Red and Green Fluorescence from Oral Biofilms.

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Volgenant, Catherine M C; Hoogenkamp, Michel A; Krom, Bastiaan P; Janus, Marleen M; Ten Cate, Jacob M; de Soet, Johannes J; Crielaard, Wim; van der Veen, Monique H

2016-01-01

Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

Red and Green Fluorescence from Oral Biofilms.

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Catherine M C Volgenant

Full Text Available Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation as compared to the sucrose grown biofilms (cariogenic simulation. Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

[Ph-Sensor Properties of a Fluorescent Protein from Dendronephthya sp].

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Pakhomov, A A; Chertkova, R V; Martynov, V I

2015-01-01

Genetically encoded biosensors based on fluorescent proteins are now widely applicable for monitoring pH changes in live cells. Here, we have shown that a fluorescent protein from Dendronephthya sp. (DendFP) exhibits a pronounced pH-sensitivity. Unlike most of known genetically encoded pH-sensors, fluorescence of the protein is not quenched upon medium acidification, but is shifting from the red to green spectral range. Therefore, quantitative measurements of intracellular pH are feasible by ratiometric comparison of emission intensities in the red and green spectral ranges, which makes DendFP advantageous compared with other genetically encoded pH-sensors.

The E1 mechanism in photo-induced beta-elimination reactions for green-to-red conversion of fluorescent proteins.

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Tsutsui, Hidekazu; Shimizu, Hideaki; Mizuno, Hideaki; Nukina, Nobuyuki; Furuta, Toshiaki; Miyawaki, Atsushi

2009-11-25

KikGR is a fluorescent protein engineered to display green-to-red photoconvertibility that is induced by irradiation with ultraviolet or violet light. Similar to Kaede and EosFP, two naturally occurring photoconvertible proteins, KikGR contains a His(62)-Tyr(63)-Gly(64) tripeptide sequence, which forms a green chromophore that can be photoconverted to a red one via formal beta-elimination and subsequent extension of a pi-conjugated system. Using a crystallizable variant of KikGR, we determined the structures of both the green and red state at 1.55 A resolution. The double bond between His(62)-C(alpha) and His(62)-C(beta) in the red chromophore is in a cis configuration, indicating that rotation along the His(62) C(alpha)-C(beta) bond occurs following cleavage of the His(62) N(alpha)-C(alpha) bond. This structural rearrangement provides evidence that the beta-elimination reaction governing the green-to-red photoconversion of KikGR follows an E1 (elimination, unimolecular) mechanism.

Using the fluorescence red edge effect to assess the long-term stability of lyophilized protein formulations.

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Qian, Ken K; Grobelny, Pawel J; Tyagi, Madhusudan; Cicerone, Marcus T

2015-04-06

Nanosecond relaxation processes in sugar matrices are causally linked through diffusional processes to protein stability in lyophilized formulations. Long-term protein degradation rates track mean-squared displacement (⟨u(2)⟩) of hydrogen atoms in sugar glasses, a parameter describing dynamics on a time scale of picoseconds to nanoseconds. However, measurements of ⟨u(2)⟩ are usually performed by neutron scattering, which is not conducive to rapid formulation screening in early development. Here, we present a benchtop technique to derive a ⟨u(2)⟩ surrogate based on the fluorescence red edge effect. Glycerol, lyophilized trehalose, and lyophilized sucrose were used as model systems. Samples containing 10(-6) mole fraction of rhodamine 6G, a fluorophore, were excited at either 532 nm (main peak) or 566 nm (red edge), and the ⟨u(2)⟩ surrogate was determined based the corresponding Stokes shifts. Results showed reasonable agreement between ⟨u(2)⟩ from neutron scattering and the surrogate from fluorescence, although deviations were observed at very low temperatures. We discuss the sources of the deviations and suggest technique improvements to ameliorate these. We expect that this method will be a valuable tool to evaluate lyophilized sugar matrices with respect to their ability to protect proteins from diffusion-limited degradation processes during long-term storage. Additionally, the method may have broader applications in amorphous pharmaceutical solids.

Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System

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Chao-Hsun Yang

2013-01-01

Full Text Available The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP at C-terminus and red fluorescent protein (RFP, DsRed at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future.

Frequency domain fluorescent diffuse tomography of small animals with DsRed2-expressed tumors

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Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Orlova, Anna G.; Sergeeva, Ekaterina A.; Kleshnin, Mikhail S.; Shirmanova, Marina V.

2006-02-01

The main applications of fluorescent proteins (FPs) are monitoring tumor growth, angiogenesis, metastases formation and effects of new classes of drugs. Different types of tomography allow fluorescence imaging of tumors located deep in human or animal tissue. These techniques were used for investigation of the distribution of near-infrared fluorescent probes, but only a few works are devoted to fluorescence tomography in visible light. In this work, preliminary results of the frequency domain fluorescent diffuse tomography (FD FDT) method in application to DsRed2 protein as a fluorescent agent are presented. For the first step of our experiments we utilized second harmonic generation of Nd:YAG laser (532 nm) modulated by low frequency (1 kHz) in the experimental setup. The transilluminative planar configuration was used in the setup. A series of model experiments has been conducted and show good agreement between theoretical and experimental fluorescence intensity. Post mortem experiments with capsules containing DsRed2 and scattering solution introduced into esophagus of rats to simulate tumor formation have been conducted. The results of these experiments show that sensitivity of the setup is sufficient to detect DsRed2 in concentrations similar to those in FP-expressed tumor, but the contrast is not enough high to separate fluorescence of DsRed2 and surrounding tissues. The setup can be significantly improved by utilizing high-frequency modulation (110 MHz using acousto-optical modulator) of the excitation light and precise phase measurements due to difference in fluorescence life-time of FPs and surrounding tissues. An algorithm of processing a fluorescent image based on calculating zero of maximum curvature was employed for detection of fluorescent inclusions boundaries in the image.

Red and green fluorescence from oral biofilms

NARCIS (Netherlands)

Volgenant, C.M.C.; Hoogenkamp, M.A.; Krom, B.P.; Janus, M.M.; ten Cate, J.M.; de Soet, J.J.; Crielaard, W.; van der Veen, M.H.

2016-01-01

Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis.

Conformational study of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) by tryptophan fluorescence and differential scanning calorimetry.

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Yin, Shou-Wei; Tang, Chuan-He; Yang, Xiao-Quan; Wen, Qi-Biao

2011-01-12

Fluorescence and differential scanning calorimetry (DSC) were used to study changes in the conformation of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) under various environmental conditions. The possible relationship between fluorescence data and DSC characteristics was also discussed. Tryptophan fluorescence and fluorescence quenching analyses indicated that the tryptophan residues in KPI, exhibiting multiple fluorophores with different accessibilities to acrylamide, are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains close to at least some of the tryptophan residues. GdnHCl was more effective than urea and SDS in denaturing KPI. SDS and urea caused variable red shifts, 2-5 nm, in the emission λ(max), suggesting the conformational compactness of KPI. The result was further supported by DSC characteristics that a discernible endothermic peak was still detected up to 8 M urea or 30 mM SDS, also evidenced by the absence of any shift in emission maximum (λ(max)) at different pH conditions. Marked decreases in T(d) and enthalpy (ΔH) were observed at extreme alkaline and/or acidic pH, whereas the presence of NaCl resulted in higher T(d) and ΔH, along with greater cooperativity of the transition. Decreases in T(d) and ΔH were observed in the presence of protein perturbants, for example, SDS and urea, indicating partial denaturation and decrease in thermal stability. Dithiothreitol and N-ethylmaleimide have a slight effect on the thermal properties of KPI. Interestingly, a close linear relationship between the T(d) (or ΔH) and the λ(max) was observed for KPI in the presence of 0-6 M urea.

Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor.

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Pakhomov, Alexey A; Martynov, Vladimir I; Orsa, Alexander N; Bondarenko, Alena A; Chertkova, Rita V; Lukyanov, Konstantin A; Petrenko, Alexander G; Deyev, Igor E

2017-12-02

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells. Copyright © 2017 Elsevier Inc. All rights reserved.

The fluorescence theatre: a cost-effective device using theatre gels for fluorescent protein and dye screening.

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Heil, John R; Nordeste, Ricardo F; Charles, Trevor C

2011-04-01

Here we report a simple cost-effective device for screening colonies on plates for expression of the monomeric red fluorescent protein mRFP1 and the fluorescent dye Nile red. This device can be built from any simple light source, in our case a Quebec Colony Counter, and cost-effective theatre gels. The device can be assembled in as little as 20 min, and it produces excellent results when screening a large number of colonies.

Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.

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Bajar, Bryce T; Wang, Emily S; Lam, Amy J; Kim, Bongjae B; Jacobs, Conor L; Howe, Elizabeth S; Davidson, Michael W; Lin, Michael Z; Chu, Jun

2016-02-16

Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.

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

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Yoko Hayashi-Takanaka

Full Text Available 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.

Spectroscopic Analysis of Red Fluorescent Proteins and Development of a Microfluidic Cell Sorter for the Generation of Improved Variants

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Lubbeck, Jennifer L.

The discovery of the green fluorescent protein (GFP) launched the development of a wide variety of fluorescent protein (FP) mutants whose spectral and photophysical diversity revolutionized in vivo imaging. The excitation and emission spectra of red fluorescent proteins (RFPs), in particular, have been ideally tuned to a window optically favorable for in vivo work. However, their quantum yields, photostabilities and fluorescence intermittency properties require improvement if they are to be broadly employed for low-copy or single-molecule measurements. Attempts to engineer improved RFPs often result in optimization of one photophysical property at the expense of others. We developed a microfluidic-based cytometer for screening HeLa cell-based genetic RFP-libraries simultaneously on the basis of fluorescence lifetime (a proxy for quantum yield), photostability, and brightness. Ten 532 nm excitation beams interrogate each cell in flow. The first is electro-optically modulated (30 MHz) to enable lifetime measurement with phase fluorimetry. The remaining beams act as a pulse sequence for isolating the irreversible photobleaching time constant. Optical-force switching is employed to sort cells based on any combination of the photophysical parameters. Screening with this instrument enables identification of regions of the structure that synergistically affect quantum yield and photostability and the sorting capability provides a new tool for accelerating the development of next generation RFPs.

Red fluorescence in reef fish: A novel signalling mechanism?

