % and 26 ±1 % .

Tank farms pump critical characteristic and specification guide

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

Titzler, P.A.

The Design Authority group for Tank Farms, in conjunction with the Construction Projects organization, have recognized that there is a need to provide consistency in the procurement and testing of pumps and to assure that known critical attributes and features are included with each pump order as well as to reduce potential confusion by pump suppliers. As a result, a panel of pump experts representing Lockheed Martin Hanford Company (LMHC), Fluor Daniel Northwest (FDNW), Numatec Hanford Corporation (NHC), SGN Eurisys Services Corporation (SESC), and ARES Corporation has been assembled to prepare a guide for pump specifications. This document contains themore » consensus listing of critical characteristics and procurement recommendations of the panel. It is intended to be used as a guide for future pump procurement activities. If followed, it will help reduce cleanup costs at the Hanford Site and promote prompt approval of pumping system designs and procurement specifications. Alternate criteria may be specified on a case by case basis if deviation from the requirements contained herein is merited due to special circumstances.« less

In vivo investigation of hybrid Paclitaxel nanocrystals with dual fluorescent probes for cancer theranostics.

PubMed

Hollis, Christin P; Weiss, Heidi L; Evers, B Mark; Gemeinhart, Richard A; Li, Tonglei

2014-06-01

To develop novel hybrid paclitaxel (PTX) nanocrystals, in which bioactivatable (MMPSense® 750 FAST) and near infrared (Flamma Fluor® FPR-648) fluorophores are physically incorporated, and to evaluate their anticancer efficacy and diagnostic properties in breast cancer xenograft murine model. The pure and hybrid paclitaxel nanocrystals were prepared by an anti-solvent method, and their physical properties were characterized. The tumor volume change and body weight change were evaluated to assess the treatment efficacy and toxicity. Bioimaging of treated mice was obtained non-invasively in vivo. The released MMPSense molecules from the hybrid nanocrystals were activated by matrix metalloproteinases (MMPs) in vivo, similarly to the free MMPSense, demonstrating its ability to monitor cancer progression. Concurrently, the entrapped FPR-648 was imaged at a different wavelength. Furthermore, when administered at 20 mg/kg, the nanocrystal formulations exerted comparable efficacy as Taxol®, but with decreased toxicity. Hybrid nanocrystals that physically integrated two fluorophores were successfully prepared from solution. Hybrid nanocrystals were shown not only exerting antitumor activity, but also demonstrating the potential of multi-modular bioimaging for diagnostics.

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.

Infrared interferometric observations of nearby exozodiacal disks: current status and perspectives

NASA Astrophysics Data System (ADS)

Defrère, D.; Absil, O.; di Folco, E.; Coudé du Foresto, V.; Mérand, A.; Augereau, J.-C.

2010-10-01

Directly detecting exozodiacal dust in the inner part of extrasolar planetary systems is nowadays feasible thanks to the advance of high-precision near-infrared interferometry. Investigating this region around nearby stars provides unique information to understand the global architecture of planetary systems and to define the population of stars suitable for future exo-Earth characterization missions. Over the last few years, a survey of nearby main-sequence stars has been ongoing at the CHARA array using the FLUOR beam combiner. The goal of this survey is to directly probe the inner part of circumstellar disks in order to detect the signature of hot dust accounting for about 1% of the near-infrared stellar flux. In this paper, we present the status of this survey and provide the first statistical results about the occurrence of bright exozodiacal disks around nearby main-sequence stars. We also report on the first H-band interferometric observations of the exozodiacal disk around Vega which have been obtained with IOTA/IONIC, and discuss the implications on the disk properties.

F18-FDG-PET for recurrent differentiated thyroid cancer: a systematic meta-analysis

PubMed Central

Haslerud, Torjan; Brauckhoff, Katrin; Reisæter, Lars; Küfner Lein, Regina; Heinecke, Achim; Varhaug, Jan Erik

2015-01-01

Background Positron emission tomography (PET) with fluor-18-deoxy-glucose (FDG) is widely used for diagnosing recurrent or metastatic disease in patients with differentiated thyroid cancer (DTC). Purpose To assess the diagnostic accuracy of FDG-PET for DTC in patients after ablative therapy. Material and Methods A systematic search was conducted in Medline/PubMed, EMBASE, Cochrane Library, Web of Science, and Open Grey looking for all English-language original articles on the performance of FDG-PET in series of at least 20 patients with DTC having undergone ablative therapy including total thyroidectomy. Diagnostic performance measures were pooled using Reitsma’s bivariate model. Results Thirty-four publications between 1996 and 2014 met the inclusion criteria. Pooled sensitivity and specificity were 79.4% (95% confidence interval [CI], 73.9–84.1) and 79.4% (95% CI, 71.2–85.4), respectively, with an area under the curve of 0.858. Conclusion F18-FDG-PET is a useful method for detecting recurrent DTC in patients having undergone ablative therapy. PMID:26163534

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

Air and smear sample calculational tool for Fluor Hanford Radiological control

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

BAUMANN, B.L.

2003-07-11

A spreadsheet calculation tool was developed to automate the calculations performed for determining the concentration of airborne radioactivity and smear counting as outlined in HNF-13536, Section 5.2.7, ''Analyzing Air and Smear Samples''. This document reports on the design and testing of the calculation tool. Radiological Control Technicians (RCTs) will save time and reduce hand written and calculation errors by using an electronic form for documenting and calculating work place air samples. Current expectations are RCTs will perform an air sample and collect the filter or perform a smear for surface contamination. RCTs will then survey the filter for gross alphamore » and beta/gamma radioactivity and with the gross counts utilize either hand calculation method or a calculator to determine activity on the filter. The electronic form will allow the RCT with a few key strokes to document the individual's name, payroll, gross counts, instrument identifiers; produce an error free record. This productivity gain is realized by the enhanced ability to perform mathematical calculations electronically (reducing errors) and at the same time, documenting the air sample.« less

Geochemical Assessment of Fluoride Pollution in Groundwater of Tribal Region in India.

PubMed

Anshumali; Kumar, Manish; Chanda, Nikki; Kumar, Abhay; Kumar, Bijendra; Venkatesh, Madavi

2018-03-01

This study assessed the fluoride (F - ) pollution in groundwater samples (n = 170) of tribal regions around Bailadila Iron Ore Mines [BIOM] Complex of Dantewada District, India. Weathering of carbonate and silicate clays were important geogenic sources of dissolved ions. A Piper diagram showed a Ca-HCO 3 water type, with positive chloro-alkaline indices illustrating the occurrence of direct base-exchange reactions. The F - concentrations varied from 0.08 to 1.95 mg L -1 with a mean value of 0.9 ± 0.3 mg L -1 . Only two groundwater samples showed F - concentrations > 1.5 mg L -1 , the drinking water guideline established by the World Health Organization (WHO) and Bureau of Indian Standards (BIS). Factor analysis showed high loadings of HCO 3 - and F - , indicating alkaline conditions, favoring the dissolution of F - in the groundwater. The K fluor value is less than 10 -10.6 , indicating that the dissociation of fluorite is very slow. As a result, groundwater locations were under-saturated with respect to fluorite.

The effect of anchovy substrate application to fluor retention rate on Sprague Dawley rat tooth email (in vivo)

NASA Astrophysics Data System (ADS)

Zabrina, S.; Puspitawati, R.; Gunawan, H. A.

2017-08-01

Usage of anchovies (Stolephorus insularis), which contain a high fluoride concentration in a CaF2 compound, needs to be examined as a topical fluoridative agent. Aim: To study the effects of an anchovy substrate application, either by chewing or smearing, in increasing fluoride retention of enamel. Fourteen Sprague Dawley rats were divided into five groups: baseline, experimental (feeding and smearing), and negative controls. After 15 days, lower incisor teeth were extracted and fluoride retention on the enamel surface was measured using EDX. Data were analyzed by the independent samples t-test, the Mann-Whitney U test, and the Kruskal-Wallis test. There was a significant increase in fluoride retention on enamel from the experimental groups compared to the negative control group (p < 0.05). Fluoride retention levels of the experimental feeding group (6.823%) were slightly higher than those of the experimental smearing group (6.783%), though the difference was not statistically significant (p < 0.05). Anchovy substrate application, either by chewing or smearing, increases fluoride retention on tooth enamel.

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.

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.

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.

Cu- and Mn-bearing tourmalines from Brazil and Mozambique: crystal structures, chemistry and correlations

NASA Astrophysics Data System (ADS)

Ertl, Andreas; Giester, Gerald; Schüssler, Ulrich; Brätz, Helene; Okrusch, Martin; Tillmanns, Ekkehart; Bank, Hermann

2013-04-01

Cu- and Mn-bearing tourmalines from Brazil and Mozambique were characterised chemically (EMPA and LA-ICP-MS) and by X-ray single-crystal structure refinement. All these samples are rich in Al, Li and F (fluor-elbaite) and contain significant amounts of CuO (up to ~1.8 wt%) and MnO (up to ~3.5 wt%). Structurally investigated samples show a pronounced positive correlation between the < Y-O> distances and the (Li + Mn2+ + Cu + Fe2+) content (apfu) at this site with R 2 = 0.90. An excellent negative correlation exists between the < Y-O> distances and the Al2O3 content ( R 2 = 0.94). The samples at each locality generally show a strong negative correlation between the X-site vacancies and the (MnO + FeO) content. The Mn content in these tourmalines depends on the availability of Mn, on the formation temperature, as well as on stereochemical constraints. Because of a very weak correlation between MnO and CuO we believe that the Cu content in tourmaline is essentially dependent on the availability of Cu and on stereochemical constraints.

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.

ShellFit: Reconstruction in the MiniCLEAN Detector

NASA Astrophysics Data System (ADS)

Seibert, Stanley

2010-02-01

The MiniCLEAN dark matter experiment is an ultra-low background liquid cryogen detector with a fiducial volume of approximately 150 kg. Dark matter candidate events produce ultraviolet scintillation light in argon at 128 nm and in neon at 80 nm. In order to detect this scintillation light, the target volume is enclosed by acrylic plates forming a spherical shell upon which an organic fluor, tetraphenyl butadiene (TPB), has been applied. TPB absorbs UV light and reemits visible light isotropically which can be detected by photomultiplier tubes. Two significant sources of background events in MiniCLEAN are decays of radon daughters embedded in the acrylic surface and external sources of neutrons, such as the photomultiplier tubes themselves. Both of these backgrounds can be mitigated by reconstructing the origin of the scintillation light and cutting events beyond a particular radius. The scrambling of photon trajectories at the TPB surface makes this task very challenging. The ``ShellFit'' algorithm for reconstructing event position and energy in a detector with a spherical wavelength-shifting shell will be described. The performance of ShellFit will be demonstrated using Monte Carlo simulation of several event types in the MiniCLEAN detector. )

Targeting personalized medicine in a non-Hodgkin lymphoma patient with 18F-FDG and 18F-choline PET/CT.

PubMed

Ribeiro, Thalles H; S, Raul; Castro, Ana Carolina G; Paulino, Eduardo; Mamede, Marcelo

2017-02-01

Early diagnosis and staging of non-Hodgkin lymphoma (NHL) is essential for therapeutic strategy decision. Positron emission tomography/computed tomography (PET/CT) with fluordeoxyglucose (FDG), a glucose analogue, labeled with fluor-18 (18F-FDG) has been used to evaluate staging, therapy response and prognosis in NHL patients. However, in some cases, 18F-FDG has shown false-positive uptake due to inflammatory reaction after chemo and/or radiation therapy. In this case report, we present a NHL patient evaluated with 18F-FDG and 18F-choline PET/CT scan imaging pre- and post-therapy. 18F-FDG and 18F-choline PET/CT were performed for the purpose of tumor staging and have shown intense uptake in infiltrative tissue as well as in the lymph node, but with some mismatching in the tumor. Post-treatment 18F-FDG and 18F-choline PET/ CT scans revealed no signs of radiotracer uptake, suggesting complete remission of the tumor. 18F-choline may be a complimentary tool for staging and assessment of therapeutic response in non-Hodgkin lymphoma, while non-18F-FDG tracer can be used for targeted therapy and patient management.

Methacrylate based cross-linkers for improved thermomechanical properties and retention of radiation detection response in plastic scintillators

NASA Astrophysics Data System (ADS)

Mahl, Adam; Lim, Allison; Latta, Joseph; Yemam, Henok A.; Greife, Uwe; Sellinger, Alan

2018-03-01

Pulse shape discrimination (PSD) is an important method that can efficiently sort and separate neutron and gamma radiation signals. PSD is currently achieved in plastic scintillators by over-doping poly(vinyl toluene) (PVT) matrices with fluorescent molecules. Meaningful separation of the signals requires addition of >20 wt% 2,5-diphenyloxazole (PPO) fluor in PVT. At these concentrations PPO acts as a plasticizer, negatively affecting the physical properties of the final plastic such as hardness, machinability, and thermomechanical stability. This work addresses these issues by implementing a cost-effective solution using cross-linking chemistry via commercially available bisphenol A dimethacrylate (BPA-DM), and a synthesized fluorinated analogue. Both improve the physical properties of over-doped PPO based plastic scintillators without degrading the measured light yield or PSD and Figure of Merit (FoM). In addition, the fluorinated analogue appears to enhance the hydrophobicity of the surface of the plastic scintillators, which may improve the scintillators' resistance to water diffusion and subsequent radiation response degradation. The new formulations improve the feasibility of widely deploying long lifetime PSD capable plastic scintillators in large area coverage assemblies.

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.

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.

Devonian climate and reef evolution: Insights from oxygen isotopes in apatite

NASA Astrophysics Data System (ADS)

Joachimski, M. M.; Breisig, S.; Buggisch, W.; Talent, J. A.; Mawson, R.; Gereke, M.; Morrow, J. R.; Day, J.; Weddige, K.

2009-07-01

Conodonts, microfossils composed of carbonate-fluor apatite, are abundant in Palaeozoic-Triassic sediments and have a high potential to preserve primary oxygen isotope signals. In order to reconstruct the palaeotemperature history of the Devonian, the oxygen isotope composition of apatite phosphate was measured on 639 conodont samples from sequences in Europe, North America and Australia. The Early Devonian (Lochkovian; 416-411 Myr) was characterized by warm tropical temperatures of around 30 °C. A cooling trend started in the Pragian (410 Myr) with intermediate temperatures around 23 to 25 °C reconstructed for the Middle Devonian (397-385 Myr). During the Frasnian (383-375 Myr), temperatures increased again with temperatures to 30 °C calculated for the Frasnian-Famennian transition (375 Myr). During the Famennian (375-359 Myr), surface water temperatures slightly decreased. Reconstructed Devonian palaeotemperatures do not support earlier views suggesting the Middle Devonian was a supergreenhouse interval, an interpretation based partly on the development of extensive tropical coral-stromatoporoid communities during the Middle Devonian. Instead, the Devonian palaeotemperature record suggests that Middle Devonian coral-stromatoporoid reefs flourished during cooler time intervals whereas microbial reefs dominated during the warm to very warm Early and Late Devonian.