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Siebeck Ulrike E

2008-09-01

Full Text Available Abstract Background At depths below 10 m, reefs are dominated by blue-green light because seawater selectively absorbs the longer, 'red' wavelengths beyond 600 nm from the downwelling sunlight. Consequently, the visual pigments of many reef fish are matched to shorter wavelengths, which are transmitted better by water. Combining the typically poor long-wavelength sensitivity of fish eyes with the presumed lack of ambient red light, red light is currently considered irrelevant for reef fish. However, previous studies ignore the fact that several marine organisms, including deep sea fish, produce their own red luminescence and are capable of seeing it. Results We here report that at least 32 reef fishes from 16 genera and 5 families show pronounced red fluorescence under natural, daytime conditions at depths where downwelling red light is virtually absent. Fluorescence was confirmed by extensive spectrometry in the laboratory. In most cases peak emission was around 600 nm and fluorescence was associated with guanine crystals, which thus far were known for their light reflecting properties only. Our data indicate that red fluorescence may function in a context of intraspecific communication. Fluorescence patterns were typically associated with the eyes or the head, varying substantially even between species of the same genus. Moreover red fluorescence was particularly strong in fins that are involved in intraspecific signalling. Finally, microspectrometry in one fluorescent goby, Eviota pellucida, showed a long-wave sensitivity that overlapped with its own red fluorescence, indicating that this species is capable of seeing its own fluorescence. Conclusion We show that red fluorescence is widespread among marine fishes. Many features indicate that it is used as a private communication mechanism in small, benthic, pair- or group-living fishes. Many of these species show quite cryptic colouration in other parts of the visible spectrum. High inter

Diversity and Ecological Correlates of Red Fluorescence in Marine Fishes

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Nils Anthes

2016-11-01

Full Text Available Marine environments at depths below -10 to -25 m are almost devoid of ambient red sunlight because water quickly attenuates long wavelengths. This stenospectral light environment presents unique opportunities for organisms that can transform ambient blue-green light into red light by fluorescence. Numerous marine fish species display intricate patterns of fluorescence. Because color vision is a key component of fish sensory ecology, several putative visual functions of red fluorescence have been proposed but are difficult to test experimentally. Here, we follow a comparative approach to assess the consistency between the phylogenetic distribution of red fluorescence with its presumed functions. We collected and analyzed the largest data set of red fluorescence in fishes to date, consisting of confirmed cases in 272 primarily diurnal fish species from 49 out of 90 surveyed fish families and 12 out of 21 surveyed fish orders, contrasted to 393 fish species with confirmed absence of red fluorescence. Based on a priori hypotheses on adaptive function, we compare the prevalence of red fluorescence among pre-defined sets of species based on ecological or biological characteristics while controlling for shared ancestry. When comparing between species, we find no evidence that red fluorescence is more prevalent in deep-water species, contrasting with our recent finding that fluorescence brightness increases with depth within species. There is also no evidence for a role in group-driven communication. Phylogenetic patterns are consistent, however, with three other predictions. First, fluorescence with a rather patchy distribution across the body occurred significantly more often among sit-and-wait predators or otherwise sedentary fish than in more mobile species, consistent with background matching for camouflage. Second, small, predatory fishes tended to show red fluorescent irides disproportionally often consistent with a proposed function in prey

Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

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Heppert, Jennifer K; Dickinson, Daniel J; Pani, Ariel M; Higgins, Christopher D; Steward, Annette; Ahringer, Julie; Kuhn, Jeffrey R; Goldstein, Bob

2016-11-07

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments. © 2016 Heppert et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Diversity and evolution of coral fluorescent proteins.

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Naila O Alieva

2008-07-01

Full Text Available GFP-like fluorescent proteins (FPs are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae and pink chromoprotein from Stylophora pistillata (Pocilloporidae, both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria. The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of

Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening

International Nuclear Information System (INIS)

Johansson, Daniel X.; Brismar, Hjalmar; Persson, Mats A.A.

2007-01-01

The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5 ± 1.5% (mean ± SEM) and 8.0 ± 0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8 ± 2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1 ± 1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals

Expression profiling of Plasmodium berghei HSP70 genes for generation of bright red fluorescent parasites.

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Marion Hliscs

Full Text Available Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite Plasmodium berghei. To allow constitutive and abundant expression of the mCherry protein we profiled expression of all members of the P. berghei heat shock protein 70 (HSP70 family. We identified PbHSP70/1, an invariant ortholog of Plasmodium falciparum HSP70-1, as the protein with the highest expression levels during Plasmodium blood, mosquito, and liver infection. Stable allelic insertion of a mCherry expression cassette into the PbHsp70/1 locus created constitutive red fluorescent P. berghei lines, termed Pbred. We show that these parasites can be used for live imaging of infected host cells and organs, including hepatocytes, erythrocytes, and whole Anopheles mosquitoes. Quantification of the fluorescence intensity of several Pbred parasite stages revealed significantly enhanced signal intensities in comparison to GFP expressed under the control of the constitutive EF1alpha promoter. We propose that systematic transcript profiling permits generation of reporter parasites, such as the Pbred lines described herein.

Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

Science.gov (United States)

Ding, Baoqing; Yuan, Yao-Wu

2016-04-01

The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

One-pot synthesis of gold nanoclusters with bright red fluorescence and good biorecognition abilities for visualization fluorescence enhancement detection of E. coli.

Science.gov (United States)

Liu, Jiali; Lu, Lili; Xu, Suying; Wang, Leyu

2015-03-01

A facile one-pot strategy was developed for the synthesis of lysozyme functionalized fluorescence gold nanoclusters (AuNCs). The lysozymes added to reduce Au(3+) ions and stabilize the AuNCs during the synthesis were coated on the AuNCs surface and retained their specific recognition ability for bacteria such as Escherichia coli (E. coli). Based on such ability, these AuNCs were specifically attached onto the surface of E. coli, which resulted in great red fluorescence enhancement. Nevertheless, the bovine serum albumin (BSA) stabilized AuNCs could not recognize E. coli and no fluorescence enhancement was observed. Upon the addition of E. coli, the red fluorescence intensity of lysozyme-AuNCs was enhanced linearly over the range of 2.4×10(4) -6.0×10(6) CFU/mL of E. coli with high sensitivity (LOD=2.0×10(4) CFU/mL, S/N=3). The visualization fluorescence evolution may enable the rapid and real-time detection of bacteria. This study may be extended to other functional proteins such as antibody, enzyme, and peptide functionalized nanoclusters while retaining the bioactivity of coating proteins and find wide applications in the fields of biochemistry and biomedicine. Copyright © 2014 Elsevier B.V. All rights reserved.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

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Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Graphitic Nitrogen Triggers Red Fluorescence in Carbon Dots.

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Holá, Kateřina; Sudolská, Mária; Kalytchuk, Sergii; Nachtigallová, Dana; Rogach, Andrey L; Otyepka, Michal; Zbořil, Radek

2017-12-26

Carbon dots (CDs) are a stable and highly biocompatible fluorescent material offering great application potential in cell labeling, optical imaging, LED diodes, and optoelectronic technologies. Because their emission wavelengths provide the best tissue penetration, red-emitting CDs are of particular interest for applications in biomedical technologies. Current synthetic strategies enabling red-shifted emission include increasing the CD particle size (sp 2 domain) by a proper synthetic strategy and tuning the surface chemistry of CDs with suitable functional groups (e.g., carboxyl). Here we present an elegant route for preparing full-color CDs with well-controllable fluorescence at blue, green, yellow, or red wavelengths. The two-step procedure involves the synthesis of a full-color-emitting mixture of CDs from citric acid and urea in formamide followed by separation of the individual fluorescent fractions by column chromatography based on differences in CD charge. Red-emitting CDs, which had the most negative charge, were separated as the last fraction. The trend in the separation, surface charge, and red-shift of photoluminescence was caused by increasing amount of graphitic nitrogen in the CD structure, as was clearly proved by XPS, FT-IR, Raman spectroscopy, and DFT calculations. Importantly, graphitic nitrogen generates midgap states within the HOMO-LUMO gap of the undoped systems, resulting in significantly red-shifted light absorption that in turn gives rise to fluorescence at the low-energy end of the visible spectrum. The presented findings identify graphitic nitrogen as another crucial factor that can red-shift the CD photoluminescence.

Evidence of green fluorescent protein and growth hormone expression in red abalone (Haliotis rufescens larvae

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Mancilla-Sánchez Edgar

2017-02-01

Full Text Available The red abalone Haliotis rufescens is a highly appreciated mollusk in the national and international markets. Due to its natural over-exploitation and low growth rate, several genetic improvements were made, however special efforts are needed to increase its production. This study presents transgenic abalone’s larvae expressing green fluorescent protein (GFP fused to Cobia (Rachycentron canadum Growth Hormone (GH using sperm media transgenesis technique (SMT, pAcGFP1-N vector under the control of cytomegalovirus (CMV promoter. Sperms were exposed to three voltages (0.5, 0.75 and 1.0 Kv using a micropulser electroporator (Bio-Rad®. The highest GFP-GH expression average (40% was obtained in abalone larvae at 0.75 v. GFP and GH transgenes were positively detected by PCR, western blot and confocal microscope, respectively.

Imaging the Antistaphylococcal Activity of CATH-2: Mechanism of Attack and Regulation of Inflammatory Response

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Schneider, Viktoria A. F.; Coorens, Maarten; Tjeerdsma-van Bokhoven, Johanna L. M.; Posthuma, George; van Dijk, Albert; Veldhuizen, Edwin J. A.

2017-01-01

ABSTRACT Chicken cathelicidin-2 (CATH-2) is a broad-spectrum antimicrobial host defense peptide (HDP) that may serve as a paradigm for the development of new antimicrobial agents. While previous studies have elucidated the mechanism by which CATH-2 kills Escherichia coli, its mode of action against Gram-positive bacteria remains to be determined. In this study, we explored the underlying antibacterial mechanism of CATH-2 against a methicillin-resistant strain of Staphylococcus aureus and the effect of CATH-2-mediated S. aureus killing on immune activation. Visualization of the antimicrobial activity of CATH-2 against S. aureus with live-imaging confocal microscopy demonstrated that CATH-2 directly binds the bacteria, which is followed by membrane permeabilization and cell shrinkage. Transmission electron microscopy (TEM) studies further showed that CATH-2 initiated pronounced morphological changes of the membrane (mesosome formation) and ribosomal structures (clustering) in a dose-dependent manner. Immunolabeling of these sections demonstrated that CATH-2 binds and passes the bacterial membrane at subminimal bactericidal concentrations (sub-MBCs). Furthermore, competition assays and isothermal titration calorimetry (ITC) analysis provided evidence that CATH-2 directly interacts with lipoteichoic acid and cardiolipin. Finally, stimulation of macrophages with S. aureus and CATH-2 showed that CATH-2 not only kills S. aureus but also has the potential to limit S. aureus-induced inflammation at or above the MBC. Taken together, it is concluded that at sub-MBCs, CATH-2 perturbs the bacterial membrane and subsequently enters the cell and binds intracellular S. aureus components, while at or above the MBC, CATH-2 causes disruption of membrane integrity and inhibits S. aureus-induced macrophage activation. IMPORTANCE Due to the high use of antibiotics in both human and veterinary settings, many bacteria have become resistant to those antibiotics that we so heavily

Light adaptation of the unicellular red alga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.

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Ueno, Yoshifumi; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

2015-08-01

Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed.

Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells.

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Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

2014-01-01

We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

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Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Selective labeling of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein

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Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Arimura, Shin-ichi; Tsutsumi, Nobuhiro; Fukui, Kiichi; Itoh, Kazuyoshi

2008-02-01

We present space-selective labeling of organelles by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. Two-photon excitation of photoconvertible fluorescent-protein, Kaede, enables space-selective labeling of organelles. We alter the fluorescence of target mitochondria in a tobacco BY-2 cell from green to red by focusing femtosecond laser pulses with a wavelength of 750 nm.

A recombinant Anticarsia gemmatalis MNPV harboring chiA and v-cath genes from Choristoneura fumiferana defective NPV induce host liquefaction and increased insecticidal activity.

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Anabele Azevedo Lima

Full Text Available One of the interesting features of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D genome is the absence of chitinase (chiA and cathepsin (v-cath genes. This characteristic may be responsible for the lack of liquefaction and melanization in A. gemmatalis larvae killed by AgMNPV-2D infection. This study aimed to test the hypothesis that CHIA and V-CATH proteins from Choristonera fumiferana DEF multiple nucleopolyhedrovirus (CfDEFNPV are able to liquefy and melanize the cuticle of A. gemmatalis larvae infected by a recombinant AgMNPV containing chiA and v-cath genes inserted in its genome. A fragment from the CfDefNPV genome containing chiA and v-cath genes was inserted into the genome of AgMNPV-2D. The recombinant virus (vAgp2100Cf.chiA/v-cath was purified and used to infect insect cells and larvae. Transcripts of v-cath and chiA genes were detected along the infection of insect cells by qRT-PCR, from early to late phases of infection. The analysis of A. gemmatalis larvae killed by vAgp2100Cf.chiA/v-cath infection confirmed the hypothesis proposed. The vAgp2100Cf.chiA/v-cath showed higher insecticidal activity against third instar A. gemmatalis larvae when compared to AgMNPV-2D. The mean time to death was also lower for the vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D at 10 days post infection. Occlusion body production was higher in A. gemmatalis larvae infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. Enzyme assays showed higher chitinase and cysteine protease activities in insect cells and insects infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. The introduction of chiA and v-cath genes into the genome of AgMNPV improves its insecticidal activity against A. gemmatalis larvae and this recombinant virus could be used as an alternative to the wild type virus to control this important insect pest.

Red fluorescence imaging for dental plaque detection and quantification: pilot study

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Liu, Zhao; Gomez, Juliana; Khan, Soniya; Peru, Debbie; Ellwood, Roger

2017-09-01

The red fluorescence of dental plaque originating from porphyrins in oral bacteria may allow visualization, detection, and scoring of plaque without disclosing agents. Two studies were conducted. The first included 24 healthy participants who abstained from oral hygiene for 24 h. Dental plaque was collected from tooth surfaces, and a 10% solution was prepared. These were scanned by a molecular spectrometer to identify the optimum excitation and emission wavelengths of plaque for developing a red fluorescence imaging system. Fourteen healthy subjects completed the second study. After a washout period (1 week), participants had a prophylaxis at baseline and abstained from oral hygiene during the study. They were monitored using the fluorescence imaging system at baseline, 24 h, and 48 h. A dentist clinically assessed plaque after disclosing and on red fluorescence images. Three descriptors were extracted from images and a RUSBoost classifier derived computer fluorescence scores through cross-validation. Red fluorescence plaque levels increased during the 48-h accumulation. Plaque progression was identified by dentist assessment and computer analysis, presenting significant differences between visits at tooth and subject levels (phygiene assessment.

A Novel Reporter Rat Strain That Conditionally Expresses the Bright Red Fluorescent Protein tdTomato.

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Hiroyuki Igarashi

Full Text Available Despite the strength of the Cre/loxP recombination system in animal models, its application in rats trails that in mice because of the lack of relevant reporter strains. Here, we generated a floxed STOP tdTomato rat that conditionally expresses a red fluorescent protein variant (tdTomato in the presence of exogenous Cre recombinase. The tdTomato signal vividly visualizes neurons including their projection fibers and spines without any histological enhancement. In addition, a transgenic rat line (FLAME that ubiquitously expresses tdTomato was successfully established by injecting intracytoplasmic Cre mRNA into fertilized ova. Our rat reporter system will facilitate connectome studies as well as the visualization of the fine structures of genetically identified cells for long periods both in vivo and ex vivo. Furthermore, FLAME is an ideal model for organ transplantation research owing to improved traceability of cells/tissues.

SEIS-PROV: Practical Provenance for Seismological Data

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Krischer, L.; Smith, J. A.; Tromp, J.

2015-12-01

It is widely recognized that reproducibility is crucial to advance science, but at the same time it is very hard to actually achieve. This results in it being recognized but also mostly ignored by a large fraction of the community. A key ingredient towards full reproducibility is to capture and describe the history of data, an issue known as provenance. We present SEIS-PROV, a practical format and data model to store provenance information for seismological data. In a seismological context, provenance can be seen as information about the processes that generated and modified a particular piece of data. For synthetic waveforms the provenance information describes which solver and settings therein were used to generate it. When looking at processed seismograms, the provenance conveys information about the different time series analysis steps that led to it. Additional uses include the description of derived data types, such as cross-correlations and adjoint sources, enabling their proper storage and exchange. SEIS-PROV is based on W3C PROV (http://www.w3.org/TR/prov-overview/), a standard for generic provenance information. It then applies an additional set of constraints to make it suitable for seismology. We present a definition of the SEIS-PROV format, a way to check if any given file is a valid SEIS-PROV document, and two sample implementations: One in SPECFEM3D GLOBE (https://geodynamics.org/cig/software/specfem3d_globe/) to store the provenance information of synthetic seismograms and another one as part of the ObsPy (http://obspy.org) framework enabling automatic tracking of provenance information during a series of analysis and transformation stages. This, along with tools to visualize and interpret provenance graphs, offers a description of data history that can be readily tracked, stored, and exchanged.

Far-Red Fluorescent Probe for Imaging of Vicinal Dithiol-Containing Proteins in Living Cells Based on a pKa Shift Mechanism.

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Zhang, Shengrui; Chen, Guojun; Wang, Yuanyuan; Wang, Qin; Zhong, Yaogang; Yang, Xiao-Feng; Li, Zheng; Li, Hua

2018-02-20

Vicinal dithiol-containing proteins (VDPs) play fundamental roles in intracellular redox homeostasis and are responsible for many diseases. In this work, we report a far-red fluorescence turn-on probe MCAs for VDPs exploiting the pK a shift of the imine functionality of the probe. MCAs is composed of a merocyanine Schiff base as the fluorescent reporter and a cyclic 1,3,2-dithiarsenolane as the specific ligand for VDPs. The imine pK a of MCAs is 4.8, and it exists predominantly in the Schiff base (SB) form at physiological pH. Due to the absence of a resonating positive charge, it absorbs at a relatively short wavelength and is essentially nonfluorescent. Upon selective binding to reduced bovine serum albumin (rBSA, selected as the model protein), MCAs was brought from aqueous media to the binding pockets of the protein, causing a large increase in pK a value of MCAs (pK a = 7.1). As a result, an increase in the protonated Schiff base (PSB) form of MCAs was observed at the physiological pH conditions, which in turn leads to a bathochromically shifted chromophore (λ abs = 634 nm) and a significant increase in fluorescence intensity (λ em = 657 nm) simultaneously. Furthermore, molecular dynamics simulations indicate that the salt bridges formed between the iminium in MCAs and the residues D72 and D517 in rBSA resist the dissociation of proton from the probe, thus inducing an increase of the pK a value. The proposed probe shows excellent sensitivity and specificity toward VDPs over other proteins and biologically relevant species and has been successfully applied for imaging of VDPs in living cells. We believe that the present pK a shift switching strategy may facilitate the development of new fluorescent probes that are useful for a wide range of applications.

Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

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Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

2017-09-01

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

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Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh; Jiang, JinJie; Tokar, Erik J; Malone, Christine C; Petrovich, Robert M; Mason, Ronald P

2017-08-01

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H 2 O 2 ) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H 2 O 2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O 2 •- ) and H 2 O 2 in the presence of NADH. Generation of the free radical O 2 •- and H 2 O 2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies. Published by Elsevier B.V.

Fluorogen-activating proteins: beyond classical fluorescent proteins

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Shengnan Xu

2018-05-01

Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

Emission shaping in fluorescent proteins: role of electrostatics and π-stacking.

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Park, Jae Woo; Rhee, Young Min

2016-02-07

For many decades, simulating the excited state properties of complex systems has been an intriguing but daunting task due to its high computational cost. Here, we apply molecular dynamics based techniques with interpolated potential energy surfaces toward calculating fluorescence spectra of the green fluorescent protein (GFP) and its variants in a statistically meaningful manner. With the GFP, we show that the diverse electrostatic tuning can shape the emission features in many different ways. By computationally modulating the electrostatic interactions between the chromophore phenoxy oxygen and its nearby residues, we demonstrate that we indeed can shift the emission to the blue or to the red side in a predictable manner. We rationalize the shifting effects of individual residues in the GFP based on the responses of both the adiabatic and the diabatic electronic states of the chromophore. We next exhibit that the yellow emitting variant, the Thr203Tyr mutant, generates changes in the electrostatic interactions and an additional π-stacking interaction. These combined effects indeed induce a red shift to emit the fluorescence into the yellow region. With the series of demonstrations, we suggest that our approach can provide sound rationales and useful insights in understanding different responses of various fluorescent complexes, which may be helpful in designing new light emitting proteins and other related systems in future studies.