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

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.

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

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

Interim Report: 100-NR-2 Apatite Treatability Test: Low Concentration Calcium Citrate-Phosphate Solution Injection for In Situ Strontium-90 Immobilization

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

Williams, Mark D.; Fritz, Brad G.; Mendoza, Donaldo P.

2008-07-11

Following an evaluation of potential Sr-90 treatment technologies and their applicability under 100-NR-2 hydrogeologic conditions, U.S. Department of Energy, Fluor Hanford, Inc., Pacific Northwest National Laboratory, and the Washington Department of Ecology agreed that the long-term strategy for groundwater remediation at 100-N Area will include apatite sequestration as the primary treatment, followed by a secondary treatment if necessary (most likely phytoremediation). Since then, the agencies have worked together to agree on which apatite sequestration technology has the greatest chance of reducing Sr-90 flux to the river at a reasonable cost. In July 2005, aqueous injection, (i.e., the introduction of apatite-formingmore » chemicals into the subsurface) was endorsed as the interim remedy and selected for field testing. Studies are in progress to assess the efficacy of in situ apatite formation by aqueous solution injection to address both the vadose zone and the shallow aquifer along the 300 ft of shoreline where Sr-90 concentrations are highest. This report describes the field testing of the shallow aquifer treatment.« less

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

The shakedown.

PubMed

Bodrock, Phil

2005-03-01

Customer Strategy Solutions, a California-based developer of order-fulfillment systems, is facing a shakedown. Six months after the firm's CEO, Pavlo Zhuk, set up a software development center in Kiev, local bureaucrats say the company hasn't filed all the tax schedules it should have. Moreover, Ukrainian tax officials claim that the company owes the government tax arrears. Zhuk is shocked; he and his colleagues have done everything by the book. This isn't the first time Zhuk has encountered trouble in Ukraine. In the process of getting the development center up and running, a state-owned telecommunications utility had made it difficult for Zhuk to get the phone lines his company needed. Senior telecom manager Vasyl Feodorovych Mylofienko had told Zhuk it would take three years to install the lines in his office-but for a certain price, Mylofienko had added, the lines could be functioning the following week. Even as the picture of rampant bribery and corruption in Ukraine becomes clear, Zhuk still doesn't want to pull out of the country. Of Ukrainian descent, he has dreams of helping to modernize the country. By paying his programmers more than they could make at any local company, he hopes to raise their standard of living so they can afford three meals a day without having to barter, stand in queues for hours, or moonlight. And yet, he isn't sure he can keep compromising his principles for the sake of the greater good. Should Customer Strategy Solutions pay off the Ukrainian tax officials? Commenting on this fictional case study are Alan L. Boeckmann, the chairman and CEO of Fluor Corporation; Rafael Di Tella, a professor at Harvard Business School; Thomas W. Dunfee, the Kolodny Professor of Social Responsibility and a professor of legal studies at Wharton; and Bozidar Djelic, the former finance and economy minister of Serbia.

[Design of an external prosthetic system with a polycentric mechanism].

PubMed

Valentino Orozco, Gerardo Alejandro; Guerrero Beltrán, Leobardo; Obregón Velasco, Dinora; Avalos Fernández, Oscar

2008-01-01

This medical and engineering work explains the process carried out to build a four-barred polycentric knee mechanism using a mechanical actuator for modular prosthetics. Imitation of the knee performance was the main goal, as well as to cover the basic needs of amputated patients with a low production cost. The polycentric knee mechanism was developed in three stages. The first one was the design and building of the four-barred mechanism using materials as stainless steel 304 and aluminum 6,061 which both have high reliability, corrosion resistance and a relatively easy modeling capability. The total mass was of 700 g. Autocad was used for design and effort analysis was done by using ANSYS. At the end of the first stage, it was observed that its density did not allow mass optimization and no assistant actuator was available for the walking movement. In the second stage, mechanism mass estimation was done by using Alumold 400; this material brings lower density than stainless steel keeping strength and fatigue resistance higher than aluminum 6061. The better ergonomic device was built; a mechanical actuator based on a spring was implemented and tested on two female patients, one geriatric and one 16 years old. The third stage will be described in a future publication, the device mass was optimized by using poly-tetra-fluor-ethylene enhanced with carbon and carbon fiber. Also, the mechanical actuator was complemented with reo-magnetic liquid addition and an electronic control. At the end of this research, an external polycentric knee mechanism was developed, by observing movement stages, velocities, ergonomics, shapes, sizes and significant weights of Mexican patients. Effort analysis by finite element was done and it was determined that the mechanism was capable of modifying its center of rotation according to the knee flexion angle making it a polycentric mechanisms. Also, it was tested on two patients, both females, one 16 and one 75 years old.

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.

Experimental investigation of detonation waves instabilities in liquid high explosives

NASA Astrophysics Data System (ADS)

Sosikov, V. A.; Torunov, S. I.; Utkin, A. V.; Mochalova, V. M.; Rapota, D. Yu

2018-01-01

Experimental investigation of unstable detonation front structure in mixtures of liquid high explosives (nitromethane and FEFO—bis-(2-fluor-2.2-dinitroethyl)-formal) with inert diluents (acetone, methanol, DETA—diethylene triamine) has been carried out. Inhomogeneities have been registered by electro-optical camera NANOGATE 4BP allowing to make 4 frames with the exposure time 10 ns. According to experimental results the detonation front in nitromethane-acetone mixture is unstable. It is evident that pulsations on detonation front do not form spatial periodic structure and their dimensions differ several times. But mean longitudinal size of pulsation is about 500 μm at 20 wt% of acetone concentration. This means that the typical size of cell equals to reaction zone width. The same structure of cellular front have been registered in 70/30 FEFO-methanol mixture. Second kind of instability, failure waves, was observed in neat nitromethane at the free surface. In this case the stability loss result in turbulent flow which is clearly detected in the shots obtained. Adding small amount of DETA (0.5 wt%) results in disappearance of the failure waves and flow stabilization. The effect is caused by the fact that DETA sharply accelerates initial rate of chemical reaction because it is sensitizer for nitromethane.

Effects of applying anchovy (Stolephorus insularis) substrates on the microhardness of tooth enamel in Sprague-Dawley rats

NASA Astrophysics Data System (ADS)

Hendrik, Y. C.; Puspitawati, R.; Gunawan, H. A.

2017-08-01

Anchovies (Stolephorus insularis) contain high levels of fluor in the form of CaF2. The aim of this study is to analyze changes in tooth enamel microhardness after application of anchovy substrates by feeding or as a topical fluoridation material. An in vivo study of the lower left incisors of nine Sprague-Dawley rats was conducted. The sample was comprised of baseline and treatment groups, including feeding application, topical application, negative control feeding, and negative control topical groups. The treatment groups were given 5% anchovy substrates through feeding and topical applications. After treatment, tooth samples were extracted from each of the rats for examination, and statistical analyses were performed after determining hardness numbers for enamel surfaces using Vickers microhardness tester. Vickers hardness numbers (VHNs) for anchovy substrate application and consumption by feeding (440.3 ± 24.72) were higher than for the negative control (315.80 ± 17.51). VHNs for the topical application group were higher than for the negative control (347.28 ± 28.56) and for the feeding group. The use of anchovy as a fluoridation material in form of topical application is potentially an effective method for increasing the microhardness of the tooth enamel surface

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.

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.

Synthesis and Live-Cell Imaging of Fluorescent Sterols for Analysis of Intracellular Cholesterol Transport.

PubMed

Modzel, Maciej; Lund, Frederik W; Wüstner, Daniel

2017-01-01

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol ® ; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Positronium signature in organic liquid scintillators for neutrino experiments

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

Franco, D.; Consolati, G.; Trezzi, D.

2011-01-15

Electron antineutrinos are commonly detected in liquid scintillator experiments via inverse {beta} decay by looking at the coincidence between the reaction products: neutrons and positrons. Prior to positron annihilation, an electron-positron pair may form an orthopositronium (o-Ps) state, with a mean lifetime of a few nanoseconds. Even if the o-Ps decay is speeded up by spin-flip or pick-off effects, it may introduce distortions in the photon emission time distribution, crucial for position reconstruction and pulse shape discrimination algorithms in antineutrino experiments. Reversing the problem, the o-Ps-induced time distortion represents a new signature for tagging antineutrinos in liquid scintillator. In thismore » article, we report the results of measurements of the o-Ps formation probability and lifetime for the most used solvents for organic liquid scintillators in neutrino physics (pseudocumene, linear alkyl benzene, phenylxylylethane, and dodecane). We characterize also a mixture of pseudocumene +1.5 g/l of 2,5-diphenyloxazole, a fluor acting as wavelength shifter. In the second part of the article, we demonstrate that the o-Ps-induced distortion of the scintillation photon emission time distributions represent an optimal signature for tagging positrons on an event by event basis, potentially enhancing the antineutrino detection.« less

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

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.

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.

Environmental Releases for Calendar Year 2001

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

DYEKMAN, D L

2002-08-01

This report fulfills the annual reporting requirements of US Department of Energy (DOE) Order 5400.1, General Environmental Protection Program. The report contains tabular data summaries on air emissions and liquid effluents released to the environment as well as nonroutine releases during calendar year (CY) 2001. These releases, bearing radioactive and hazardous substances, were from Bechtel Hanford, Inc. (BHI), CH2M HILL Hanford Group, Inc. (CHG), and Fluor Hanford (FH) managed facilities and activities. These data were obtained from direct sampling and analysis and from estimates based upon approved release factors. This report further serves as a supplemental resource to the Hanfordmore » Site Environmental Report (HSER PNNL-13910), published by the Pacific Northwest National Laboratory. HSER includes a yearly accounting of the impacts on the surrounding populace and environment from major activities at the Hanford Site. HSER also summarizes the regulatory compliance status of the Hanford Site. Tables ES-1 through ES-5 display comprehensive data summaries of CY2001 air emission and liquid effluent releases. The data displayed in these tables compiles the following: Radionuclide air emissions; Nonradioactive air emissions; Radionuclides in liquid effluents discharged to ground; Total volumes and flow rates of radioactive liquid effluents discharged to ground; and Radionuclides discharged to the Columbia River.« less

Bismuth- and lithium-loaded plastic scintillators for gamma and neutron detection (Conference Presentation)

NASA Astrophysics Data System (ADS)

Martinez, H. Paul; Cherepy, Nerine J.; Sanner, Robert D.; Beck, Patrick R.; Swanberg, Eric L.; Payne, Stephen A.

2016-09-01

Plastic scintillators are widely deployed for ionizing radiation detection, as they can be fabricated in large sizes, for high detection efficiency. However commercial plastics are limited in use for gamma spectroscopy, since their photopeak is too weak, due to low Z, and they are also limited in use for neutron detection, since proton recoils are indistinguishable from other ionizing radiation absorption events in standard plastics. We are working on scale up and production of transparent plastic scintillators based on polystyrene (PS) with high loading of bismuth metallorganics for gamma spectroscopy, and with lithium metallorganics for neutron detection. When activated with standard organic fluors, PS scintillators containing 8 wt% bismuth provide energy resolution of 11% at 662 keV. A PS plastic formulation including 1.3 wt% lithium-6 provides a neutron capture peak at 525 keVee, with 11% resolution for the capture peak and 90% efficiency for thermal neutron capture in 2mm thickness. Acknowledgements This work was performed under the auspices of the U.S. DOE by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344, and has been supported by the US DOE National Nuclear Security Administration, Defense Nuclear Nonproliferation Research and Development under Contract No. DE-AC03-76SF00098

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.

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

Mono-Amine Functionalized Phthalocyanines: Mwave-Assisted Solid-Phase Synthesis and Bioconjugation Strategies

PubMed Central

Erdem, S. Sibel; Nesterova, Irina V.; Soper, Steven A.; Hammer, Robert P.

2009-01-01

Phthalocyanines (Pcs) are excellent candidates for use as fluors for near-infrared (near-IR) fluorescent tagging of biomolecules for a wide variety of bioanalytical applications. Mono-functionalized Pcs, having two different types of peripheral substitutents; one for covalent conjugation of the Pc to biomolecules and others to improve the solubility of the macrocycle, ideally suit for the desired applications. To date, difficulties faced during the purification of the mono-functionalized Pcs limited their usage in various types of applications. Herein are reported a new synthetic method for rapid synthesis of the target Pcs and bioconjugation techniques for labeling of the oligonucleotides with the near-IR flours. A novel synthetic route was developed utilizing a hydrophilic, polyethylene glycol-based (PEG) support with an acid labile Rink Amide linker. The Pcs were functionalized with an amine group for covalent conjugation purposes and were decorated with short PEG chains, serving as solubilizing groups. Mwave-assisted solid-phase synthetic method was successfully applied to obtain pure asymmetrically-substituted mono-amine functionalized Pcs in a short period of time. Three different bioconjugation techniques, reductive amination, amidation and Huisgen cycloaddition, were employed for covalent conjugation of Pcs to oligonucleotides. The described μwave-assisted bioconjugation methods give an opportunity to synthesize and isolate the Pc-oligonucleotide conjugate in a few hours. PMID:19911767

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.

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.

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.

Hanford Borehole Geologic Information System (HBGIS) Updated User’s Guide for Web-based Data Access and Export

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

Mackley, Rob D.; Last, George V.; Allwardt, Craig H.

2008-09-24

The Hanford Borehole Geologic Information System (HBGIS) is a prototype web-based graphical user interface (GUI) for viewing and downloading borehole geologic data. The HBGIS is being developed as part of the Remediation Decision Support function of the Soil and Groundwater Remediation Project, managed by Fluor Hanford, Inc., Richland, Washington. Recent efforts have focused on improving the functionality of the HBGIS website in order to allow more efficient access and exportation of available data in HBGIS. Users will benefit from enhancements such as a dynamic browsing, user-driven forms, and multi-select options for selecting borehole geologic data for export. The need formore » translating borehole geologic data into electronic form within the HBGIS continues to increase, and efforts to populate the database continue at an increasing rate. These new web-based tools should help the end user quickly visualize what data are available in HBGIS, select from among these data, and download the borehole geologic data into a consistent and reproducible tabular form. This revised user’s guide supersedes the previous user’s guide (PNNL-15362) for viewing and downloading data from HBGIS. It contains an updated data dictionary for tables and fields containing borehole geologic data as well as instructions for viewing and downloading borehole geologic data.« less

Acetanilide and bromoacetyl-lysine derivatives as activators for human histone deacetylase 8.

PubMed

Mukhtar, Yusif M; Huang, Yajun; Liu, Jiajia; Chen, Di; Zheng, Weiping

2017-06-01

In the current study, seven compounds (i.e. 1-7) were found to be novel activators for the N ε -acetyl-lysine deacetylation reaction catalyzed by human histone deacetylase 8 (HDAC8). When assessed with the commercially available HDAC8 peptide substrate Fluor-de-Lys®-HDAC8 that harbors the unnatural 7-amino-4-methylcoumarin (AMC) residue immediately C-terminal to the N ε -acetyl-lysine residue to be deacetylated, our compounds exhibited comparable activation potency to that of TM-2-51, the strongest HDAC8 activator reported in the current literature. However, when assessed with an AMC-less peptide substrate derived from the native HDAC8 non-histone substrate protein Zinc finger protein ZNF318, while our compounds were all found to be able to activate HDAC8 deacetylation reaction, TM-2-51 was found not to be able to. Our compounds also seemed to be largely selective for HDAC8 over other classical HDACs. Moreover, treatment with the strongest activator among our compounds (i.e. 7) was found to decrease the K M of the above AMC-less HDAC8 substrate, while nearly maintaining the k cat of the HDAC8-catalyzed deacetylation on this substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Do they have to fluoridate water for public usage?].

PubMed

Vargas Marcos, F; Robledo de Dios, T

1993-01-01

In order to facilitate the convenience of fluorating or not the public use waters, a bibliographic review about the positive or negative aspects of this public health measure has been made. The different epidemiologic surveys about dental health conditions in our country, since 1969 till the present time, have been reviewed. From the analysis of these works, it can be deduced that the degree of caries severity index (DMF) is situated at a moderate to low level, according to the classification of the World Health Organization. The advantages, inconvenience, cost and percentage of caries reduction of the different methods to prevent dental caries are studied. It is considered positive to go on with the policy of public use waters fluoration, provided that is is based on a series of prerequisites which guarantee the highest level of effectiveness and efficiency. Among the different objective criteria to fluorate waters, the following ones stand out: a high prevalence of caries, a good system of waters out: a high prevalence of caries, a good system of waters diffusion, a natural fluor concentration lower than 0.7 grams/litre, the cost-benefit analysis, the population size. The elaboration of studies to determine the total quantity of fluorides, ingested by the population from various sources, is recommended.

Mitogenic signaling of urokinase receptor-deficient kidney fibroblasts: actions of an alternative urokinase receptor and LDL receptor-related protein.

PubMed

Zhang, Guoqiang; Cai, Xiaohe; López-Guisa, Jesús M; Collins, Sarah J; Eddy, Allison A

2004-08-01

The urokinase receptor (uPAR) attenuates myofibroblast recruitment and fibrosis in the kidney. This study examined the role of uPAR and its co-receptor LDL receptor-related protein (LRP) in the regulation of kidney fibroblast proliferation and extracellular signal-regulated kinase (ERK) signaling. Compared with uPAR+/+ cells, uPAR-/- kidney fibroblasts were hyperproliferative. UPAR-/- fibroblast proliferation was 60% inhibited by an ERK kinase inhibitor. LRP protein was reduced and extracellular accumulation of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) proteins were greater in uPAR-/- cultures. Addition of functional uPA protein or LRP antisense RNA significantly increased ERK signaling and cell mitosis in both genotypes. Enhanced uPAR-/- fibroblast proliferation was reversed by a recombinant nonfunctional uPA peptide. The density of cell-bound fluor-uPA was similar between uPAR-/- and uPAR+/+ fibroblasts (78 +/- 6 versus 92 +/- 16 units). These data suggest that uPAR-deficient kidney fibroblasts express lower levels of its scavenger co-receptor LRP, resulting in greater extracellular accumulation of uPA and PAI-1. Enhanced proliferation of uPAR-/- fibroblasts seems to be mediated by uPA-dependent ERK signaling via an alternative urokinase receptor.

Advanced Oxidation Protein Products-Modified Albumin Induces Differentiation of RAW264.7 Macrophages into Dendritic-Like Cells Which Is Modulated by Cell Surface Thiols.

PubMed

Garibaldi, Silvano; Barisione, Chiara; Marengo, Barbara; Ameri, Pietro; Brunelli, Claudio; Balbi, Manrico; Ghigliotti, Giorgio

2017-01-10

Local accumulation of Advanced Oxidation Protein Products (AOPP) induces pro-inflammatory and pro-fibrotic processes in kidneys and is an independent predictor of renal fibrosis and of rapid decline of eGFR in patients with chronic kidney disease (CKD). In addition to kidney damage, circulating AOPP may be regarded as mediators of systemic oxidative stress and, in this capacity, they might play a role in the progression of atherosclerotic damage of arterial walls. Atherosclerosis is a chronic inflammatory disease that involves activation of innate and adaptive immunity. Dendritic cells (DCs) are key cells in this process, due to their role in antigen presentation, inflammation resolution and T cell activation. AOPP consist in oxidative modifications of proteins (such as albumin and fibrinogen) that mainly occur through myeloperoxidase (MPO)-derived hypochlorite (HOCl). HOCl modified proteins have been found in atherosclerotic lesions. The oxidizing environment and the shifts in cellular redox equilibrium trigger inflammation, activate immune cells and induce immune responses. Thus, surface thiol groups contribute to the regulation of immune functions. The aims of this work are: (1) to evaluate whether AOPP-proteins induce activation and differentiation of mature macrophages into dendritic cells in vitro; and (2) to define the role of cell surface thiol groups and of free radicals in this process. AOPP-proteins were prepared by in vitro incubation of human serum albumin (HSA) with HOCl. Mouse macrophage-like RAW264.7 were treated with various concentrations of AOPP-HSA with or without the antioxidant N -acetyl cysteine (NAC). Following 48 h of HSA-AOPP treatment, RAW264.7 morphological changes were evaluated by microscopic observation, while markers of dendritic lineage and activation (CD40, CD86, and MHC class II) and allogeneic T cell proliferation were evaluated by flow cytometry. Cell surface thiols were measured by AlexaFluor-maleimide binding, and ROS production was assessed as DCF fluorescence by flow cytometry. HSA-AOPP induced the differentiation of RAW264.7 cells into a dendritic-like phenotype, as shown by morphological changes, by increased CD40, CD86 and MHC class II surface expression and by induction of T cell proliferation. The cell surface thiols dose dependently decreased following HSA-AOPP treatment, while ROS production increased. NAC pre-treatment enhanced the amount of cell surface thiols and prevented their reduction due to treatment with AOPP. Both ROS production and RAW264.7 differentiation into DC-like cells induced by HSA-AOPP were reduced by NAC. Our results highlight that oxidized plasma proteins modulate specific immune responses of macrophages through a process involving changes in the thiol redox equilibrium. We suggest that this mechanism may play a role in determining the rapid progression of the atherosclerotic process observed in CKD patients.

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

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

Probing Cherenkov and Scintillation Light Separation for Next-Generation Neutrino Detectors

NASA Astrophysics Data System (ADS)

Caravaca, J.; Descamps, F. B.; Land, B. J.; Orebi Gann, G. D.; Wallig, J.; Yeh, M.

2017-09-01

The ability to separate Cherenkov and scintillation signals in liquid scintillator detectors would enable outstanding background rejection for next-generation neutrino experiments. Reconstruction of directional information, ring imaging, and sub-Cherenkov threshold detection all have the potential to substantially improve particle and event identification. The Cherenkov-Scintillation Separation (CHESS) experiment uses an array of small, fast photomultipliers (PMTs) and state-of-the-art electronics to demonstrate the reconstruction of a Cherenkov ring in a scintillation medium based on photon hit times and detected charge. This setup has been used to characterize the ability to detect Cherenkov light in a range of target media. We show results with pure organic scintillator (LAB) and the prospects with scintillators with a secondary fluor (LAB/PPO). There are future plans to deploy the newly developed water-based liquid scintillator, a medium with a higher Cherenkov/Scintillation light yield ratio than conventional pure liquid scintillators, enhancing the visibility of the less abundant Cherenkov light in the presence of scintillation light. These results can inform the development of future large-scale detectors, such as the proposed Theia experiment, or other large detectors at underground laboratories such as the far-site of the new Long Baseline Neutrino Facility at the Sanford Underground Research Facility. CHESS detector calibrations and commissioning will be discussed, and the latest results will be presented.

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.

324 Building B-Cell Pressurized Water Reactor Spent Fuel Packaging & Shipment RL Readiness Assessment Final Report [SEC 1 Thru 3

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

HUMPHREYS, D C

A parallel readiness assessment (RA) was conducted by independent Fluor Hanford (FH) and U. S. Department of Energy, Richland Operations Office (RL) team to verify that an adequate state of readiness had been achieved for activities associated with the packaging and shipping of pressurized water reactor fuel assemblies from B-Cell in the 324 Building to the interim storage area at the Canister Storage Building in the 200 Area. The RL review was conducted in parallel with the FH review in accordance with the Joint RL/FH Implementation Plan (Appendix B). The RL RA Team members were assigned a FH RA Teammore » counterpart for the review. With this one-on-one approach, the RL RA Team was able to assess the FH Team's performance, competence, and adherence to the implementation plan and evaluate the level of facility readiness. The RL RA Team agrees with the FH determination that startup of the 324 Building B-Cell pressurized water reactor spent nuclear fuel packaging and shipping operations can safely proceed, pending completion of the identified pre-start items in the FH final report (see Appendix A), completion of the manageable list of open items included in the facility's declaration of readiness, and execution of the startup plan to operations.« less

Antibacterial effects of fluoride varnish compared with chlorhexidine plus fluoride in disabled children.

PubMed

Baygin, Ozgul; Tuzuner, Tamer; Kusgoz, Adem; Senel, Ahmet Can; Tanriver, Mehmet; Arslan, Ipek

2014-01-01

To evaluate the effects of fluoride varnish vs a combination of chlorhexidine-thymol varnish plus a gel containing chlorhexidine and fluoride on oral hygiene and caries prevention in disabled children. Ninety patients aged 3-17 years who were treated under general anaesthesia were randomly assigned into three groups as follows: group 1: Fluor Protector (0.1% fluoride varnish); group 2: Cervitec Plus (1% chlorhexidine- 1% thymol varnish) + Cervitec Gel (0.2% chlorhexidine-0.2% sodium fluoride); group 3: control (toothbrushing only). Mutans streptococci (MS) and lactobacilli (LB) levels, visible plaque index (VPI) and gingival bleeding index (GBI) were evaluated at four stages: T0, before general anaesthesia; T1, one month after treatment; T2, six months after treatment; T3, twelve months after treatment. The data were evaluated using Kruskal-Wallis and Mann-Whitney U-tests (P < 0.05). Groups 1 and 2 showed significantly lower scores than group 3 for all parameters at T1 and T2. No statistically significant difference was detected among any of the the groups at T3 (P > 0.05). The use of materials that include both fluoride and chlorhexidine as routine treatment of children with disability may increase the success of restorations by improving oral hygiene, reduce the need for future restorative treatments and thus the need for general anaesthesia.

Amphibole ceramics: conceptual development and preliminary experiments

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

Lauf, R.J.

1985-08-01

Certain natural silicate minerals, commonly called jade, are well known for their resistance to brittle fracture. One type of jade, nephrite, is a compacted form of the amphibole mineral series tremolite-actinolite. Nephrite is tough because the naturally acicular crystal habit of these amphibole minerals produces an interwoven fibrous microstructure that impedes crack propagation. Object of this work was to duplicate the fibrous microstructure of nephrite by pulverizing natural tremolite or actinolite and then compacting it by hot pressing to form a dense body. Two other materials were also investigated, namely, clinochrysotile (serpentine asbestos) and synthetic fluor-tremolite. For each material, themore » milling characteristics and densification were studied. The resulting microstructures were characterized, and fracture toughness was measured for a limited number of samples. The most ''fibrous'' microstructure was obtained by hot pressing clinochrysotile 15 min a 1000/sup 0/C. Actinolite, hot pressed 15 min at 1100/sup 0/C, had a critical fracture toughness K/sub IC/ = 4.5 MPa m/sup 1/2/, which compares favorably with the toughness of nephrite jade (K/sub IC/ = 3.6). Decomposition of tremolite and actinolite to more stable phases occurred to some degree during hot pressing. Results suggest that hot isostatic pressing at high water vapor pressure should yield a dense product without causing amphibole decomposition. 28 figs., 7 tabs.« less

Synthesis of Single-walled Carbon Nanotubes Coated with Thiol-reactive Gel via Emulsion Polymerization.

PubMed

Nagai, Yukiko; Tsutsumi, Yusuke; Nakashima, Naotoshi; Fujigaya, Tsuyohiko

2018-06-15

Single-walled carbon nanotubes (SWNTs) have unique near-infrared absorption and photoemission properties that are attractive for in vivo biological applications such as photothermal cancer treatment and bioimaging. Therefore, a smart functionalization strategy for SWNTs to create biocompatible surfaces and introduce various ligands to target active cancer cells without losing the unique optical properties of the SWNTs is strongly desired. This paper reports the de-sign and synthesis of a SWNT/gel hybrid containing maleimide groups, which react with various thiol compounds through Michael addition reactions. In this hybrid, the method called carbon nanotube micelle polymerization was used to non-covalently modify the surface of SWNTs with a cross-linked polymer gel layer. This method can form an extremely stable gel layer on SWNTs; such stability is essential for in vivo biological applications. The monomer used to form the gel layer contained a maleimide group, which was protected with furan in endo-form. The resulting hybrid was treated in water to induce deprotection via retro Diels-Alder reaction and then functionalized with thiol com-pounds through Michael addition. The functionalization of the hybrid was explored using a thiol-containing fluores-cent dye as a model thiol and the formation of the SWNT-dye conjugate was confirmed by energy transfer from the dye to SWNTs. Our strategy offers a promising SWNT-based platform for biological functionalization for cancer targeting, imaging, and treatment.

Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells

PubMed Central

Pessoto, Felipe S.; Yokomizo, Cesar H.; Prieto, Tatiana; Fernandes, Cleverton S.; Silva, Alan P.; Kaiser, Carlos R.; Basso, Ernani A.; Nantes, Iseli L.

2015-01-01

A series of thiosemicarbazone (TSC) p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics. PMID:26075034

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.

Visualizing DOM super-spectrum covariance in vanKrevelen space

NASA Astrophysics Data System (ADS)

Fatland, D. R.; Kalawe, J.; Stubbins, A.; Spencer, R. G.; Sleighter, R. L.; Abdulla, H. A.; Dittmar, T.

2011-12-01

We investigate the fate of terrigenous organic matter, DOM exported to the coastal marine environ. Many methods (fluor., FT-ICR-MS, NMR, 13C, lignin, etc) help characterize this DOM. We define a 'super spectrum' as amalgamation of analyses to a data stack and we search for physically significant patterns therein beginning with covariance across 31 samples from six circum-Arctic rivers: The Ob, Kolyma, Mackenzie, Yukon, Lena, and Yenisey sampled five times throughout the year. A vanKrevelen diagram is convenient to view distributions of molecules provided by Fourier Transform Ion Cyclotron Resonance Mass Spectometry (FT-ICR-MS). We augment this distribution space in the vertical dimension, for example to show peak height, molecular mass, principle component weighting or covariance. We use Worldwide Telescope, a virtual globe with strong data support from Microsoft Research to explore covariance results along 3+ dimensions (adding brightness, color and a parameter slide). The results show interesting covariance e.g. between molecules and PARAFAC peaks, a step towards fluorophore and cohort identification in the terrigenous DOM spectrum. Given the geoscience explosion in data volume and data complexity we feel these results should survive beyond the end point of a journal article. We are building a cloud-based Library on the Microsoft Azure platform to support this and subsequent analyses to enable data and methods to carry over and benefit other research groups and objectives.