A new approach to dual-color two-photon microscopy with fluorescent proteins

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Rebane Aleks

2010-02-01

Full Text Available Abstract Background Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA efficiency. Results Here we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein. Conclusion Our method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

Fluorescent IgG fusion proteins made in E. coli

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Luria, Yael; Raichlin, Dina; Benhar, Itai

2012-01-01

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals. PMID:22531449

Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

Energy Technology Data Exchange (ETDEWEB)

Zhou, Changhua; Mao, Mao [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Yuan, Hang [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Shen, Huaibin [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Wu, Feng; Ma, Lan, E-mail: malan@sz.tsinghua.edu.cn [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Li, Lin Song, E-mail: lsli@henu.edu.cn [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China)

2013-09-15

In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 Degree-Sign C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

Graphitic Nitrogen Triggers Red Fluorescence in Carbon Dots

Czech Academy of Sciences Publication Activity Database

Holá, K.; Sudolská, M.; Kalytchuk, S.; Nachtigallová, Dana; Rogach, A. L.; Otyepka, M.; Zbořil, R.

2017-01-01

Roč. 11, č. 12 (2017), s. 12402-12410 ISSN 1936-0851 R&D Projects: GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388963 Keywords : nitrogen-doped * graphene dots * red fluorescence * fluorescence mechanism * band-gap tuning Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 13.942, year: 2016

Murine leukemia virus (MLV replication monitored with fluorescent proteins

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Bittner Alexandra

2004-12-01

Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

Highly thermostable fluorescent proteins

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Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

2011-03-22

Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling

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Glick Benjamin S

2009-04-01

Full Text Available Abstract Background Whole-cell labeling is a common application of fluorescent proteins (FPs, but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties. Results Building on previous studies of DsRed mutants, we created two DsRed-Express2 derivatives: E2-Orange, an orange FP, and E2-Red/Green, a dual-color FP with both red and green emission. We show that these new FPs retain the low cytotoxicity of DsRed-Express2. In addition, we show that these new FPs are useful as second or third colors for flow cytometry and fluorescence microscopy. Conclusion E2-Orange and E2-Red/Green will facilitate the production of healthy, stably fluorescent cell lines and transgenic organisms for multi-color labeling studies.

Green Fluorescence of Cytaeis Hydroids Living in Association with Nassarius Gastropods in the Red Sea

KAUST Repository

Prudkovsky, Andrey A.; Ivanenko, Viatcheslav N.; Nikitin, Mikhail A.; Lukyanov, Konstantin A.; Belousova, Anna; Reimer, James D.; Berumen, Michael L.

2016-01-01

Green Fluorescent Proteins (GFPs) have been reported from a wide diversity of medusae, but only a few observations of green fluorescence have been reported for hydroid colonies. In this study, we report on fluorescence displayed by hydroid polyps of the genus Cytaeis Eschscholtz, 1829 (Hydrozoa: Anthoathecata: Filifera) found at night time in the southern Red Sea (Saudi Arabia) living on shells of the gastropod Nassarius margaritifer (Dunker, 1847) (Neogastropoda: Buccinoidea: Nassariidae). We examined the fluorescence of these polyps and compare with previously reported data. Intensive green fluorescence with a spectral peak at 518 nm was detected in the hypostome of the Cytaeis polyps, unlike in previous reports that reported fluorescence either in the basal parts of polyps or in other locations on hydroid colonies. These results suggest that fluorescence may be widespread not only in medusae, but also in polyps, and also suggests that the patterns of fluorescence localization can vary in closely related species. The fluorescence of polyps may be potentially useful for field identification of cryptic species and study of geographical distributions of such hydroids and their hosts.

Green Fluorescence of Cytaeis Hydroids Living in Association with Nassarius Gastropods in the Red Sea

KAUST Repository

Prudkovsky, Andrey A.

2016-02-03

Green Fluorescent Proteins (GFPs) have been reported from a wide diversity of medusae, but only a few observations of green fluorescence have been reported for hydroid colonies. In this study, we report on fluorescence displayed by hydroid polyps of the genus Cytaeis Eschscholtz, 1829 (Hydrozoa: Anthoathecata: Filifera) found at night time in the southern Red Sea (Saudi Arabia) living on shells of the gastropod Nassarius margaritifer (Dunker, 1847) (Neogastropoda: Buccinoidea: Nassariidae). We examined the fluorescence of these polyps and compare with previously reported data. Intensive green fluorescence with a spectral peak at 518 nm was detected in the hypostome of the Cytaeis polyps, unlike in previous reports that reported fluorescence either in the basal parts of polyps or in other locations on hydroid colonies. These results suggest that fluorescence may be widespread not only in medusae, but also in polyps, and also suggests that the patterns of fluorescence localization can vary in closely related species. The fluorescence of polyps may be potentially useful for field identification of cryptic species and study of geographical distributions of such hydroids and their hosts.

Protein subcellular localization assays using split fluorescent proteins

Science.gov (United States)

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2009-09-08

The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

Influence of different phenolic fractions on red wine astringency based on polyphenol/protein binding

OpenAIRE

Ren, M.; Wang, X.; Du, G.; Tian, C.; Zhang, J.; Song, X.; Zhu, D.

2017-01-01

The presence of phenolic compounds can make a great contribution to the perception of astringency in red wines based on their interactions with proteins. Human salivary protein and bovine serum albumin were used in this study to investigate the relationship between astringency and polyphenol composition. The interactions between polyphenols and proteins were analysed by means of electrophoresis and fluorescence spectra, and they were further confirmed by sensory analysis. The results indicate...

Recombinant canine distemper virus strain Snyder Hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret.

Science.gov (United States)

Ludlow, M; Nguyen, D T; Silin, D; Lyubomska, O; de Vries, R D; von Messling, V; McQuaid, S; De Swart, R L; Duprex, W P

2012-07-01

The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.

Fluorescent sensors based on bacterial fusion proteins

International Nuclear Information System (INIS)

Mateu, Batirtze Prats; Pum, Dietmar; Sleytr, Uwe B; Toca-Herrera, José L; Kainz, Birgit

2014-01-01

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

Incidence and outcome of retained Port-A-Cath fragments during removal.

Science.gov (United States)

Aworanti, Olugbenga Michael; Linnane, Niall; Tareen, Farhan; Mortell, Alan

2017-07-01

Port-A-Cath devices are frequently used for long-term venous access. We postulate that long-term use predisposes them to getting stuck and retained in a central vein at the time of removal. We aim to report the incidence and outcome of this complication. Between January 2006 and July 2016, a retrospective review of all Port-A-Cath removals that were performed at our centre was conducted. At the time of removal, catheters that could not be removed from the vein were considered retained. During the study period, 107 children had 174 episodes of silicone Sitimplant (Vygon, Ecouen, France) Port-A-Cath insertions. These children required 135 removal episodes and there were 3 (2.2%) instances whereby the catheter fragment was retained. These episodes of retained catheters only occurred in children with factor VIII deficiency (4.1% incidence in this cohort). For each episode of catheter insertion and removal, the catheters had been left in situ for a mean duration of 43 months in children with factor deficiency and no retained fragments, and the mean duration was 91 months in children with factor deficiency and retained catheter fragments (p = 0.0011). Port-A-Caths that are retained after attempted removal is a complication encountered predominantly in catheters that have been in use for a prolonged duration. Furthermore, factor replacement therapy in haemophiliacs may be a risk factor for this complication.

The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

International Nuclear Information System (INIS)

Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

2010-01-01

The crystal structure of a monomeric mutant of Azami-Green (mAG) from G. fascicularis was determined at 2.2 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants

Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo

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Jing Zhou

2017-12-01

Full Text Available Far-red fluorescent reporter genes can be used for tracking cells non-invasively in vivo using fluorescence imaging. Here, we investigate the effectiveness of the far-red fluorescent protein, E2-Crimson (E2C, for tracking mouse embryonic cells (mESCs in vivo following subcutaneous administration into mice. Using a knock-in strategy, we introduced E2C into the Rosa26 locus of an E14-Bra-GFP mESC line, and after confirming that the E2C had no obvious effect on the phenotype of the mESCs, we injected them into mice and imaged them over nine days. The results showed that fluorescence intensity was weak, and cells could only be detected when injected at high densities. Furthermore, intensity peaked on day 4 and then started to decrease, despite the fact that tumour volume continued to increase beyond day 4. Histopathological analysis showed that although E2C fluorescence could barely be detected in vivo at day 9, analysis of frozen sections indicated that all mESCs within the tumours continued to express E2C. We hypothesise that the decrease in fluorescence intensity in vivo was probably due to the fact that the mESC tumours became more vascular with time, thus leading to increased absorbance of E2C fluorescence by haemoglobin. We conclude that the E2C reporter has limited use for tracking cells in vivo, at least when introduced as a single copy into the Rosa26 locus.

Red fluorescence increases with depth in reef fishes, supporting a visual function, not UV protection

Science.gov (United States)

Meadows, Melissa G.; Anthes, Nils; Dangelmayer, Sandra; Alwany, Magdy A.; Gerlach, Tobias; Schulte, Gregor; Sprenger, Dennis; Theobald, Jennifer; Michiels, Nico K.

2014-01-01

Why do some marine fishes exhibit striking patterns of natural red fluorescence? In this study, we contrast two non-exclusive hypotheses: (i) that UV absorption by fluorescent pigments offers significant photoprotection in shallow water, where UV irradiance is strongest; and (ii) that red fluorescence enhances visual contrast at depths below −10 m, where most light in the ‘red’ 600–700 nm range has been absorbed. Whereas the photoprotection hypothesis predicts fluorescence to be stronger near the surface and weaker in deeper water, the visual contrast hypothesis predicts the opposite. We used fluorometry to measure red fluorescence brightness in vivo in individuals belonging to eight common small reef fish species with conspicuously red fluorescent eyes. Fluorescence was significantly brighter in specimens from the −20 m sites than in those from −5 m sites in six out of eight species. No difference was found in the remaining two. Our results support the visual contrast hypothesis. We discuss the possible roles fluorescence may play in fish visual ecology and highlight the possibility that fluorescent light emission from the eyes in particular may be used to detect cryptic prey. PMID:25030989

Red fluorescent proteins for gene expression and protein localization studies in Streptococcus pneumoniae and efficient transformation with Gibson assembled DNA

NARCIS (Netherlands)

Beilharz, Katrin; van Raaphorst, Renske; Kjos, Morten; Veening, Jan-Willem

2015-01-01

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single cell level. Recently, we have benchmarked a set of green fluorescent

Single Molecule Spectroscopy of Fluorescent Proteins

NARCIS (Netherlands)

Blum, Christian; Subramaniam, Vinod

2009-01-01

The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

Fluorescent proteins as singlet oxygen photosensitizers: mechanistic studies in photodynamic inactivation of bacteria

Science.gov (United States)

Ruiz-González, Rubén.; White, John H.; Cortajarena, Aitziber L.; Agut, Montserrat; Nonell, Santi; Flors, Cristina

2013-02-01

Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer, light and oxygen to produce reactive oxygen species (ROS), mainly singlet oxygen (1O2), to photo-oxidize important biomolecules and induce cell death. aPDT is a promising alternative to standard antimicrobial strategies, but its mechanisms of action are not well understood. One of the reasons for that is the lack of control of the photosensitizing drugs location. Here we report the use of geneticallyencoded fluorescent proteins that are also 1O2 photosensitizers to address the latter issue. First, we have chosen the red fluorescent protein TagRFP as a photosensitizer, which unlike other fluorescent proteins such as KillerRed, is able to produce 1O2 but not other ROS. TagRFP photosensitizes 1O2 with a small, but not negligible, quantum yield. In addition, we have used miniSOG, a more efficient 1O2 photosensitizing fluorescent flavoprotein that has been recently engineered from phototropin 2. We have genetically incorporated these two photosensitizers into the cytosol of E. coli and demonstrated that intracellular 1O2 is sufficient to kill bacteria. Additional assays have provided further insight into the mechanism of cell death. Photodamage seems to occur primarily in the inner membrane, and extends to the outer membrane if the photosensitizer's efficiency is high enough. These observations are markedly different to those reported for external photosensitizers, suggesting that the site where 1O2 is primarily generated proves crucial for inflicting different types of cell damage.

Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

Science.gov (United States)

Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

2012-08-01

Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

A polarizable embedding DFT study of one-photon absorption in fluorescent proteins

DEFF Research Database (Denmark)

Beerepoot, Maarten; Steindal, Arnfinn H.; Kongsted, Jacob

2013-01-01

mutants (BFP, eGFP, YFP and eCFP). The observed trends in excitation energies among the FPs are reproduced by our approach when performing calculations directly on the crystal structures or when using structures extracted from a molecular dynamics simulations. However, in the former case, QM/MM geometry......A theoretical study of the one-photon absorption of five fluorescent proteins (FPs) is presented. The absorption properties are calculated using a polarizable embedding approach combined with density functional theory (PE-DFT) on the wild-type green fluorescent protein (wtGFP) and several of its...... optimization of the chromophores within a frozen protein environment is needed in order to reproduce the experimental trends. Explicit account of polarization in the force field is not needed to yield the correct trend between the different FPs, but is necessary for reproducing the experimentally observed red...

The use of port-a-caths in adult patients with Lysosomal Storage Disorders receiving Enzyme Replacement Therapy-one centre experience

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Mairead McLoughlin

2017-12-01

Full Text Available Port-a-cath is a widely used device in patients with long-term venous access demand such as frequent or continuous administration of medications such as Enzyme Replacement Therapy (ERT, chemotherapy delivery, blood transfusions, blood products, and fluids. Patients with Lysosomal Storage Diseases (LSDs often require recurrent courses of ERT. We reviewed our experience of using port-a-caths in patients with LSDs with the focus on challenges and complications associated with these catheters. Among 245 adult patients who were treated with ERT, twenty patients (8.2% had a port-a-cath inserted due to poor venous access. Six patients were using their first port whereas five other patients had their port-a-caths replaced at least once. The remaining six patients had inactive port-a-caths. The majority of patients with active port-a-caths never missed more than one consecutive infusion, although one patient missed 2 consecutive infusions whilst on holiday. We identified significant gaps in patients' and their families' understanding of the management of port-a-caths and risks associated with them. It resulted in producing a leaflet and designing an educational program for our LSD patients.

Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

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Mann David GJ

2012-05-01

Full Text Available Abstract Background The expression of fluorescent protein (FP genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

Optimization of fluorescent proteins

NARCIS (Netherlands)

Bindels, D.S.; Goedhart, J.; Hink, M.A.; van Weeren, L.; Joosen, L.; Gadella (jr.), T.W.J.; Engelborghs, Y.; Visser, A.J.W.G.

2014-01-01

Nowadays, fluorescent protein (FP) variants have been engineered to fluoresce in all different colors; to display photoswitchable, or photochromic, behavior; or to show yet other beneficial properties that enable or enhance a still growing set of new fluorescence spectroscopy and microcopy

Fluorescent Protein Approaches in Alpha Herpesvirus Research

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Ian B. Hogue

2015-11-01

Full Text Available In the nearly two decades since the popularization of green fluorescent protein (GFP, fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1 and pseudorabies virus (PRV structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

Photoconversion and fluorescence properties of a red/green-type cyanobacteriochrome AM1_C0023g2 that binds not only phycocyanobilin but also biliverdin.

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Keiji eFushimi

2016-04-01

Full Text Available Cyanobacteriochromes (CBCRs are distantly related to the red/far-red responsive phytochromes. Red/green-type CBCRs are widely distributed among various cyanobacteria. The red/green-type CBCRs covalently bind phycocyanobilin (PCB and show red/green reversible photoconversion. Recent studies revealed that some red/green-type CBCRs from chlorophyll d-bearing cyanobacterium Acaryochloris marina covalently bind not only PCB but also biliverdin (BV. The BV-binding CBCRs show far-red/orange reversible photoconversion. Here, we identified another CBCR (AM1_C0023g2 from A. marina that also covalently binds not only PCB but also BV with high binding efficiencies, although BV chromophore is unstable in the presence of urea. Replacement of Ser334 with Gly resulted in significant improvement in the yield of the BV-binding holoprotein, thereby ensuring that the mutant protein is a fine platform for future development of optogenetic switches. We also succeeded in detecting near-infrared fluorescence from mammalian cells harboring PCB-binding AM1_C0023g2 whose fluorescence quantum yield is 3.0%. Here the PCB-binding holoprotein is shown as a platform for future development of fluorescent probes.

Accuracy of image-guided radiotherapy of prostate cancer based on the BeamCath urethral catheter technique

DEFF Research Database (Denmark)

Poulsen, Per Rugaard; Fokdal, Lars; Petersen, Jørgen B.B.

2007-01-01

BACKGROUND AND PURPOSE: To examine the accuracy of the BeamCath urethral catheter technique for prostate localization during radiotherapy. MATERIALS AND METHODS: Sixty-four patients were CT scanned twice with the BeamCath catheter, and once without the catheter. The catheter contains radiopaque...

Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic vesicles.

Science.gov (United States)

Pierzyńska-Mach, Agnieszka; Janowski, Paweł A; Dobrucki, Jurek W

2014-08-01

Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis. © 2014 International Society for Advancement of Cytometry.

Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

International Nuclear Information System (INIS)

Zhang, Yi; Keegan, Gemma L.; Stranik, Ondrej; Brennan-Fournet, Margaret E.; McDonagh, Colette

2015-01-01

Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs

Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

Energy Technology Data Exchange (ETDEWEB)

Zhang, Yi; Keegan, Gemma L., E-mail: gemmakeegan@gmail.com [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland); Stranik, Ondrej [Leibniz Institute of Photonic Technology, Department of NanoBiophotonics (Germany); Brennan-Fournet, Margaret E. [CMP-EMSE, MOC, Department of Bioelectronics, Ecole Nationale Superieure des Mines (France); McDonagh, Colette [Dublin City University, School of Physical Sciences, Biomedical Diagnostics Institute (Ireland)

2015-07-15

Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of ∼19-fold compared to a control assay without AgNPs.

Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment.

Directory of Open Access Journals (Sweden)

David F Gruber

Full Text Available We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs. Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp., two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II. We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.

A red fluorescent nude mouse model of human endometriosis: advantages of a non-invasive imaging method.

Science.gov (United States)

Wang, Ningning; Hong, Shanshan; Tan, Jinfeng; Ke, Peiqi; Liang, Lili; Fei, Hui; Liu, Bin; Liu, Liqun; Liu, Yongdong; Yu, Bingjun

2014-05-01

To establish red fluorescent human endometriosis lesions in a nude mouse model and dynamically and non-invasively to compare intraperitoneal and subcutaneous injection models. Primary cultures of endometrial stromal cells (ESCs) and epithelial cells (EECs) isolated from 24 patients with a normal uterine cavity were transfected with 2.5×10(8) (Group 1) and 1.25×10(8) (Group 2) plaque-forming units (PFU) of adenovirus encoding red fluorescent protein (Ad-RFP). Transfection efficiencies, fluorescence intensity and apoptosis rate of the two types of cells were compared in vitro. A mixture of 2.5×10(8) PFU Ad-RFP-infected approximately 400 EECs cell mass and 2×10(6) ESCs for 36h was injected individually into 24 female nude mice subcutaneously (Group A) or intraperitoneally (Group B). From Day 5 after injection, an in vivo imaging system (IVIS) was used to non-invasively observe and compare the lesions of the two groups every week until Day 33. Specifically, the fluorescent intensity, positive rates, persistence time and lesion weight in the implanted human endometriosis lesions were compared. A parametric Student's t-test and two-way analysis of variance were used for statistical analysis. Compared with 1.25×10(8) PFU RFP, a titre of 2.5×10(8) PFU RFP ESCs and EECs incubated for 36h exhibited higher transfection efficiencies and higher fluorescence intensities in vitro. In vivo imaging of the fluorescent human endometriosis lesions originating from an RFP titre of 2.5×10(8) PFU showed that the intensity and lesion weight in Group A were significantly higher than in Group B. However, the two groups had the same RFP-positive rates and fluorescence persistence. The structure of each lesion was evaluated by immunohistochemistry to confirm its human endometrial origin. The red fluorescent human endometriosis model established by subcutaneously injecting 2.5×10(8) PFU RFP-transfected stromal cells and epithelial cells into nude mice had a higher fluorescent positive

Quenched carbonaceous composite - Fluorescence spectrum compared to the extended red emission observed in reflection nebulae

Science.gov (United States)

Sakata, Akira; Wada, Setsuko; Narisawa, Takatoshi; Asano, Yoichi; Iijima, Yutaka; Onaka, Takashi; Tokunaga, Alan T.