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.

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

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

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.

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.

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.

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.

Cytotoxicity assessment of porous silicon microparticles for ocular drug delivery.

PubMed

Korhonen, Eveliina; Rönkkö, Seppo; Hillebrand, Satu; Riikonen, Joakim; Xu, Wujun; Järvinen, Kristiina; Lehto, Vesa-Pekka; Kauppinen, Anu

2016-03-01

Porous silicon (PSi) is a promising material for the delivery and sustained release of therapeutic molecules in various tissues. Due to the constant rinsing of cornea by tear solution as well as the short half-life of intravitreal drugs, the eye is an attractive target for controlled drug delivery systems, such as PSi microparticles. Inherent barriers ensure that PSi particles are retained in the eye, releasing drugs at the desired speed until they slowly break down into harmless silicic acid. Here, we have examined the in vitro cytotoxicity of positively and negatively charged thermally oxidized (TOPSi) and thermally carbonized (TCPSi) porous silicon microparticles on human corneal epithelial (HCE) and retinal pigment epithelial (ARPE-19) cells. In addition to ocular assessment under an inverted microscope, cellular viability was evaluated using the CellTiter Blue™, CellTiter Fluor™, and lactate dehydrogenase (LDH) assays. CellTiter Fluor proved to be a suitable assay but due to non-specific and interfering responses, neither CellTiter Blue nor LDH assays should be used when evaluating PSi particles. Our results suggest that the toxicity of PSi particles is concentration-dependent, but at least at concentrations less than 200μg/ml, both positively and negatively charged PSi particles are well tolerated by human corneal and retinal epithelial cells and therefore applicable for delivering drug molecules into ocular tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Simultaneous separation and detection of actinides in acidic solutions using an extractive scintillating resin.

PubMed

Roane, J E; DeVol, T A

2002-11-01

An extractive scintillating resin was evaluated for the simultaneous separation and detection of actinides in acidic solutions. The transuranic extractive scintillating (TRU-ES) resin is composed of an inert macroporous polystyrene core impregnated with organic fluors (diphenyloxazole and 1,4-bis-(4-methyl-5-phenyl-2-oxazolyl)benzene) and an extractant (octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide in tributyl phosphate). The TRU-ES resin was packed into FEP Teflon tubing to produce a flow cell (0.2-mL free column volume), which is placed into a scintillation detection system to obtain pulse height spectra and time series data during loading and elution of actinides onto/from the resin. The alpha-particle absolute detection efficiencies ranged from 77% to 96.5%, depending on the alpha energy and quench. In addition to the on-line analyses, off-line analyses of the effluent can be conducted using conventional detection methods. The TRU-ES resin was applied to the quantification of a mixed radionuclide solution and two actual waste samples. The on-line characterization of the mixed radionuclide solution was within 10% of the reported activities whereas the agreement with the waste samples was not as good due to sorption onto the sample container walls and the oxidation state of plutonium. Agreement between the on-line and off-line analyses was within 35% of one another for both waste samples.

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

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.

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.

Canopy Level Solar Induced Fluorescence for Vegetation in Controlled Experiments

NASA Technical Reports Server (NTRS)

Middleton, E. M.; Corp, L. A.; Campbell, P. K. Entcheva

2007-01-01

Solar induced chlorophyll fluorescence (SIF) was retrieved from high resolution reflectance spectra acquired one meter above saplings of three deciduous tree species during springtime (three weeks after leaf flush) and in late summer when foliage was mature. SIF was determined by application of the Fraunhofer Line Depth (FLD) Principal to above-canopy spectra acquired with an Analytical Spectral Devices (ASD) Fieldspec spectroradiometer (3.2 nm resolution with 1.2 nm sampling interval). SIF retrievals were made at the two atmospheric oxygen (O2) absorption features that occur in the chlorophyll fluorescence (ChlF) region (660 -780 nm). These telluric features are 02V, the broader and deeper feature centered at 760 nm, but located on the shoulder of the far-red ChlF peak at 740 nm; and 023, a narrow feature centered at 688 nm that is positioned near the red ChlF peak at 685 nm. Supporting, coincident leaf level fluorescence, reflectance, photochemical and other measurements were also made. At the leaf level, these measurements included in situ photosynthetic capacity (Pmax) and light adapted total chlorophyll fluorescence (Fs') collected at steady state under high light and controlled chamber conditions (e.g., temperature, PAR, humidity, and COz); optical properties (reflectance, transmittance, absorptance); chlorophyll and carotenoid content; specific leaf mass; carbon (C) and nitrogen (N) content; fluorescence emission spectra at multiple excitation wavelengths; the ChlF contribution to red (R) and far-red (FR) reflectance; fluorescence imagery; and fluorescence excitation-emission matrices (EEMs). The tree species examined were tulip poplar (Liriodendron tulipifera L.), red maple (Acer rubrum L.), and sweetgum (Liquidambar styraczflua L.), and each had been provided four levels of N augmentation (0, 19, 37, and 75 kg Nhectare seasonally) to simulate atmospheric deposition from air pollution. Whole-plant SIF measurements of these species were compared with SIF estimates derived using FluorMOD, a radiative transfer model that includes fluorescence properties of foliage, when provided with our supporting measurements. Simulated values for SIF were also compared with similar estimates made over three years for corn (Zea mays L) crops under N treatments (20, 50,70, and 140 kg Nhectare) and with corn and pepper (Capsicum annuum L.) plants provided dimethyl urea (DCMU, 0 and 5 x 10" M) which were grown in pots and grouped in artificial canopies for spectral measurements. For near-surface measurements of healthy and stressed vegetation, red SIF retrieved at 688 nm (023) varied between 2-7 mW/m(exp 2)/nm/sr while far-red SIF retrieved at 760 nm (O2A) varied between 0.5 and 4.0 mW/m(exp 2)/nm/sr. Typical values for the SIF red/far-red ratio ranged between 1.75 and 4.0. Relationships of SIF to spectral reflectance indices and foliar photochemical indices were examined, such as photosynthetic light use efficiency. Initial investigations of the variability in these measurements at the same leaf temperature for sunlit and shaded foliage showed us that shaded foliage produced higher ChlF and light use efficiency. Other factors that affect SIF determinations are discussed. These results will assist in determine the expected intensity of the SIF signal from vegetation near the surface, as well as to identify enhancements needed for FluorMOD (or other such models), and will assist in determining relationships of SIF to reflectance indices and carbon dynamics.

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.

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.

Hydrothermal karst and associated breccias in Neoproterozoic limestone from the Barker-Villa Cacique area (Tandilia belt), Argentina

NASA Astrophysics Data System (ADS)

Dristas, Jorge A.; Martínez, Juan C.; van den Kerkhof, Alfons M.; Massonne, Hans-Joachim; Theye, Thomas; Frisicale, María C.; Gregori, Daniel A.

2017-07-01

In the Barker-Villa Cacique area (Tandilia belt), remarkable megabreccias, limestone breccias and phosphate-bearing breccias hosted in black limestone and along the contact with the upper section of the sedimentary succession are exposed. These rocks are the result of extensive hydrothermal alteration of the original micritic limestone and other fine-grained clastic sediments. Typical alteration minerals are sericite, chlorite, interstratified chlorite/K-white mica, kaolinite, dickite, pyrite, chalcopyrite, goethite, quartz, calcite, Fe-calcite, dolomite, ankerite, fluor-apatite, barite and aluminium-phosphate-sulfate (APS) minerals. Quartz and calcite cements from hydraulic breccias in the limestone contain low-salinity aqueous fluid inclusions. Corresponding homogenization temperatures display 200-220 °C and 110-140 °C in hydrothermal quartz, and 130-150 °C in late calcite cement. Carbon and oxygen stable isotope analyses of carbonates from the Loma Negra quarry (LNQ) support the major role of hydrothermal activity. A significant difference was found between δ18Ocar values from unaltered micritic limestone (ca. 23.8‰ SMOW) and secondary calcite (ca. 18.5‰ SMOW). The lower δ18Ocar values are interpreted as a result of calcite precipitation from hot hydrothermal fluids. At a late stage, the hydrothermal fluid containing H2S mixed with descending and oxidizing meteoric waters. Circulation of the ensuing acid fluids resulted in the partly dissolution and collapse brecciation of the Loma Negra Formation. The hydrothermal stage can be tentatively dated ca. 590-620 Ma corresponding to the Brasiliano orogeny.

Preconditioning results in S-nitrosylation of proteins involved in regulation of mitochondrial energetics and calcium transport.

PubMed

Sun, Junhui; Morgan, Meghan; Shen, Rong-Fong; Steenbergen, Charles; Murphy, Elizabeth

2007-11-26

Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca2+ channel alpha1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca2+-ATPase, alpha-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase alpha1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart.

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.

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

[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.

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

Efficacy of fluoride varnish for preventing white spot lesions and gingivitis during orthodontic treatment with fixed appliances-a prospective randomized controlled trial.

PubMed

Kirschneck, Christian; Christl, Jan-Joachim; Reicheneder, Claudia; Proff, Peter

2016-12-01

The development of white spot lesions around orthodontic brackets and gingivitis is a common problem during orthodontic treatment with fixed appliances. This prospective randomized double-blind controlled clinical trial investigated the preventive efficacy of a one-time application of two commonly used fluoride varnishes in patients with low to moderate caries risk. Ninety adolescent orthodontic patients with a low to moderate caries risk were prospectively randomized to three groups of 30 patients each: (1) standardized dental hygiene with fluoride toothpaste and one-time application of placebo varnish (control) or (2) of elmex® fluid or (3) of Fluor Protector S on all dental surfaces at the start of fixed therapy. The extent of enamel demineralization and gingivitis was determined with the ICDAS and the gingivitis index (GI) at baseline and after 4, 12, and 20 weeks. Each treatment group showed a significant increase of the ICDAS index, but not of the GI over the course of time with no significant intergroup differences detectable. A one-time application of fluoride varnish at the start of orthodontic treatment did not provide any additional preventive advantage over sufficient dental hygiene with fluoride toothpaste with regard to formation of white spots and gingivitis in patients with a low to moderate caries risk. In dental practice, patients often receive an application of fluoride varnish at the start of orthodontic treatment with fixed appliances. However, the efficacy of this procedure is still unclear.

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.

Crystal structure and electrochemical properties of [Ni(bztmpen)(CH3CN)](BF4)2 {bztmpen is N-benzyl-N,N',N'-tris-[(6-methyl-pyridin-2-yl)meth-yl]ethane-1,2-di-amine}.

PubMed

Chen, Lin; Ren, Gan; Guo, Yakun; Sang, Ge

2017-06-01

The mononuclear nickel title complex (acetonitrile-κ N ){ N -benzyl- N , N ', N '-tris-[(6-methyl-pyridin-2-yl)meth-yl]ethane-1,2-di-amine}-nickel(II) bis-(tetra-fluor-ido-borate), [Ni(C 30 H 35 N 5 )(CH 3 CN)](BF 4 ) 2 , was prepared from the reaction of Ni(BF 4 ) 2 ·6H 2 O with N -benzyl- N , N ', N '-tris-[(6-methyl-pyridin-2-yl)meth-yl]ethane-1,2-di-amine ( bztmpen ) in aceto-nitrile at room temperature. With an open site occupied by the aceto-nitrile mol-ecule, the nickel(II) atom is chelated by five N-atom sites from the ligand and one N atom from the ligand, showing an overall octa-hedral coordination environment. Compared with analogues where the 6-methyl substituent is absent, the bond length around the Ni 2+ cation are evidently longer. Upon reductive dissociation of the acetro-nitrile mol-ecule, the title complex has an open site for a catalytic reaction. The title complex has two redox couples at -1.50 and -1.80 V ( versus F c +/0 ) based on nickel. The F atoms of the two BF 4 - counter-anions are split into two groups and the occupancy ratios refined to 0.611 (18):0.389 (18) and 0.71 (2):0.29 (2).

Dual-Color Fluorescence Imaging of EpCAM and EGFR in Breast Cancer Cells with a Bull's Eye-Type Plasmonic Chip.

PubMed

Izumi, Shota; Yamamura, Shohei; Hayashi, Naoko; Toma, Mana; Tawa, Keiko

2017-12-19

Surface plasmon field-enhanced fluorescence microscopic observation of a live breast cancer cell was performed with a plasmonic chip. Two cell lines, MDA-MB-231 and Michigan Cancer Foundation-7 (MCF-7), were selected as breast cancer cells, with two kinds of membrane protein, epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR), observed in both cells. The membrane proteins are surface markers used to differentiate and classify breast cancer cells. EGFR and EpCAM were detected with Alexa Fluor ® 488-labeled anti-EGFR antibody (488-EGFR) and allophycocyanin (APC)-labeled anti-EpCAM antibody (APC-EpCAM), respectively. In MDA-MB231 cells, three-fold plus or minus one and seven-fold plus or minus two brighter fluorescence of 488-EGFR were observed on the 480-nm pitch and the 400-nm pitch compared with that on a glass slide. Results show the 400-nm pitch is useful. Dual-color fluorescence of 488-EGFR and APC-EpCAM in MDA-MB231 was clearly observed with seven-fold plus or minus two and nine-fold plus or minus three, respectively, on the 400-nm pitch pattern of a plasmonic chip. Therefore, the 400-nm pitch contributed to the dual-color fluorescence enhancement for these wavelengths. An optimal grating pitch of a plasmonic chip improved a fluorescence image of membrane proteins with the help of the surface plasmon-enhanced field.

Office of Inspector General audit report on credit card usage at the Ohio Field Office and the Fernald and Miamisburg Environmental Management Projects

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

NONE

1999-03-01

In 1994 the Department of Energy (Department) obtained the services of Rocky Mountain BankCard System, through the use of a General Services Administration contract, as a means for the Department and its contractors to make small purchases. The use of credit cards was expected to simplify small purchase procedures and improve cash management. The Ohio Field Office (Field Office) uses the credit card system and oversees usage by its area offices. Contractors under the Field Office also use the credit card system to make small purchases. The Office of Inspector General (OIG) has issued one audit report concerning the usemore » of credit cards. In April 1996, the OIG issued Report WR-B-96-06, Audit of Bonneville Power Administration`s Management of Information Resources. The audit concluded that improvements could be made in implementing credit card and property procedures in Bonneville`s management of computer-related equipment. Specifically, many credit card purchases were made by employees whose authority to buy was not properly documented, and the purchasing files often lacked invoices that would show what was purchased. Additionally, some cardholders split purchases to avoid credit card limits. The objective of this audit was to determine whether the Field Office, Fernald and Miamisburg Environmental Management Projects, Fluor Daniel, and B and W were using credit cards for the appropriate purposes and within the limitations established by Federal and Departmental regulations.« less

Effect of two fluoride varnishes on the color stability of three resin-based restorative materials: an in vitro study.

PubMed

Tafaroji, Raha; Biria, Mina; Ameri, Farhad; Torabzadeh, Hassan; Qahari, Pasha; Akbarzadeh Baghban, Alireza

2016-11-01

The aim of the present study was to evaluate the effect of two fluoride varnishes on color stability of three resin-based restorative materials. Fifty-four discs (14.5 × 1.7 mm) were fabricated from A2 and A3 shades of a compomer (F2000), a flowable composite (Z350), and a hybrid composite (Z250), and incubated at 37°C for 48 h. Dura Shield (colored) and Fluor Protector (colorless) fluoride varnishes were applied onto the discs. The coating was cleaned using a low-speed handpiece and nylon bristle brush after 24 h of storage in distilled water. A second coating was then applied. A control group with no coating was immersed in distilled water and used. The CIE L*a*b* color scale was measured before the treatments and following each cleaning utilizing a spectrophotometer. The colored fluoride varnish exhibited the highest overall color change (∆E) after the first and the second cleaning procedures in all the materials. Among these, the greatest color change was observed in the A3 shade of F2000, followed by the A3 shade of Z-250. The ∆E was less than 3.3 in all groups, and was therefore clinically acceptable. Color changes following the application of fluoride varnishes were found to be clinically acceptable in all groups. © 2015 Wiley Publishing Asia Pty Ltd.

Natural dye sensitizer from cassava (Manihot utilissima) leaves extract and its adsorption onto TiO2 photo-anode

NASA Astrophysics Data System (ADS)

Nurlela; Wibowo, R.; Gunlazuardi, J.

2017-04-01

Interaction between TiO2 and dyes sensitizer have been studied. The chlorophyll presents in the crude leave extract (CLE-dye) from cassava (Manihot utilissima) was immobilized on to the photo-anode, consists of TiO2 supported by fluor doped Tin oxide (SnO2-F) Glass. The TiO2 was prepared by Rapid Breakdown Anodization (RBA) method then immobilized on to glass coated by SnO2-F using doctor blade technique, to give CLE-dye/TiO2/SnO2-F/Glass photo-anode. The prepared photo-anode was characterized by UV-Vis-DRS, FTIR, XRD, SEM, electrochemical and spectro-electrochemical systems. In this study, the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energy level of the CLE-dye were empirically determined by cyclic voltammetry method, while spectro-electro-chemistry method was used to determine the coefficient of degradation and formation of the dyes, and diffusion coefficient of the hole recombination as well. Good anchoring between TiO2 with dye extracts (CLE-dye) can be seen from value of dye LUMO energy level (-4.26 eV), which is approaching the conduction band of TiO2 (-4.3 eV). The coefficient of degradation and formation of the CLE-dye showed a quasi reversible and diffusion coefficient hole recombination values were small, indicated that it is quite suitable as a sensitizer in a dyes sensitized solar cell.

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

Analysis of short tandem repeat polymorphisms using infrared fluorescence with M18 tailed primers

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

Oetting, W.S.; Wiesner, G.; Laken, S.

The use of short tandem repeat polymorphisms (STRPs) are becoming increasingly important as markers for linkage analysis due to their large numbers of the human genome and their high degree of polymorphism. Fluorescence based detection of the STRP pattern using the LI-COR model 4000S automated DNA sequencer eliminates the need for radioactivity and produces a digitized image that can be used for the analysis of the polymorphisms. In an effort to reduce the cost of STRP analysis, we have synthesized primers with a 19 bp extension complementary to the sequence of the M13 primer on the 5{prime} end of onemore » of the two primers used in the amplification of the STRP instead of using primers with direct conjugation of the infrared fluorescent dye. Up to 5 primer pairs can be multiplexed together with the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye. Comparisons between primers that have been directly conjugated to the fluor with those having the M13 sequence extension show no difference in the ability to determine the STRP pattern. At present, the entire Weber 4A set of STRP markers is available with the M13 5{prime} extension. We are currently using this technique for linkage analysis of familial breast cancer and asthma. The combination of STRP analysis using fluorescence detection will allow this technique to be fully automated for allele scoring and linkage analysis.« less

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.

Surface biofunctionalization of β-TCP blocks using aptamer 74 for bone tissue engineering.

PubMed

Ardjomandi, N; Huth, J; Stamov, D R; Henrich, A; Klein, C; Wendel, H-P; Reinert, S; Alexander, D

2016-10-01

Successful bone regeneration following oral and maxillofacial surgeries depends on efficient functionalization strategies that allow the recruitment of osteogenic progenitor cells at the tissue/implant interface. We have previously identified aptamer 74, which exhibited a binding affinity for osteogenically induced jaw periosteal cells (JPCs). In the present study, this aptamer was used for the surface biofunctionalization of β-tricalcium phosphate (β-TCP) blocks. Atomic force microscopy (AFM) measurements showed increased binding activity of aptamer 74 towards osteogenically induced JPCs compared to untreated controls. The immobilization efficiency of aptamer 74 was analyzed using the QuantiFluor ssDNA assay for 2D surfaces and by amino acid analysis for 3D β-TCP constructs. Following the successful immobilization of aptamer 74 in 2D culture wells and on 3D constructs, in vitro assays showed no significant differences in cell proliferation compared to unmodified surfaces. Interestingly, JPC mineralization was significantly higher on the 2D surfaces and higher cell adhesion was detected on the 3D constructs with immobilized aptamer. Herein, we report an established, biocompatible β-TCP matrix with surface immobilization of aptamer 74, which enhances properties such as cell adhesion on 3D constructs and mineralization on 2D surfaces. Further studies need to be performed to improve the immobilization efficiency and to develop a suitable approach for JPC mineralization growing within 3D β-TCP constructs. Copyright © 2016 Elsevier B.V. All rights reserved.

Voltage-sensitive rhodol with enhanced two-photon brightness.

PubMed

Kulkarni, Rishikesh U; Kramer, Daniel J; Pourmandi, Narges; Karbasi, Kaveh; Bateup, Helen S; Miller, Evan W

2017-03-14

We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using two-photon (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue.

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.

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.

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.

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.

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.

Ecological Characterization Data for the 2004 Composite Analysis

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

Downs, Janelle L.; Simmons, Mary A.; Stegen, Jennifer A.

2004-11-01

A composite analysis is required by U.S. Department of Energy (DOE) Order 435.1 to ensure public safety through the management of active and planned low-level radioactive waste disposal facilities associated with the Hanford Site. The original Hanford Site Composite Analysis of 1998 must be revised and submitted to DOE Headquarters (DOE-HQ) in 2004 because of revisions to waste site information in the 100, 200, and 300 Areas, updated performance assessments and environmental impact statements (EIS), changes in inventory estimates for key sites and constituents, and a change in the definition of offsite receptors. Beginning in fiscal year (FY) 2003, themore » DOE Richland Operations Office (DOE-RL) initiated activities, including the development of data packages, to support the 2004 Composite Analysis. This report describes the data compiled in FY 2003 to support ecological site assessment modeling for the 2004 Composite Analysis. This work was conducted as part of the Characterization of Systems Task of the Groundwater Remediation Project (formerly the Groundwater Protection Program) managed by Fluor Hanford, Inc., Richland, Washington. The purpose of this report is to provide summaries of the characterization information and available spatial data on the biological resources and ecological receptors found in the upland, riparian, aquatic, and island habitats on the Hanford Site. These data constitute the reference information used to establish parameters for the ecological risk assessment module of the System Assessment Capability and other assessment activities requiring information on the presence and distribution of biota on the Hanford Site.« less

EVALUATION OF VADOSE ZONE TREATMENT TECHNOLOGIES TO IMMOBILIZE TECHNETIUM-99

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

PETERSEN, S.W.

2006-03-15

The Hanford Site End State Vision document (DOE/RL-2003-59) states: ''There should be an aggressive plan to develop technology for remediation of the contamination that could get to the groundwater (particularly the technetium [{sup 99}Tc])''. In addition, there is strong support from the public and regulatory agencies for the above statement, with emphasis on investigation of treatment alternatives. In July 2004, PNNL completed a preliminary evaluation of remediation technologies with respect to their effectiveness and implementability for immobilization of {sup 99}Tc beneath the BC Cribs in the 200 West Area (Truex, 2004). As a result of this evaluation, PNNL recommended treatabilitymore » testing of in situ soil desiccation, because it has the least uncertainty of those technologies evaluated in July 2004 (Treatability Test Outline, September 30, 2004). In 2005, DOE-RL and Fluor Hanford convened an independent technical panel to review alternative remediation technologies, including desiccation, at a three-day workshop in Richland, Washington. The panel was composed of experts in vadose-zone transport, infiltration control, hydrology, geochemistry, environmental engineering, and geology. Their backgrounds include employment in academia, government laboratories, industry, and consulting. Their review, presented in this document, is based upon written reports from Hanford, oral presentations from Hanford staff, and each panel members' years of experience in their particular field of expertise. The purpose of this report is to document the panel's evaluation of various treatment alternatives with potential for minimizing contaminant migration in the deep vadose zone at the Department of Energy Hanford Site. The panel was tasked with assessing the most viable and practical approach and making recommendations for testing. The evaluation of vadose-zone treatment alternatives was conducted to be broadly applicable at a variety of locations at Hanford. However, because of limitations of time, the panel was asked to focus on one example, {sup 99}Tc contamination below the BC Cribs and Trenches. It is well recognized that conditions at BC Cribs and Trenches are not the same as those at other Hanford locations, but it was selected so that the panel could develop an understanding of site conditions at one location. The recommendations in this report are not intended to address the regulatory decision process for this site.« less

Observations of Jupiter From Cassini, Galileo and Hst

NASA Astrophysics Data System (ADS)

West, R. A.

This report summarizes recent scientific results for JupiterSs atmosphere from instru- ments sensing ultraviolet and visible wavelengths (to the CCD sensitivity limit near 1000 nm) on the Hubble Space Telescope and the Galileo and Cassini spacecraft. Most prominent among these have been images of the aurora which show the morphology and temporal behavior of the main oval as well as active regions inside the oval and Galilean satellite flux tube and wake interactions. Galileo and especially Cassini ul- traviolet spectrometers added to this picture by revealing auroral brightenings and, along with in situ plasma instruments establish a link between solar wind events and jovian auroral activity. Cassini spectra of the quiescent day and night glow provide compelling evidence for a dominating influence of soft electron excitation (probably secondary electrons) at high altitude and limit the possible contribution of fluores- cence to about 15 percent of the short-wave UV flux. Although fluorescence does not dominate the emission process sunlight is the ultimate source of the emission via photo excitation of vibrationally excited H2. Energetic H2 molecules can be excited by more abundant longer wavelength solar photons. This new insight goes a long way toward resolving the mystery of how the abundant UV flux is produced. At longer wave- lengths (200-300 nm) images by HST and by the Cassini ISS instrument reveal haze morphology and motions in the polar stratosphere. The most striking new discovery in that realm proved to be the formation and evolution of a large dark oval near latitude +60, about the same size and shape as JupiterSs Great Red Spot but ephemeral and invisible at longer wavelengths. Galileo and Cassini made new observations of light- ning. Lightning on the night side can be mapped to cloud features seen on the day side and illuminated by light from Io on the night side. High spatial resolution images in methane bands made by Galileo and Cassini are contributing to new analyses of cloud structure. Galileo and Cassini also made measurements of how the clouds polarize the light. Only a small fraction of these data have been analyzed to date. This work summarizes results from individuals and instrument teams on the Hubble Space Tele- scope, and the Galileo and Cassini spacecraft. Part of this work was performed at the Jet Propulsion Laboratory of the California Institute of Technology and was funded by NASA.

First synthesis of 2'-deoxyfluoropuromycin analogues: experimental insight into the mechanism of the Staudinger reaction.

PubMed

Charafeddine, Adib; Dayoub, Wissam; Chapuis, Hubert; Strazewski, Peter

2007-01-01

The N(6),N(6)-dedimethyl-2'-deoxyfluoro analogue of puromycin (= 3'-deoxy-N(6),N(6)-dimethyl-3'-[O-methyltyrosylamido]adenosine), its 2',3'-regioisomer and a 3'-cytidyl-5'-(2'-deoxyfluoro)puromycyl dinucleotide analogue were synthesized following an approach involving i) the diastereospecific nitrite-assisted formation of a lyxo nucleosidic 2',3'-epoxide from an adenosine-2',3'-ditriflate derivative in a biphasic solvent mixture; ii) the regio- and stereoselective epoxide ring opening with sodium azide under mildly acidic aqueous conditions, iii) the stereospecific introduction of the fluor atom using DAST and iv) the reaction between the nucleosidyl or dinucleotidyl azide and an active ester of the N-protected amino acid using highly efficient solution conditions for the Staudinger-Vilarrasa coupling, to obtain the corresponding carboxamide directly from the in situ formed iminophosphorane. This coupling reaction furnished sterically quite demanding amides in 94 % isolated yields under very mild conditions and should therefore be of a more general value. Under certain reaction conditions we isolated (amino)acyltriazene derivatives from which dinitrogen was not eliminated. These secondary products are trapped and stabilized witnesses of the first intermediate of the Staudinger reaction, the phosphatriazenes (phosphazides, triazaphosphadienes) which usually eliminate dinitrogen in situ and rapidly rearrange into iminophosphoranes, unless they are derived from conjugated or sterically bulky azides and phosphines. The acyltriazenes could either be thermally decomposed or converted to the corresponding N-alkyl carboxamides through proton-assisted elimination of dinitrogen. All compounds were carefully characterized through MS spectrometry, (1)H, (19)F, (31)P and (13)C NMR spectroscopy.

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.

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.

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.

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.

Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization

PubMed Central

DeLong, Edward F.; Taylor, Lance Trent; Marsh, Terence L.; Preston, Christina M.

1999-01-01

Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4′,6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50–56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 105/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or “ghosts,” as was suggested in a previous report. PMID:10584017

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.

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin.

PubMed Central

Waud, J P; Bermúdez Fajardo, A; Sudhaharan, T; Trimby, A R; Jeffery, J; Jones, A; Campbell, A K

2001-01-01

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (alpha-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20--60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as 'Rainbow' proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2 microM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor. PMID:11463339

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

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.

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.

Voltage-sensitive rhodol with enhanced two-photon brightness

PubMed Central

Kulkarni, Rishikesh U.; Kramer, Daniel J.; Pourmandi, Narges; Karbasi, Kaveh; Bateup, Helen S.

2017-01-01

We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using two-photon (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue. PMID:28242676

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.

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.

Development of consistent hazard controls for DOE transuranic waste operations

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

Woody, W.J.