1992-01-01

The photoluminescence (fluorescence) of a film of the laboratory-synthesized quenched carbonaceous composite (filmy QCC) is shown to have a single broad emission feature with a peak wavelength that varies from 670 to 725 nm, and coincides with that of the extended red emission observed in reflection nebulae. The rapid decay of the filmy QCC red fluorescence in air and of the stable blue fluorescence of the filmy QCC dissolved in liquid Freon suggests that the red fluorescence originates from the interaction of active chemical species and aromatic components in the filmy QCC. A material similar in nature to that of the filmy QCC may be a major component of interstellar dust.

Red-light absorption and fluorescence of phytochrome chromophores: A comparative theoretical study

Energy Technology Data Exchange (ETDEWEB)

Falklöf, Olle; Durbeej, Bo, E-mail: bodur@ifm.liu.se

2013-11-08

Highlights: • Calculation of red-light absorption and emission of phytochrome chromophores. • Comparison of TD-DFT and ab initio methods. • Pure functionals show better accuracy than hybrid functionals. - Abstract: Currently, much experimental effort is being invested in the engineering of phytochromes, a large superfamily of photoreceptor proteins, into fluorescent proteins suitable for bioimaging in the near-infrared regime. In this work, we gain insight into the potential of computational methods to contribute to this development by investigating how well representative quantum chemical methods reproduce recently recorded red-light absorption and emission maxima of synthetic derivatives of the bilin chromophores of phytochromes. Focusing on the performance of time-dependent density functional theory but using also the ab initio CIS(D), CC2 and CASPT2 methods, we explore how various methodological considerations influence computed spectra and find, somewhat surprisingly, that density functionals lacking exact exchange reproduce the experimental measurements with smaller errors than functionals that include exact exchange. Thus, for the important class of chromophores that bilins constitute, the widely established trend that hybrid functionals give more accurate excitation energies than pure functionals does not apply.

Astringency reduction in red wine by whey proteins.

Science.gov (United States)

Jauregi, Paula; Olatujoye, Jumoke B; Cabezudo, Ignacio; Frazier, Richard A; Gordon, Michael H

2016-05-15

Whey is a by-product of cheese manufacturing and therefore investigating new applications of whey proteins will contribute towards the valorisation of whey and hence waste reduction. This study shows for the first time a detailed comparison of the effectiveness of gelatin and β-lactoglobulin (β-LG) as fining agents. Gelatin was more reactive than whey proteins to tannic acid as shown by both the astringency method (with ovalbumin as a precipitant) and the tannins determination method (with methylcellulose as a precipitant). The two proteins showed similar selectivity for polyphenols but β-LG did not remove as much catechin. The fining agent was removed completely or to a trace level after centrifugation followed by filtration which minimises its potential allergenicity. In addition, improved understanding of protein-tannin interactions was obtained by fluorescence, size measurement and isothermal titration calorimetry (ITC). Overall this study demonstrates that whey proteins have the potential of reducing astringency in red wine and can find a place in enology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fluorescence Imaging in the Red and Far-Red Region during Growth of Sunflower Plantlets. Diagnosis of the Early Infection by the Parasite Orobanche cumana

Science.gov (United States)

Ortiz-Bustos, Carmen M.; Pérez-Bueno, María L.; Barón, Matilde; Molinero-Ruiz, Leire

2016-01-01

Broomrape, caused by the root holoparasite Orobanche cumana, is the main biotic constraint to sunflower oil production worldwide. By the time broomrape emerges, most of the metabolic imbalance has been produced by O. cumana to sunflower plants. UV-induced multicolor fluorescence imaging (MCFI) provides information on the fluorescence emitted by chlorophyll (Chl) a of plants in the spectral bands with peaks near 680 nm (red, F680) and 740 nm (far-red, F740). In this work MCFI was extensively applied to sunflowers, either healthy or parasitized plants, for the first time. The distribution of red and far-red fluorescence was analyzed in healthy sunflower grown in pots under greenhouse conditions. Fluorescence patterns were analyzed across the leaf surface and throughout the plant by comparing the first four leaf pairs (LPs) between the second and fifth week of growth. Similar fluorescence patterns, with a delay of 3 or 4 days between them, were obtained for LPs of healthy sunflower, showing that red and far-red fluorescence varied with the developmental stage of the leaf. The use of F680 and F740 as indicators of sunflower infection by O. cumana during underground development stages of the parasite was also evaluated under similar experimental conditions. Early increases in F680 and F740 as well as decreases in F680/F740 were detected upon infection by O. cumana. Significant differences between inoculated and control plants depended on the LP that was considered at any time. Measurements of Chl contents and final total Chl content supported the results of MCFI, but they were less sensitive in differentiating healthy from inoculated plants. Sunflower infection was confirmed by the presence of broomrape nodules in the roots at the end of the experiment. The potential of MCFI in the red and far-red region for an early detection of O. cumana infection in sunflower was revealed. This technique might have a particular interest for early phenotyping in sunflower breeding

Analysis of Red and Far-Red Sun-Induced Chlorophyll Fluorescence and Their Ratio in Different Canopies Based on Observed and Modeled Data

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Micol Rossini

2016-05-01

Full Text Available Sun-induced canopy chlorophyll fluorescence in both the red (FR and far-red (FFR regions was estimated across a range of temporal scales and a range of species from different plant functional types using high resolution radiance spectra collected on the ground. Field measurements were collected with a state-of-the-art spectrometer setup and standardized methodology. Results showed that different plant species were characterized by different fluorescence magnitude. In general, the highest fluorescence emissions were measured in crops followed by broadleaf and then needleleaf species. Red fluorescence values were generally lower than those measured in the far-red region due to the reabsorption of FR by photosynthetic pigments within the canopy layers. Canopy chlorophyll fluorescence was related to plant photosynthetic capacity, but also varied according to leaf and canopy characteristics, such as leaf chlorophyll concentration and Leaf Area Index (LAI. Results gathered from field measurements were compared to radiative transfer model simulations with the Soil-Canopy Observation of Photochemistry and Energy fluxes (SCOPE model. Overall, simulation results confirmed a major contribution of leaf chlorophyll concentration and LAI to the fluorescence signal. However, some discrepancies between simulated and experimental data were found in broadleaf species. These discrepancies may be explained by uncertainties in individual species LAI estimation in mixed forests or by the effect of other model parameters and/or model representation errors. This is the first study showing sun-induced fluorescence experimental data on the variations in the two emission regions and providing quantitative information about the absolute magnitude of fluorescence emission from a range of vegetation types.

Protective effect of in ovo treatment with the chicken cathelicidin analog D-CATH-2 against avian pathogenic E. coli

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Cuperus, Tryntsje; van Dijk, Albert; Matthijs, Mieke G. R.; Veldhuizen, Edwin J. A.; Haagsman, Henk P.

2016-01-01

Increasing antibiotic resistance and ever stricter control on antibiotic use are a driving force to develop alternatives to antibiotics. One such strategy is the use of multifunctional Host Defense Peptides. Here we examined the protective effect of prophylactic treatment with the D analog of chicken cathelicidin-2 (D-CATH-2) against a respiratory E. coli infection. Chickens were treated with D-CATH-2 in ovo at day 18 of embryonic development or intramuscularly at days 1 and 4 after hatch. At 7 days of age, birds were challenged intratracheally with avian pathogenic E. coli. Protection was evaluated by recording mortality, morbidity (Mean Lesion Score) and bacterial swabs of air sacs at 7 days post-infection. In ovo D-CATH-2 treatment significantly reduced morbidity (63%) and respiratory bacterial load (>90%), while intramuscular treatment was less effective. D-CATH-2 increased the percentage of peripheral blood lymphocytes and heterophils by both administration routes. E. coli specific IgM levels were lower in in ovo treated animals compared to intramuscular D-CATH-2 treatment. In short, in ovo treatment with the Host Defense Peptide derived D-CATH-2 can partially protect chickens from E. coli infection, making this peptide an interesting starting point to develop alternatives to antibiotics for use in the poultry sector. PMID:27229866

Using a Specific RNA-Protein Interaction To Quench the Fluorescent RNA Spinach.

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Roszyk, Laura; Kollenda, Sebastian; Hennig, Sven

2017-12-15

RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labeled. "Spinach" is a genetically encodable RNA aptamer that starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyze RNA-protein interactions by quenching the spinach aptamer.

2D fluorescence spectroscopy for monitoring ion-exchange membrane based technologies - Reverse electrodialysis (RED).

Science.gov (United States)

Pawlowski, Sylwin; Galinha, Claudia F; Crespo, João G; Velizarov, Svetlozar

2016-01-01

Reverse electrodialysis (RED) is one of the emerging, membrane-based technologies for harvesting salinity gradient energy. In RED process, fouling is an undesirable operation constraint since it leads to a decrease of the obtainable net power density due to increasing stack electric resistance and pressure drop. Therefore, early fouling detection is one of the main challenges for successful RED technology implementation. In the present study, two-dimensional (2D) fluorescence spectroscopy was used, for the first time, as a tool for fouling monitoring in RED. Fluorescence excitation-emission matrices (EEMs) of ion-exchange membrane surfaces and of natural aqueous streams were acquired during one month of a RED stack operation. Fouling evolvement on the ion-exchange membrane surfaces was successfully followed by 2D fluorescence spectroscopy and quantified using principal components analysis (PCA). Additionally, the efficiency of cleaning strategy was assessed by measuring the membrane fluorescence emission intensity before and after cleaning. The anion-exchange membrane (AEM) surface in contact with river water showed to be significantly affected due to fouling by humic compounds, which were found to cross through the membrane from the lower salinity (river water) to higher salinity (sea water) stream. The results obtained show that the combined approach of using 2D fluorescence spectroscopy and PCA has a high potential for studying fouling development and membrane cleaning efficiency in ion exchange membrane processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice.

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Noah Saederup

2010-10-01

Full Text Available Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents.We created CCR2-red fluorescent protein (RFP knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C(hi/CCR2(hi monocytes. Surprisingly, neutrophils, not Ly6C(lo monocytes, largely replaced Ly6C(hi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia.These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.