2007-07-01

This paper describes the results of a re-engineering initiative undertaken with the Department of Energy's (DOE) Office of Environmental Management (EM) in order to standardize hazard analysis assumptions and methods and resulting safety controls applied to multiple transuranic (TRU) waste operations located across the United States. A wide range of safety controls are historically applied to transuranic waste operations, in spite of the fact that these operations have similar operational characteristics and hazard/accident potential. The re-engineering effort supported the development of a DOE technical standard with specific safety controls designated for accidents postulated during waste container retrieval, staging/storage, venting, onsitemore » movements, and characterization activities. Controls cover preventive and mitigative measures; include both hardware and specific administrative controls; and provide protection to the facility worker, onsite co-located workers and the general public located outside of facility boundaries. The Standard development involved participation from all major DOE sites conducting TRU waste operations. Both safety analysts and operations personnel contributed to the re-engineering effort. Acknowledgment is given in particular to the following individuals who formed a core working group: Brenda Hawks, (DOE Oak Ridge Office), Patrice McEahern (CWI-Idaho), Jofu Mishima (Consultant), Louis Restrepo (Omicron), Jay Mullis (DOE-ORO), Mike Hitchler (WSMS), John Menna (WSMS), Jackie East (WSMS), Terry Foppe (CTAC), Carla Mewhinney (WIPP-SNL), Stephie Jennings (WIPP-LANL), Michael Mikolanis (DOESRS), Kraig Wendt (BBWI-Idaho), Lee Roberts (Fluor Hanford), and Jim Blankenhorn (WSRC). Additional acknowledgment is given to Dae Chung (EM) and Ines Triay (EM) for leadership and management of the re-engineering effort. (authors)« less

Interferometric Gravity Darkening Observations of Vega with the CHARA Array

NASA Astrophysics Data System (ADS)

Aufdenberg, J. P.; Merand, A.; Coude Foresto, V.; Absil, O.; Di Folco, E.; Kervella, P.; Ridgway, S. T.; Sturmann, J.; Sturmann, L.; ten Brummelaar, T. A.; Turner, N. H.; Berger, D. H.; McAlister, H. A.

2005-12-01

We have obtained high-precision interferometric measurements of the A0 V standard star Vega with the Center for High Angular Resolution Astronomy (CHARA) Array and the Fiber Linked Unit for Optical Recombination (FLUOR) beam combiner in the K' band at projected baselines between 103 m and 273 m. The measured squared visibility amplitudes beyond the first lobe are significantly weaker than expected for a slowly rotating star and provide strong evidence for the model of Vega as a rapidly rotating star viewed very nearly pole on. We have constructed a Roche-von Zeipel gravity-darkened model atmosphere which is in generally good agreement with both our interferometric data and archival spectrophotometry. Our model indicates Vega is rotating at ˜92% of its angular break-up rate with an equatorial velocity of ˜275 km s-1. We find a polar effective temperature of ˜10150 K and a pole-to-equator effective temperature difference of ˜2500 K, much larger than the ˜300 K derived by Gulliver, Hill, and Adelman. Our model suggests that Vega's cool equatorial atmosphere may have significant convective flux and predicts a significantly cooler spectral energy distribution for Vega as seen by its surrounding debris disk. This work was performed in part under contract with the Jet Propulsion Laboratory (JPL) funded by NASA through the Michelson Fellowship Program. JPL is managed for NASA by the California Institute of Technology. The CHARA Array is operated by the Center for High Angular Resolution Astronomy with support from Georgia State University and the National Science Foundation, the Keck Foundation and the Packard Foundation.

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

Dendritic Cytoskeletal Architecture Is Modulated by Combinatorial Transcriptional Regulation in Drosophila melanogaster.

PubMed

Das, Ravi; Bhattacharjee, Shatabdi; Patel, Atit A; Harris, Jenna M; Bhattacharya, Surajit; Letcher, Jamin M; Clark, Sarah G; Nanda, Sumit; Iyer, Eswar Prasad R; Ascoli, Giorgio A; Cox, Daniel N

2017-12-01

Transcription factors (TFs) have emerged as essential cell autonomous mediators of subtype specific dendritogenesis; however, the downstream effectors of these TFs remain largely unknown, as are the cellular events that TFs control to direct morphological change. As dendritic morphology is largely dictated by the organization of the actin and microtubule (MT) cytoskeletons, elucidating TF-mediated cytoskeletal regulatory programs is key to understanding molecular control of diverse dendritic morphologies. Previous studies in Drosophila melanogaster have demonstrated that the conserved TFs Cut and Knot exert combinatorial control over aspects of dendritic cytoskeleton development, promoting actin and MT-based arbor morphology, respectively. To investigate transcriptional targets of Cut and/or Knot regulation, we conducted systematic neurogenomic studies, coupled with in vivo genetic screens utilizing multi-fluor cytoskeletal and membrane marker reporters. These analyses identified a host of putative Cut and/or Knot effector molecules, and a subset of these putative TF targets converge on modulating dendritic cytoskeletal architecture, which are grouped into three major phenotypic categories, based upon neuromorphometric analyses: complexity enhancer, complexity shifter, and complexity suppressor. Complexity enhancer genes normally function to promote higher order dendritic growth and branching with variable effects on MT stabilization and F-actin organization, whereas complexity shifter and complexity suppressor genes normally function in regulating proximal-distal branching distribution or in restricting higher order branching complexity, respectively, with spatially restricted impacts on the dendritic cytoskeleton. Collectively, we implicate novel genes and cellular programs by which TFs distinctly and combinatorially govern dendritogenesis via cytoskeletal modulation. Copyright © 2017 by the Genetics Society of America.

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

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

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.

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).

The Mars Hand Lens Imager (MAHLI) aboard the Mars rover, Curiosity

NASA Astrophysics Data System (ADS)

Edgett, K. S.; Ravine, M. A.; Caplinger, M. A.; Ghaemi, F. T.; Schaffner, J. A.; Malin, M. C.; Baker, J. M.; Dibiase, D. R.; Laramee, J.; Maki, J. N.; Willson, R. G.; Bell, J. F., III; Cameron, J. F.; Dietrich, W. E.; Edwards, L. J.; Hallet, B.; Herkenhoff, K. E.; Heydari, E.; Kah, L. C.; Lemmon, M. T.; Minitti, M. E.; Olson, T. S.; Parker, T. J.; Rowland, S. K.; Schieber, J.; Sullivan, R. J.; Sumner, D. Y.; Thomas, P. C.; Yingst, R. A.

2009-08-01

The Mars Science Laboratory (MSL) rover, Curiosity, is expected to land on Mars in 2012. The Mars Hand Lens Imager (MAHLI) will be used to document martian rocks and regolith with a 2-megapixel RGB color CCD camera with a focusable macro lens mounted on an instrument-bearing turret on the end of Curiosity's robotic arm. The flight MAHLI can focus on targets at working distances of 20.4 mm to infinity. At 20.4 mm, images have a pixel scale of 13.9 μm/pixel. The pixel scale at 66 mm working distance is about the same (31 μm/pixel) as that of the Mars Exploration Rover (MER) Microscopic Imager (MI). MAHLI camera head placement is dependent on the capabilities of the MSL robotic arm, the design for which presently has a placement uncertainty of ~20 mm in 3 dimensions; hence, acquisition of images at the minimum working distance may be challenging. The MAHLI consists of 3 parts: a camera head, a Digital Electronics Assembly (DEA), and a calibration target. The camera head and DEA are connected by a JPL-provided cable which transmits data, commands, and power. JPL is also providing a contact sensor. The camera head will be mounted on the rover's robotic arm turret, the DEA will be inside the rover body, and the calibration target will be mounted on the robotic arm azimuth motor housing. Camera Head. MAHLI uses a Kodak KAI-2020CM interline transfer CCD (1600 x 1200 active 7.4 μm square pixels with RGB filtered microlenses arranged in a Bayer pattern). The optics consist of a group of 6 fixed lens elements, a movable group of 3 elements, and a fixed sapphire window front element. Undesired near-infrared radiation is blocked using a coating deposited on the inside surface of the sapphire window. The lens is protected by a dust cover with a Lexan window through which imaging can be ac-complished if necessary, and targets can be illuminated by sunlight or two banks of two white light LEDs. Two 365 nm UV LEDs are included to search for fluores-cent materials at night. DEA and Onboard Processing. The DEA incorpo-rates the circuit elements required for data processing, compression, and buffering. It also includes all power conversion and regulation capabilities for both the DEA and the camera head. The DEA has an 8 GB non-volatile flash memory plus 128 MB volatile storage. Images can be commanded as full-frame or sub-frame and the camera has autofocus and autoexposure capa-bilities. MAHLI can also acquire 720p, ~7 Hz high definition video. Onboard processing includes options for Bayer pattern filter interpolation, JPEG-based compression, and focus stack merging (z-stacking). Malin Space Science Systems (MSSS) built and will operate the MAHLI. Alliance Spacesystems, LLC, designed and built the lens mechanical assembly. MAHLI shares common electronics, detector, and software designs with the MSL Mars Descent Imager (MARDI) and the 2 MSL Mast Cameras (Mastcam). Pre-launch images of geologic materials imaged by MAHLI are online at: http://www.msss.com/msl/mahli/prelaunch_images/.

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.

Ischaemic preconditioning preferentially increases protein S-nitrosylation in subsarcolemmal mitochondria

PubMed Central

Sun, Junhui; Nguyen, Tiffany; Aponte, Angel M.; Menazza, Sara; Kohr, Mark J.; Roth, David M.; Patel, Hemal H.; Murphy, Elizabeth; Steenbergen, Charles

2015-01-01

Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischaemic preconditioning (IPC)-induced acute cardioprotection. The majority of proteins that show increased SNO following IPC are localized to the mitochondria, and our recent studies suggest that caveolae transduce acute NO/SNO cardioprotective signalling in IPC hearts. Due to the close association between subsarcolemmal mitochondria (SSM) and the sarcolemma/caveolae, we tested the hypothesis that SSM, rather than the interfibrillar mitochondria (IFM), are major targets for NO/SNO signalling derived from caveolae-associated eNOS. Following either control perfusion or IPC, SSM and IFM were isolated from Langendorff perfused mouse hearts, and SNO was analysed using a modified biotin switch method with fluorescent maleimide fluors. In perfusion control hearts, the SNO content was higher in SSM compared with IFM (1.33 ± 0.19, ratio of SNO content Perf-SSM vs. Perf-IFM), and following IPC SNO content significantly increased preferentially in SSM, but not in IFM (1.72 ± 0.17 and 1.07 ± 0.04, ratio of SNO content IPC-SSM vs. Perf-IFM, and IPC-IFM vs. Perf-IFM, respectively). Consistent with these findings, eNOS, caveolin-3, and connexin-43 were detected in SSM, but not in IFM, and IPC resulted in a further significant increase in eNOS/caveolin-3 levels in SSM. Interestingly, we did not observe an IPC-induced increase in SNO or eNOS/caveolin-3 in SSM isolated from caveolin-3−/− mouse hearts, which could not be protected with IPC. In conclusion, these results suggest that SSM may be the preferential target of sarcolemmal signalling-derived post-translational protein modification (caveolae-derived eNOS/NO/SNO), thus providing an important role in IPC-induced cardioprotection. PMID:25694588

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

Post-PET ultrasound improves specificity of 18F-FDG-PET for recurrent differentiated thyroid cancer while maintaining sensitivity

PubMed Central

Kråkenes, Jostein; Brauckhoff, Katrin; Haugland, Hans Kristian; Heinecke, Achim; Akslen, Lars A; Varhaug, Jan Erik; Brauckhoff, Michael

2015-01-01

Background Positron emission tomography (PET) using fluor-18-deoxyglucose (18F-FDG) with or without computed tomography (CT) is generally accepted as the most sensitive imaging modality for diagnosing recurrent differentiated thyroid cancer (DTC) in patients with negative whole body scintigraphy with iodine-131 (I-131). Purpose To assess the potential incremental value of ultrasound (US) over 18F-FDG-PET-CT. Material and Methods Fifty-one consecutive patients with suspected recurrent DTC were prospectively evaluated using the following multimodal imaging protocol: (i) US before PET (pre-US) with or without fine needle biopsy (FNB) of suspicious lesions; (ii) single photon emission computed tomography (≥3 GBq I-131) with co-registered CT (SPECT-CT); (iii) 18F-FDG-PET with co-registered contrast-enhanced CT of the neck; (iv) US in correlation with the other imaging modalities (post-US). Postoperative histology, FNB, and long-term follow-up (median, 2.8 years) were taken as composite gold standard. Results Fifty-eight malignant lesions were identified in 34 patients. Forty lesions were located in the neck or upper mediastinum. On receiver operating characteristics (ROC) analysis, 18F-FDG-PET had a limited lesion-based specificity of 59% at a set sensitivity of 90%. Pre-US had poor sensitivity and specificity of 52% and 53%, respectively, increasing to 85% and 94% on post-US, with knowledge of the PET/CT findings (P < 0.05 vs. PET and pre-US). Multimodal imaging changed therapy in 15 out of 51 patients (30%). Conclusion In patients with suspected recurrent DTC, supplemental targeted US in addition to 18F-FDG-PET-CT increases specificity while maintainin sensitivity, as non-malignant FDG uptake in cervical lesions can be confirmed. PMID:25770086

Expression of the hypoxia-inducible monocarboxylate transporter MCT4 is increased in triple negative breast cancer and correlates independently with clinical outcome

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

Doyen, J.; Trastour, C.; Ettore, F.

2014-08-15

Highlights: • Glycolytic markers are highly expressed in triple negative breast cancers. • Lactate/H{sup +} symporter MCT4 demonstrated the strongest deleterious impact on survival. • MCT4 should serve as a new prognostic factor in node-negative breast cancers. - Abstract: Background: {sup 18}Fluor-deoxy-glucose PET-scanning of glycolytic metabolism is being used for staging in many tumors however its impact on prognosis has never been studied in breast cancer. Methods: Glycolytic and hypoxic markers: glucose transporter (GLUT1), carbonic anhydrase IX (CAIX), monocarboxylate transporter 1 and 4 (MCT1, 4), MCT accessory protein basigin and lactate-dehydrogenase A (LDH-A) were assessed by immunohistochemistry in two cohortsmore » of breast cancer comprising 643 node-negative and 127 triple negative breast cancers (TNBC) respectively. Results: In the 643 node-negative breast tumor cohort with a median follow-up of 124 months, TNBC were the most glycolytic (≈70%), followed by Her-2 (≈50%) and RH-positive cancers (≈30%). Tumoral MCT4 staining (without stromal staining) was a strong independent prognostic factor for metastasis-free survival (HR = 0.47, P = 0.02) and overall-survival (HR = 0.38, P = 0.002). These results were confirmed in the independent cohort of 127 cancer patients. Conclusion: Glycolytic markers are expressed in all breast tumors with highest expression occurring in TNBC. MCT4, the hypoxia-inducible lactate/H{sup +} symporter demonstrated the strongest deleterious impact on survival. We propose that MCT4 serves as a new prognostic factor in node-negative breast cancer and can perhaps act soon as a theranostic factor considering the current pharmacological development of MCT4 inhibitors.« less

Development Of A Dynamic Radiographic Capability Using High-Speed Video

NASA Astrophysics Data System (ADS)

Bryant, Lawrence E.

1985-02-01

High-speed video equipment can be used to optically image up to 2,000 full frames per second or 12,000 partial frames per second. X-ray image intensifiers have historically been used to image radiographic images at 30 frames per second. By combining these two types of equipment, it is possible to perform dynamic x-ray imaging of up to 2,000 full frames per second. The technique has been demonstrated using conventional, industrial x-ray sources such as 150 Kv and 300 Kv constant potential x-ray generators, 2.5 MeV Van de Graaffs, and linear accelerators. A crude form of this high-speed radiographic imaging has been shown to be possible with a cobalt 60 source. Use of a maximum aperture lens makes best use of the available light output from the image intensifier. The x-ray image intensifier input and output fluors decay rapidly enough to allow the high frame rate imaging. Data are presented on the maximum possible video frame rates versus x-ray penetration of various thicknesses of aluminum and steel. Photographs illustrate typical radiographic setups using the high speed imaging method. Video recordings show several demonstrations of this technique with the played-back x-ray images slowed down up to 100 times as compared to the actual event speed. Typical applications include boiling type action of liquids in metal containers, compressor operation with visualization of crankshaft, connecting rod and piston movement and thermal battery operation. An interesting aspect of this technique combines both the optical and x-ray capabilities to observe an object or event with both external and internal details with one camera in a visual mode and the other camera in an x-ray mode. This allows both kinds of video images to appear side by side in a synchronized presentation.

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.

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.

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.

Extended Envelopes around Galactic Cepheids. III. Y Ophiuchi and α Persei from Near-Infrared Interferometry with CHARA/FLUOR

NASA Astrophysics Data System (ADS)

Mérand, Antoine; Aufdenberg, Jason P.; Kervella, Pierre; Foresto, Vincent Coudé du; ten Brummelaar, Theo A.; McAlister, Harold A.; Sturmann, Laszlo; Sturmann, Judit; Turner, Nils H.

2007-08-01

Unbiased angular diameter measurements are required for accurate distances to Cepheids using the interferometric Baade-Wesselink method (IBWM). The precision of this technique is currently limited by interferometric measurements at the 1.5% level. At this level, the center-to-limb darkening (CLD) and the presence of circumstellar envelopes (CSE) seem to be the two main sources of bias. The observations we performed aim at improving our knowledge of the interferometric visibility profile of Cepheids. In particular, we assess the systematic presence of CSE around Cepheids in order determine accurate distances with the IBWM free from CSE biased angular diameters. We observed a Cepheid (Y Oph) for which the pulsation is well resolved and a nonpulsating yellow supergiant (α Per) using long-baseline near-infrared interferometry. We interpreted these data using a simple CSE model we previously developed. We found that our observations of α Per do not provide evidence for a CSE. The measured CLD is explained by an hydrostatic photospheric model. Our observations of Y Oph, when compared to smaller baseline measurements, suggest that it is surrounded by a CSE with characteristics similar to CSEs found previously around other Cepheids. We have determined the distance to Y Oph to be d=491+/-18 pc. Additional evidence points toward the conclusion that most Cepheids are surrounded by faint CSEs, detected by near-infrared interferometry: after observing four Cepheids, all show evidence for a CSE. Our CSE nondetection around a nonpulsating supergiant in the instability strip, α Per, provides confidence in the detection technique and suggests a pulsation driven mass-loss mechanism for the Cepheids.

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.

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.

Remote-Reading Safety and Safeguards Surveillance System for 3013 Containers

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

Lechelt, W. M.; Skorpik, J. R.; Silvers, K. L.

2002-02-26

At Hanford's Plutonium Finishing Plant (PFP), plutonium oxide is being loaded into stainless steel containers for long-term storage on the Hanford Site. These containers consist of two weld-sealed stainless steel cylinders nested one within the other. A third container holds the plutonium within the inner cylinder. This design meets the U.S. Department of Energy (DOE) storage standard, DOE-STD- 3013-2000, which anticipates a 50-year storage lifetime. The 3013 standard also requires a container surveillance program to continuously monitor pressure and to assure safeguards are adequate. However, the configuration of the container system makes using conventional measurement and monitoring methods difficult. Tomore » better meet the 3013 monitoring requirements, a team from Fluor Hanford (who manages the PFP), Pacific Northwest National Laboratory (PNNL), and Vista Engineering Technologies, LLC, developed a safer, cost-efficient, remote PFP 3013 container surveillance system. This new surveillance system is a combination of two successfully deployed technologies: (1) a magnetically coupled pressure gauge developed by Vista Engineering and (2) a radio frequency (RF) tagging device developed by PNNL. This system provides continuous, 100% monitoring of critical parameters with the containers in place, as well as inventory controls. The 3013 container surveillance system consists of three main elements: (1) an internal magnetic pressure sensor package, (2) an instrument pod (external electronics package), and (3) a data acquisition storage and display computer. The surveillance system described in this paper has many benefits for PFP and DOE in terms of cost savings and reduced personnel exposure. In addition, continuous safety monitoring (i.e., internal container pressure and temperature) of every container is responsible nuclear material stewardship and fully meets and exceeds DOE's Integrated Surveillance Program requirements.« less

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

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.

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.

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.

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.

Progressive hearing loss and degeneration of hair cell stereocilia in taperin gene knockout mice

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

Chen, Mo; Wang, Qin; Zhu, Gang-Hua

The TPRN gene encodes taperin, which is prominently present at the taper region of hair cell stereocilia. Mutations in TPRN have been reported to cause autosomal recessive nonsyndromic deafness 79(DFNB 79). To investigate the role of taperin in pathogenesis of hearing loss, we generated TPRN knockout mice using TALEN technique. Sanger sequencing confirmed an 11 bp deletion at nucleotide 177–187 in exon 1 of TPRN, which results in a truncated form of taperin protein. Heterozygous TPRN{sup +/−} mice showed apparently normal auditory phenotypes to their wide-type (WT) littermates. Homozygous TPRN{sup −/−} mice exhibited progressive sensorineural hearing loss as reflected bymore » auditory brainstem response to both click and tone burst stimuli at postnatal days 15 (P15), 30 (P30), and 60 (P60). Alex Fluor-594 phalloidin labeling showed no obvious difference in hair cell numbers in the cochlea between TPRN{sup −/−} mice and WT mice under light microscope. However, scanning electronic microscopy revealed progressive degeneration of inner hair cell stereocilia, from apparently normal at postnatal days 3 (P3) to scattered absence at P15 and further to substantial loss at P30. The outer hair cell stereocilia also showed progressive degeneration, though much less severe, Collectively, we conclude that taperin plays an important role in maintenance of hair cell stereocilia. Establishment of TPRN knockout mice enables further investigation into the function of this gene. - Highlights: • TPRN{sup −/−} mice were generated using TALEN technique. • TPRN{sup −/−} mice presented progressive hearing loss. • WT and TPRN{sup −/−} mice showed no difference in hair cell numbers. • TPRN{sup −/−} mice showed progressive degeneration of hair cell stereocilia.« less

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.

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

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.

Effects of six different preventive treatments on the shear bond strength of orthodontic brackets: in vitro study.

PubMed

Cossellu, Gianguido; Lanteri, Valentina; Butera, Andrea; Sarcina, Michele; Farronato, Giampietro

2015-01-01

Objective : The aim of this study is to evaluate the effect of six different prophylactic agents on shear bond strength (SBS) of orthodontic brackets. Materials and methods : One hundred twenty-six freshly extracted mandibular bovine incisors were used. Teeth were randomly divided into 7 equal groups (18 per group) as follows: group-1 served as control with no pre-treatment; group-2 enamel treated with fluoride varnish (Fluor Protector, Ivoclar Vivadent); group-3 containing casein-phosphopeptide-amorphous calcium-phosphate (CPP-ACP) paste (GC Tooth Mousse, RECALDENT™); group-4 with ozone (HealOzone, Kavo ) ; group-5 with glycine powder (Perio Flow, EMS); group-6 with hydroxyapatite powder 99.5% (Coswell S.p.A.); group-7 with a toothpaste made of hydroxyapatite nanocrystals (BioRepair® Plus, Coswell S.p.A). Brackets were all bonded using the same technique with transbond XT (3 M Unitek, Monrovia, CA). All the bonded specimens were stored for 24 h in deionized water (37 °C) and subjected to thermal cycling for 1000 cycles. The SBS was measured with an Instron Universal Testing machine and the adhesive remnant was assessed with the adhesive remnant index (ARI) using a stereomicroscope at 10× magnification. Results : Statistical differences (ANOVA) were found among the seven investigated groups ( F = 12.226, p < 0.001). SBS of groups 2, 5 and 6 were significantly lower than the control group ( p < 0.05). ARI scores (chi-square test) were correlated with the differences of SBS values. Conclusion: CPP-ACP paste, ozone or BioRepair® did not compromise on bracket bond strength. Fluoride, glycine or hydroxyapatite significantly decreased the SBS; only the fluoride group showed significant clinically low (<6 MPa) SBS values.

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

Anna-Liisa Brownell

Adult progenitor cells hold promise for therapeutic treatment where there has been a disabling loss of function due to death of cells from trauma, disease or aging. However, it will be essential in clinical application to be able to follow the fate of the transplanted cells over time using in vivo tracking methods. We have developed protocol for labeling of progenitor cells to monitor cell trafficking by high resolution magnetic resonance imaging (MRI) and super high resolution positron emission tomography (PET). We have transfected rat subventricular zone stem cells (SVZ, progenitor cell line) and another control cell line (PC12, pheochromocytomamore » cells) utilizing super paramagnetic iron oxide and poly-L-lysine complex for MR imaging or radiolabeling with 18F-fluor deoxy-D- glucose for PET imaging. The labeled cells were transplanted into the rostral migratory stream (RMS) or striatum of normal or 6-hydroxydopamine lesioned Spraque-Dawley rats. Longitudinal MRI studies (up to 40 days) showed that transplantation site has significant impact to the fate of the cells; when SVZ cells were transplanted into the RMS, cells migrated several centimeter into the olfactory bulb; after transplantation into the striatum, the migration was minimal, only 2 mm. PC 12 cells grew a massive tumor after the striatal implantation and significantly smaller tumor after the RMS implantation. PET studies conducted immediately after transplantation verified the transplantation site. MRI studies were able to show the whole path of migration in one image, since part of the cells die during migration and will get detected because of iron content. Endpoint histological studies verified the cell survival and immunohistochemical studies revealed the differentiation of the transplanted cells into astrocytes and neurons.« 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.

Highly Preorganized Ligand 1,10-Phenanthroline-2,9-dicarboxylic Acid for the Selective Recovery of Uranium from Seawater in the Presence of Competing Vanadium Species

DOE PAGES

Lashley, Mark A.; Ivanov, Alexander S.; Bryantsev, Vyacheslav S.; ...

2016-09-30

Studies of the complexation of new promising ligands with uranyl (UO 2 2+) and other seawater cations can aid the development of more efficient, selective, and robust sorbents for the recovery of uranium from seawater. Here, we propose that the ligand design principles based on structural preorganization can be successfully applied to obtain a dramatic enhancement in UO 2 2+ ion binding affinity and selectivity. This concept is exemplified through the investigation of the com-plexes of UO 2 2+, VO 2+, and VO 2+ with the highly preorganized ligand PDA (1,10-phenanthroline-2,9-dicarboxylic acid) using a combination of fluores-cence and absorbance techniques,more » along with den-sity functional theory (DFT) calculations. Moreover, the measured stability constant value, log K1, of 16.5 for the UO 2 2+/PDA complex is very high compared to uranyl complexes with other dicarboxylic ligands. Moreover, PDA exhibits strong selectivity for uranyl over vanadium ions, since the determined sta-bility constant values of the PDA complexes of the vanadium ions are quite low (V(IV) log K1 = 7.4, V(V) = 7.3). Finally, the structures of the corresponding UO 2 2+, VO 2+, and VO 2+ complexes with PDA were identified by systematic DFT calculations, and helped to interpret the stronger binding affinity for uranium over the vanadium ions. Due to its high chemical stability, selectivity, and structural preor-ganization for UO 2 2+ complexation, PDA is a very promising candidate that can be potentially used in the development of novel adsorbent materials for the selective extraction of uranium from sea-water.« less

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.

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.

Highly Preorganized Ligand 1,10-Phenanthroline-2,9-dicarboxylic Acid for the Selective Recovery of Uranium from Seawater in the Presence of Competing Vanadium Species

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

Lashley, Mark A.; Ivanov, Alexander S.; Bryantsev, Vyacheslav S.

Studies of the complexation of new promising ligands with uranyl (UO 2 2+) and other seawater cations can aid the development of more efficient, selective, and robust sorbents for the recovery of uranium from seawater. Here, we propose that the ligand design principles based on structural preorganization can be successfully applied to obtain a dramatic enhancement in UO 2 2+ ion binding affinity and selectivity. This concept is exemplified through the investigation of the com-plexes of UO 2 2+, VO 2+, and VO 2+ with the highly preorganized ligand PDA (1,10-phenanthroline-2,9-dicarboxylic acid) using a combination of fluores-cence and absorbance techniques,more » along with den-sity functional theory (DFT) calculations. Moreover, the measured stability constant value, log K1, of 16.5 for the UO 2 2+/PDA complex is very high compared to uranyl complexes with other dicarboxylic ligands. Moreover, PDA exhibits strong selectivity for uranyl over vanadium ions, since the determined sta-bility constant values of the PDA complexes of the vanadium ions are quite low (V(IV) log K1 = 7.4, V(V) = 7.3). Finally, the structures of the corresponding UO 2 2+, VO 2+, and VO 2+ complexes with PDA were identified by systematic DFT calculations, and helped to interpret the stronger binding affinity for uranium over the vanadium ions. Due to its high chemical stability, selectivity, and structural preor-ganization for UO 2 2+ complexation, PDA is a very promising candidate that can be potentially used in the development of novel adsorbent materials for the selective extraction of uranium from sea-water.« less

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

Timing considerations on the shear bond strength of orthodontic brackets after topical fluoride varnish applications

PubMed Central

Cossellu, Gianguido; Lanteri, Valentina; Butera, Andrea; Laffi, Nicola; Merlini, Alberto; Farronato, Giampietro

2017-01-01

Objectives: To assess the best temporal association between the application of a fluoride varnish on enamel and bonding procedures. Materials and Methods: Eighty mandibular bovine incisors were used. Teeth were divided into 4 groups (20 per group); Groups 1–3 were treated with fluoride varnish (Fluor Protector, Ivoclar Vivadent, Schaan, Liechtenstein), and Group 4 served as control with no pretreatment. Tooth were stored in deionized water (37°C) and subjected to thermal cycling for 400 (Group 1), 800 (Group 2), and 2500 (Group 3) cycles corresponding, respectively, to 15, 30, and 90 days in order to simulate the three different timing of bracket bonding. Shear bond strength (SBS) was measured using an Instron Universal Testing machine. Tooth surfaces were examined under a stereomicroscope at 10× magnification to assess the amount of adhesive remnant index (ARI). One-way analysis of variance (ANOVA) and Tukey's honestly significant difference post-hoc test were used for the comparison of SBS values between groups (P < 0.05). The Chi-square test was used to examine differences among ARI scores. (P < 0.05). Results: One-way ANOVA and Tukey post-hoc test showed that the SBS of different groups were significantly different and was impacted by different timing of bonding (P < 0.05). The main differences were between the control group (17.02 ± 6.38 MPa) and Group 1 (6.93 ± 4.3 MPa). The ARI scores showed that there were no significant differences between the four tested groups. Conclusions: The SBS of the brackets bonded 15 days after the application of the fluoride was set back to an optimal value. PMID:28197397

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.