Red fluorescence of dental plaque in children -A cross-sectional study.

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Volgenant, Catherine M C; Zaura, Egija; Brandt, Bernd W; Buijs, Mark J; Tellez, Marisol; Malik, Gayatri; Ismail, Amid I; Ten Cate, Jacob M; van der Veen, Monique H

2017-03-01

The relation between the presence of red fluorescent plaque and the caries status in children was studied. In addition, the microbial composition of dental plaque from sites with red fluorescent plaque (RFP) and from sites with no red fluorescent plaque (NFP) was assessed. Fluorescence photographs were taken from fifty children (6-14 years old) with overnight plaque. Full-mouth caries scores (ICDAS II) were obtained. The composition of a saliva sample and two plaque samples (RFP and NFP) was assessed using 16S rDNA sequencing. At the site level, no clinically relevant correlations were found between the presence of RFP and the caries status. At the subject level, a weak correlation was found between RFP and the caries status when non-cavitated lesions were included (r s =0.37, p=0.007). The microbial composition of RFP differed significantly from NFP. RFP had more anaerobes and more Gram-negative bacterial taxa. The most discriminative operational taxonomic units (OTUs) for RFP were Corynebacterium, Leptotrichia, Porphyromonas and Selenomonas, while the most discriminative OTUs for NFP were Neisseria, Actinomyces, Streptococcus and Rothia. There were no clinical relevant correlations in this cross-sectional study between the presence of RFP and (early) caries lesions. There were differences in the composition of these phenotypically different plaque samples: RFP contained more Gram-negative, anaerobic taxa and was more diverse than NFP. The study outcomes provide more insight in the possibilities to use plaque fluorescence in oral health risk assessments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same

Science.gov (United States)

Tsien, Roger Y [La Jolla, CA; Wang, Lei [San Diego, CA

2008-07-01

Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters.

OpenAIRE

Paul Carroll; Lise J Schreuder; Julian Muwanguzi-Karugaba; Siouxsie Wiles; Brian D Robertson; Jorge Ripoll; Theresa H Ward; Gregory J Bancroft; Ulrich E Schaible; Tanya Parish

2010-01-01

Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycoba...

Ultrafast proton shuttling in Psammocora cyan fluorescent protein.

Science.gov (United States)

Kennis, John T M; van Stokkum, Ivo H M; Peterson, Dayna S; Pandit, Anjali; Wachter, Rebekka M

2013-09-26

Cyan, green, yellow, and red fluorescent proteins (FPs) homologous to green fluorescent protein (GFP) are used extensively as model systems to study fundamental processes in photobiology, such as the capture of light energy by protein-embedded chromophores, color tuning by the protein matrix, energy conversion by Förster resonance energy transfer (FRET), and excited-state proton transfer (ESPT) reactions. Recently, a novel cyan fluorescent protein (CFP) termed psamFP488 was isolated from the genus Psammocora of reef building corals. Within the cyan color class, psamFP488 is unusual because it exhibits a significantly extended Stokes shift. Here, we applied ultrafast transient absorption and pump-dump-probe spectroscopy to investigate the mechanistic basis of psamFP488 fluorescence, complemented with fluorescence quantum yield and dynamic light scattering measurements. Transient absorption spectroscopy indicated that, upon excitation at 410 nm, the stimulated cyan emission rises in 170 fs. With pump-dump-probe spectroscopy, we observe a very short-lived (110 fs) ground-state intermediate that we assign to the deprotonated, anionic chromophore. In addition, a minor fraction (14%) decays with 3.5 ps to the ground state. Structural analysis of homologous proteins indicates that Glu-167 is likely positioned in sufficiently close vicinity to the chromophore to act as a proton acceptor. Our findings support a model where unusually fast ESPT from the neutral chromophore to Glu-167 with a time constant of 170 fs and resulting emission from the anionic chromophore forms the basis of the large psamFP488 Stokes shift. When dumped to the ground state, the proton on neutral Glu is very rapidly shuttled back to the anionic chromophore in 110 fs. Proton shuttling in excited and ground states is a factor of 20-4000 faster than in GFP, which probably results from a favorable hydrogen-bonding geometry between the chromophore phenolic oxygen and the glutamate acceptor, possibly

Photophysical studies on the interaction of amides with Bovine Serum Albumin (BSA) in aqueous solution: Fluorescence quenching and protein unfolding

International Nuclear Information System (INIS)

Kumaran, R.; Ramamurthy, P.

2014-01-01

Addition. of amides containing a H-CO(NH 2 ) or CH 3 -CO(NH 2 ) framework to BSA results in a fluorescence quenching. On the contrary, fluorescence enhancement with a shift in the emission maximum towards the blue region is observed on the addition of dimethylformamide (DMF) (H-CON(CH 3 ) 2 ). Fluorescence quenching accompanied initially with a shift towards the blue region and a subsequent red shift in the emission maximum of BSA is observed on the addition of formamide (H-CO(NH 2 )), whereas a shift in the emission maximum only towards the red region results on the addition of acetamide (CH 3 -CONH 2 ). Steady state emission spectral studies reveal that amides that possess a free NH 2 and N(CH 3 ) 2 moiety result in fluorescence quenching and enhancement of BSA respectively. The 3D contour spectral studies of BSA with formamide exhibit a shift in the emission towards the red region accompanied with fluorescence quenching, which indicates that the tryptophan residues of the BSA are exposed to a more polar environment. Circular Dichroism (CD) studies of BSA with amides resulted in a gradual decrease in the α-helical content of BSA at 208 nm, which confirms that there is a conformational change in the native structure of BSA. Time-resolved fluorescence studies illustrate that the extent of buried trytophan moieties exposed to the aqueous phase on the addition of amides follows the order DMF 2 hydrogen and the carbonyl oxygen of amide form a concerted hydrogen-bonding network with the carbonyl oxygen and the amino moieties of amino acids respectively is established from fluorescence methods. -- Highlights: • The manuscript deals with the absorption, emission and fluorescence lifetime studies of Bovine Serum Albumin with amides in aqueous medium. • Fluorescence is correlated to the presence of fluorescing amino acid, tryptophan located in a heterogeneous environment. • This article provides an insight about the fluorescence spectral characteristics of a protein

Adsorption properties of BSA and DsRed proteins deposited on thin SiO2 layers: optically non-absorbing versus absorbing proteins

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Scarangella, A.; Soumbo, M.; Villeneuve-Faure, C.; Mlayah, A.; Bonafos, C.; Monje, M.-C.; Roques, C.; Makasheva, K.

2018-03-01

Protein adsorption on solid surfaces is of interest for many industrial and biomedical applications, where it represents the conditioning step for micro-organism adhesion and biofilm formation. To understand the driving forces of such an interaction we focus in this paper on the investigation of the adsorption of bovine serum albumin (BSA) (optically non-absorbing, model protein) and DsRed (optically absorbing, naturally fluorescent protein) on silica surfaces. Specifically, we propose synthesis of thin protein layers by means of dip coating of the dielectric surface in protein solutions with different concentrations (0.01-5.0 g l-1). We employed spectroscopic ellipsometry as the most suitable and non-destructive technique for evaluation of the protein layers’ thickness and optical properties (refractive index and extinction coefficient) after dehydration, using two different optical models, Cauchy for BSA and Lorentz for DsRed. We demonstrate that the thickness, the optical properties and the wettability of the thin protein layers can be finely controlled by proper tuning of the protein concentration in the solution. These results are correlated with the thin layer morphology, investigated by AFM, FTIR and PL analyses. It is shown that the proteins do not undergo denaturation after dehydration on the silica surface. The proteins arrange themselves in a lace-like network for BSA and in a rod-like structure for DsRed to form mono- and multi-layers, due to different mechanisms driving the organization stage.

As-CATH1-6, novel cathelicidins with potent antimicrobial and immunomodulatory properties from Alligator sinensis, play pivotal roles in host antimicrobial immune responses.

Science.gov (United States)

Chen, Yan; Cai, Shasha; Qiao, Xue; Wu, Mali; Guo, Zhilai; Wang, Renping; Kuang, Yi-Qun; Yu, Haining; Wang, Yipeng

2017-08-10

Crocodilians are regarded as possessing a powerful immune system. However, the composition and action of the crocodilian immune system have remained unclear until now. Cathelicidins, the principal family of host defense peptides, play pivotal roles in vertebrate immune defense against microbial invasions. However, cathelicidins from crocodilians have not been extensively studied to date. In the present study, six novel cathelicidins (As-CATH1-6) were identified and characterized from the endangered Chinese alligator ( Alligator sinensis ). As-CATH1-6 exhibit no sequence similarity with any of the known cathelicidins. Structure analysis indicated that As-CATH1-3 adopt a random coil secondary conformation, whereas As-CATH4-6 were predicted to mainly adopt an amphipathic α-helix conformation. Among them, As-CATH4-6 exhibited potent, broad-spectrum and rapid antimicrobial activity by inducing the disruption of cell membrane integrity. They also exhibited strong ability to prevent the formation of bacterial biofilms and eradicate preformed biofilms. Furthermore, As-CATH4-6 exhibited potent anti-inflammatory activity by inhibiting the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and pro-inflammatory cytokines in mouse peritoneal macrophages. They directly neutralized LPS toxicity and therefore inhibited the binding of LPS to the TLR4 receptor and the subsequent activation of inflammatory response pathways. In a peritonitis mice model, As-CATH2-6 provided effective protection against bacterial infection through enhanced immune cell recruitment. In the host Chinese alligator, As-CATH1-6 are mainly expressed in immune organs and epithelial tissues. Bacterial infection significantly enhances their expression, which implies an important role in host anti-infective response. Taken together, the diversity and multiple functions of As-CATH1-6 partially reveal the powerful immune system of the Chinese alligator. © 2017 The Author(s). Published by Portland

Acclimatization of symbiotic corals to mesophotic light environments through wavelength transformation by fluorescent protein pigments.