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

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

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.

Hot exozodiacal dust resolved around Vega with IOTA/IONIC

NASA Astrophysics Data System (ADS)

Defrère, D.; Absil, O.; Augereau, J.-C.; di Folco, E.; Berger, J.-P.; Coudé du Foresto, V.; Kervella, P.; Le Bouquin, J.-B.; Lebreton, J.; Millan-Gabet, R.; Monnier, J. D.; Olofsson, J.; Traub, W.

2011-10-01

Context. Although debris discs have been detected around a significant number of main-sequence stars, only a few of them are known to harbour hot dust in their inner part where terrestrial planets may have formed. Thanks to infrared interferometric observations, it is possible to obtain a direct measurement of these regions, which are of prime importance for preparing future exo-Earth characterisation missions. Aims: We resolve the exozodiacal dust disc around Vega with the help of infrared stellar interferometry and estimate the integrated H-band flux originating from the first few AUs of the debris disc. Methods: Precise H-band interferometric measurements were obtained on Vega with the 3-telescope IOTA/IONIC interferometer (Mount Hopkins, Arizona). Thorough modelling of both interferometric data (squared visibility and closure phase) and spectral energy distribution was performed to constrain the nature of the near-infrared excess emission. Results: Resolved circumstellar emission within ~6 AU from Vega is identified at the 3-σ level. The most straightforward scenario consists in a compact dust disc producing a thermal emission that is largely dominated by small grains located between 0.1 and 0.3 AU from Vega and accounting for 1.23 ± 0.45% of the near-infrared stellar flux for our best-fit model. This flux ratio is shown to vary slightly with the geometry of the model used to fit our interferometric data (variations within ± 0.19%). Conclusions: The presence of hot exozodiacal dust in the vicinity of Vega, initially revealed by K-band CHARA/FLUOR observations, is confirmed by our H-band IOTA/IONIC measurements. Whereas the origin of the dust is still uncertain, its presence and the possible connection with the outer disc suggest that the Vega system is currently undergoing major dynamical perturbations.

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.

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

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.

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.

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.

Ischaemic preconditioning preferentially increases protein S-nitrosylation in subsarcolemmal mitochondria.

PubMed

Sun, Junhui; Nguyen, Tiffany; Aponte, Angel M; Menazza, Sara; Kohr, Mark J; Roth, David M; Patel, Hemal H; Murphy, Elizabeth; Steenbergen, Charles

2015-05-01

Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischaemic preconditioning (IPC)-induced acute cardioprotection. The majority of proteins that show increased SNO following IPC are localized to the mitochondria, and our recent studies suggest that caveolae transduce acute NO/SNO cardioprotective signalling in IPC hearts. Due to the close association between subsarcolemmal mitochondria (SSM) and the sarcolemma/caveolae, we tested the hypothesis that SSM, rather than the interfibrillar mitochondria (IFM), are major targets for NO/SNO signalling derived from caveolae-associated eNOS. Following either control perfusion or IPC, SSM and IFM were isolated from Langendorff perfused mouse hearts, and SNO was analysed using a modified biotin switch method with fluorescent maleimide fluors. In perfusion control hearts, the SNO content was higher in SSM compared with IFM (1.33 ± 0.19, ratio of SNO content Perf-SSM vs. Perf-IFM), and following IPC SNO content significantly increased preferentially in SSM, but not in IFM (1.72 ± 0.17 and 1.07 ± 0.04, ratio of SNO content IPC-SSM vs. Perf-IFM, and IPC-IFM vs. Perf-IFM, respectively). Consistent with these findings, eNOS, caveolin-3, and connexin-43 were detected in SSM, but not in IFM, and IPC resulted in a further significant increase in eNOS/caveolin-3 levels in SSM. Interestingly, we did not observe an IPC-induced increase in SNO or eNOS/caveolin-3 in SSM isolated from caveolin-3(-/-) mouse hearts, which could not be protected with IPC. In conclusion, these results suggest that SSM may be the preferential target of sarcolemmal signalling-derived post-translational protein modification (caveolae-derived eNOS/NO/SNO), thus providing an important role in IPC-induced cardioprotection. Published by Oxford University Press on behalf of the European Society of Cardiology 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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

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.

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.

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.

Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes.

PubMed

Hodge, Greg; Jersmann, Hubertus; Tran, Hai B; Holmes, Mark; Reynolds, Paul N; Hodge, Sandra

2015-01-09

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR). Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy. COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = -.615; ) and with FEV1 in COPD (R = -.777). COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.

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.

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization▿

PubMed Central

Graczyk, Thaddeus K.; Johansson, Michael A.; Tamang, Leena; Visvesvara, Govinda S.; Moura, Laci S.; DaSilva, Alexandre J.; Girouard, Autumn S.; Matos, Olga

2007-01-01

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 × 103 to 4.4 × 105 for E. bieneusi (mean, 8.8 × 104/ml), 2.3 × 102 to 7.8 × 104 (mean, 1.5 × 104/ml) for E. intestinalis, and 1.8 × 102 to 3.6 × 102 for E. hellem (mean, 2.7 × 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings. PMID:17287331

Prognostic value of pre-treatment 18F-FDG PET uptake for nasopharyngeal carcinoma.

PubMed

Aktan, Meryem; Kanyilmaz, Gul; Yavuz, Berrin Benli; Koc, Mehmet; Eryılmaz, Mehmet Akif; Adli, Mustafa

2017-11-25

To evaluate the prognostic value of maximal standardized uptake values (SUV max ) from serial fluor-18-fluorodeoxyglucose positron emission tomography/computed tomography ( 18 F-FDG PET/CT) in patients with nasopharyngeal carcinoma (NPC). Fifty-two patients with NPC who underwent 18 F-FDG PET/CT scan before radiotherapy with/without chemotherapy were reviewed retrospectively. Twenty-seven patients (52%) were applied 3-D conformal radiotherapy and 25 patients (48%) applied intensity-modulated radiotherapy (IMRT). Fourteen (27%) patients were given neoadjuvant chemotherapy and forty-four (84.6%) patients were given concomitant and adjuvant chemotherapy. Median follow-up time was 34 months (range 5.6-66.4 months). Forty-four (84.6%) patients were alive at last follow-up and eight (15.4%) had died. The best cut-off value of the SUV max for the primary tumor site (SUV max -PT) was 13 and 9 for the lymph nodes (SUV max -LN). Patients with SUV max -PT ≥ 13.0 and SUV max -LN ≥ 9 had a significantly higher risk for the development of the distant metastases (p = 0.044 and p = 0.038). DFS was affected in patients with SUV max -PT ≥ 13 (log rank χ 2  = 2.54, p = 0.017) and was significantly lower in patients with SUV max -LN ≥ 9 for the lymph nodes (log rank χ 2  = 5.81, p = 0.013). OS was not affected by SUV levels. A multivariate Cox proportional hazard model of DFS included age (≥ 40), SUV max -LN (< 9), T stage (T1-2) and neoadjuvant chemotherapy are significantly better prognosis for the DFS. 18 F-FDG PET/CT uptake before treatment, as determined by SUV max , may be a valuable tool to evaluate prognosis in NPC patients.

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

Group II Xenoliths from Lunar Crater Volcanic Field, Central Nevada: Evidence for a Kinked Geotherm

NASA Astrophysics Data System (ADS)

Roden, M.; Mosely, J.; Norris, J.

2015-12-01

Group II xenoliths associated with the 140 Ka Easy Chair Crater, Lunar Crater volcanic field, NV, consist of amphibole rich-inclusions including amphibolites, pyroxenites, and gabbros. Abundant minerals in these inclusions are kaersutite, aluminous (7.3-9.7 wt% Al2O3), calcic clinopyroxene, primarily diopside, and olivine (Mg# 69-73) with accessory spinel, sulfide and apatite. Although most apatites are fluor-hydroxyapatite solid solutions, one xenolith contains Cl- and OH-rich apatite suggesting that Cl may have been an important constituent in the parent magma(s) . The xenoliths show abundant evidence for equilibration at relatively low temperatures including amphibole and orthopyroxene exsolution in clinopyroxene, and granules of magnetite in hercynite hosts. If latter texture is due to exsolution, then this particular Group II xenolith equilibrated at temperatures near or below 500oC or at a depth of about 15 km along a conductive geotherm. It may be that all the Group II xenoliths equilibrated at low temperatures given the abundant exsolution textures although Fe-Mg exchange relations suggest equilibration at temperatures in excess of 800oC. Low equilibration temperatures are in conflict with the unusually high equilibration temperatures, >1200oC (Smith, 2000) displayed by Group I xenoliths from this same volcanic field. Taken at face value, the geothermometric results indicate unusually high temperatures in the upper mantle, normal temperatures in the crust and the possibility of a kinked geotherm in the region. Curiously the LCVF lies in an area of "normal" heat flow, south of the Battle Mountain area of high heat flow but the number of heat flow measurements in the Lunar Crater area is very low (Humphreys et al., 2003; Sass, 2005). References: Humphreys et al., 2003, Int. Geol. Rev. 45: 575; Sass et al., 2005, http://pubs.usgs.gov/of/2005/1207/; Smith, 2000, JGR 105: 16769.

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.

Influence of dose rate on the induction of simple and complex chromosome exchanges by gamma rays.

PubMed

Loucas, Bradford D; Eberle, Richard; Bailey, Susan M; Cornforth, Michael N

2004-10-01

Single-color painting of whole chromosomes, or protocols in which only a few chromosomes are distinctively painted, will always fail to detect a proportion of complex exchanges because they frequently produce pseudosimple painting patterns that are indistinguishable from those produced by bona fide simple exchanges. When 24-color multi-fluor FISH (mFISH) was employed for the purpose of distinguishing (truly) simple from pseudosimple exchanges, it was confirmed that the acute low-LET radiation dose-response relationship for simple exchanges lacked significant upward curvature. This result has been interpreted to indicate that the formation of simple exchanges requires only one chromosome locus be damaged (e.g. broken) by radiation to initiate an exchange-not two, as classical cytogenetic theory maintains. Because a one-lesion mechanism implies single-track action, it follows that the production of simple exchanges should not be influenced by changes in dose rate. To examine this prediction, we irradiated noncycling primary human fibroblasts with graded doses of (137)Cs gamma rays at an acute dose rate of 1.10 Gy/min and compared, using mFISH, the yield of simple exchanges to that observed after exposure to the same radiation delivered at a chronic dose rate of 0.08 cGy/min. The shape of the dose response was found to be quasi-linear for both dose rates, but, counter to providing support for a one-lesion mechanism, the yield of simple aberrations was greatly reduced by protracted exposure. Although chronic doses were delivered at rates low enough to produce damage exclusively by single-track action, this did not altogether eliminate the formation of complex aberrations, an analysis of which leads to the conclusion that a single track of low-LET radiation is capable of inducing complex exchanges requiring up to four proximate breaks for their formation. For acute exposures, the ratio of simple reciprocal translocations to simple dicentrics was near unity.

Analyse de la dégradation thermique du Poly(éther éther cétone)

NASA Astrophysics Data System (ADS)

Courvoisier, Emilie; Bicaba, Yoann; Colin, Xavier

2018-03-01

La dégradation thermique du PEEK a été étudiée à l'état caoutchoutique dans de larges intervalles de température (entre 180 et 320 °C) et de pression partielle d'oxygène (entre 0,21 et 50 bars). Tout d'abord, les mécanismes de vieillissement thermique ont été analysés et élucidés par spectroscopie IRTF et par calorimétrie différentielle (DSC) sur des films de PEEK suffisamment minces (entre 10 et 60 μm d'épaisseur) pour s'affranchir totalement des effets de la diffusion d'oxygène. L'oxydation se produit sur les cycles aromatiques provoquant la croissance de cinq nouvelles bandes d'absorption IR centrées à 3650, 3525, 1780, 1740 et 1718 cm-1 et attribuées aux vibrations d'élongation des liaisons O-H du phénol et de l'acide benzoïque, et des liaisons C-O de l'anhydride benzoïque, du benzoate de phényle et de la fluorénone respectivement. De plus, l'oxydation conduit à une large prédominance de la réticulation sur les coupures de chaîne (augmentation de Tg) empêchant le recuit du PEEK, en particulier lorsque la température d'exposition est supérieure au pied du pic de fusion. Enfin, les conséquences de l'oxydation sur les propriétés élastiques ont été analysées et élucidées par micro-indentation sur des sections droites préalablement polies de plaquettes de PEEK de 3 mm d'épaisseur. Les variations du module d'Young et du taux de cristallinité se corrèlent parfaitement, vérifiant ainsi la relation de Tobolsky.

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.

Élimination du bore du silicium par plasma inductif sous champ électrique

NASA Astrophysics Data System (ADS)

Combes, R.; Morvan, D.; Picard, G.; Amouroux, J.

1993-05-01

We analyzed purification mechanisms of silicon by inductive plasma with a fluoride slag. The aim is to study boron elimination from doped electronic grade silicon in function of the nature of the slag to obtain a photovoltaic grade silicon. The steady began with the calculation and the comparison of the stability diagram of boron compounds in presence of CaF2, BaF2 and MgF2. This study led us to conclude that BaF2 is the better slag for silicon purification. This has been confirmed by experience. In a second time, we made purifications under electric bias to enhance slag efficiency. We noticed that BaF2 is more sensitive to electric bias than other slags. Nous avons analysé le mécanisme de purification du silicium sous plasma inductif en présence d'un laitier fluoré. L'objectif principal est d'étudier l'élimination du bore du silicium électronique dopé en fonction de la nature du fluorure pour obtenir un silicium de qualité photovoltaïque. L'étude a commencé par l'établissement et la comparaison de diagrammes des composés du bore en présence de CaF2, de MgF2 et de BaF2. Nous avons déduit de cette première étude que BaF2 est le meilleur laitier pour la purification du silicium. Ceci a été corroboré par l'expérience. Nous avons ensuite opéré en présence d'un champ électrique dans le but d'améliorer encore l'efficacité des laitiers. Nous avons constaté que BaF2 est plus sensible au champ électrique que les deux autres laitiers utilisés.

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

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.