Science.gov (United States)

Smith, Edward G; D'Angelo, Cecilia; Sharon, Yoni; Tchernov, Dan; Wiedenmann, Joerg

2017-07-12

The depth distribution of reef-building corals exposes their photosynthetic symbionts of the genus Symbiodinium to extreme gradients in the intensity and spectral quality of the ambient light environment. Characterizing the mechanisms used by the coral holobiont to respond to the low intensity and reduced spectral composition of the light environment in deeper reefs (greater than 20 m) is fundamental to our understanding of the functioning and structure of reefs across depth gradients. Here, we demonstrate that host pigments, specifically photoconvertible red fluorescent proteins (pcRFPs), can promote coral adaptation/acclimatization to deeper-water light environments by transforming the prevalent blue light into orange-red light, which can penetrate deeper within zooxanthellae-containing tissues; this facilitates a more homogeneous distribution of photons across symbiont communities. The ecological importance of pcRFPs in deeper reefs is supported by the increasing proportion of red fluorescent corals with depth (measured down to 45 m) and increased survival of colour morphs with strong expression of pcRFPs in long-term light manipulation experiments. In addition to screening by host pigments from high light intensities in shallow water, the spectral transformation observed in deeper-water corals highlights the importance of GFP-like protein expression as an ecological mechanism to support the functioning of the coral- Symbiodinium association across steep environmental gradients. © 2017 The Authors.

Protein- protein interaction detection system using fluorescent protein microdomains

Science.gov (United States)

Waldo, Geoffrey S.; Cabantous, Stephanie

2010-02-23

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants.

Science.gov (United States)

Palencia, Edwin Rene; Glenn, Anthony Elbie; Hinton, Dorothy Mae; Bacon, Charles Wilson

2013-09-01

Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli-maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP1) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri. © 2013.

C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

Science.gov (United States)

Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

2012-01-01

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

Directory of Open Access Journals (Sweden)

Angela Brieger

Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

Radiation levels in Cath Lab and occupational exposures during manual 192Ir intracoronary brachytherapy

International Nuclear Information System (INIS)

Sharma, S.D.; Shanta, A.; Tripathi, U.B.; Bhatt, B.C.

2001-01-01

Intracoronary brachytherapy is a new modality of radiation therapy and is being used to reduce the rate of restenosis after angioplasty. Clinical trials for evaluation of safety and efficacy of manually implanted 192 Ir seed ribbons are underway at various cardiology centres in India. 192 Ir emits high energy gamma rays (0.136 -1.06 MeV), which causes concern regarding safety of the personnel when these sources are manually used in the cardiac catheterization laboratory (Cath Lab) for intracoronary irradiation. Radiation levels in Cath Lab and exposures to personnel have been measured at 6 different cardiology centres in the country during 8 different clinical trials using radiation survey meter, personnel monitoring badges and pocket dosimeters. Activities of 192 Ir seed ribbons used in these clinical trials were in the range of 5.55 - 14.8 GBq. Measured radiation levels behind the mobile lead shields, at the top of lead shields, near the patient head, near the patient toes and at the main door of the Cath Lab were in the range of 2.6-20, 50-256, 385-450, 22-225 and 2-16 μSv/hr/3.7GBq, respectively. Measured effective doses to occupational workers were in range of 14-100 μSv/procedure/3.7GBq. Based on these measurements, user institutions have been advised to use lead glass mounted L-shaped mobile lead shields with proper orientation during clinical trials, avoid unwanted occupancy in the Cath Lab and around the patient during irradiation and use conveniently long forceps or tongs for implantation and removal of sources. (author)

Fluorescence of Alexa fluor dye tracks protein folding.

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Simon Lindhoud

Full Text Available Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488, which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.

Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation.

Science.gov (United States)

Malkani, Naila; Schmid, Johannes A

2011-04-07

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins. When we applied a commonly used FRET microscopy technique--the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10-15% after illumination at the YFP-excitation wavelength--a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor. Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.

pH-dependent fluorescence property of methyl red isomers in silver colloids

International Nuclear Information System (INIS)

Wong, Jian-How; Lee, Szetsen

2012-01-01

We report the use of silver (Ag) colloids in the spectroscopic differentiation of methyl red (MR) isomers (o-MR, m-MR, p-MR) by fluorescence techniques. Under different pH conditions, the formation of MR-Ag complex has an impact on the fluorescence band shapes and peak position shift, which are distinctive between MR isomers. The fluorescence quenching between 400 and 414 nm accompanied by simultaneous enhancement between 510 and 541 nm changes with pH are closely related to energy transfer efficiency and the interaction between the MR isomers and the Ag surface.

Silole-Based Red Fluorescent Organic Dots for Bright Two-Photon Fluorescence In vitro Cell and In vivo Blood Vessel Imaging.

Science.gov (United States)

Chen, Bin; Feng, Guangxue; He, Bairong; Goh, Chiching; Xu, Shidang; Ramos-Ortiz, Gabriel; Aparicio-Ixta, Laura; Zhou, Jian; Ng, Laiguan; Zhao, Zujin; Liu, Bin; Tang, Ben Zhong

2016-02-10

Robust luminescent dyes with efficient two-photon fluorescence are highly desirable for biological imaging applications, but those suitable for organic dots fabrication are still rare because of aggregation-caused quenching. In this work, a red fluorescent silole, 2,5-bis[5-(dimesitylboranyl)thiophen-2-yl]-1-methyl-1,3,4-triphenylsilole ((MesB)2 DTTPS), is synthesized and characterized. (MesB)2 DTTPS exhibits enhanced fluorescence efficiency in nanoaggregates, indicative of aggregation-enhanced emission (AEE). The organic dots fabricated by encapsulating (MesB)2 DTTPS within lipid-PEG show red fluorescence peaking at 598 nm and a high fluorescence quantum yield of 32%. Upon excitation at 820 nm, the dots show a large two-photon absorption cross section of 3.43 × 10(5) GM, which yields a two-photon action cross section of 1.09 × 10(5) GM. These (MesB)2 DTTPS dots show good biocompatibility and are successfully applied to one-photon and two-photon fluorescence imaging of MCF-7 cells and two-photon in vivo visualization of the blood vascular of mouse muscle in a high-contrast and noninvasive manner. Moreover, the 3D blood vasculature located at the mouse ear skin with a depth of over 100 μm can also be visualized clearly, providing the spatiotemporal information about the whole blood vascular network. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescence quantum yield measurements of fluorescent proteins: a laboratory experiment for a biochemistry or molecular biophysics laboratory course.

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Wall, Kathryn P; Dillon, Rebecca; Knowles, Michelle K

2015-01-01

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper-level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum. © 2014 The International Union of Biochemistry and Molecular Biology.

Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

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Davidson Michael W

2008-03-01

Full Text Available Abstract Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP, the expanding set of fluorescent protein (FP variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1, a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His. Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP. To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our

Determination of trace aluminum by fluorescence quenching method based on catalysis of potassium chlorate oxidizing alizarin red

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Shao-Qin, Lin; Xuan, Lin; Shi-Rong, Hu; Li-Qing, Zeng; Yan, Wang; Li, Chen; Jia-Ming, Liu; Long-Di, Li

2005-11-01

A new method for the determination of trace aluminum has been proposed. It is based on the fact that alizarin red can emit strong and stable fluorescence at 80 °C for 30 min and Al 3+ can effectively catalyze potassium chlorate oxidizing alizarin red to form non-fluorescence complex which cause the fluorescence quenching. The linear dynamic range of this method is 0.040-4.00 ng l -1 with a detection limit of 5.3 pg l -1. The regression equation can be expressed as Δ If = 8.731 + 21.73 c (ng l -1), with the correlation coefficient r = 0.9992 ( n = 6). This sensitive, rapid and accurate method has been applied to the determination of trace aluminum(III) in human hair and tea samples successfully. What is more, the mechanism of catalyzing potassium chlorate oxidizing alizarin red by the fluorescence quenching method is also discussed.

Thermal precipitation fluorescence assay for protein stability screening.

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Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

2011-09-01

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Vivenciando um mundo de procedimentos e preocupações: experiência da criança com Port-a-Cath Vivenciando un mundo de procedimientos y preocupaciones: experiencia del niño con Port-a-Cath A world of procedures and worries: experience of children with a Port-a-Cath

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Circéa Amalia Ribeiro

2009-01-01

Full Text Available OBJETIVOS: Compreender como é para a criança com câncer a vivência de ser portadora de Port-a-Cath a partir de suas manifestações numa sessão de Brinquedo Terapêutico Dramático e propiciar a ela um meio de alívio. MÉTODOS: Estudo descritivo qualitativo realizado com seis crianças escolares e uma adolescente, cujos dados foram coletados numa sessão de Brinquedo Terapêutico Dramático e submetidos à análise qualitativa de conteúdo. RESULTADOS: Permitiram compreender que os procedimentos intrusivos geram ansiedade, preocupação, medo e dor às crianças, assim como que elas reconhecem a importância dos procedimentos, dos medicamentos, da realização dos exames físico e laboratoriais para o tratamento; reconhecem as vantagens da utilização do Port-a-Cath, mas que sua utilização é fonte de ansiedade, limitações e preocupações, especialmente as relacionadas ao risco de infecção, e que se sentiram felizes, confortadas e fortalecidas com o brincar. CONSIDERAÇÕES FINAIS: Os enfermeiros precisam estar preparados para assistirem a essas crianças integralmente e para utilizarem sistematicamente o Brinquedo Terapêutico como instrumento de comunicação e intervenção de enfermagem.OBJETIVOS: Comprender cómo es para el niño con cáncer la vivencia de ser portador de Port-a-Cath a partir de sus manifestaciones en una sesión de Juego Terapéutico Dramático y propiciarle un medio de alivio. MÉTODOS: Estudio descriptivo cualitativo realizado con seis niños escolares y una adolescente, cuyos datos fueron recolectados en una sesión de Juego Terapéutico Dramático y sometidos al análisis cualitativo de contenido. RESULTADOS: Se pudo comprender que los procedimientos intrusivos generan ansiedad, preocupación, miedo y dolor a los niños, así como que ellos reconocen la importancia de los procedimientos, de los medicamentos, de la realización de los exámenes físico y de laboratorio para el tratamiento; reconocen las

Cathepsin D Specifically Cleaves the Chemokines Macrophage Inflammatory Protein-1α, Macrophage Inflammatory Protein-1β, and SLC That Are Expressed in Human Breast Cancer

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Wolf, Marlene; Clark-Lewis, Ian; Buri, Caroline; Langen, Hanno; Lis, Maddalena; Mazzucchelli, Luca

2003-01-01

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1α and MIP-1β degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu58 to Trp59 bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1β hybrids indicated that processing of MIP-1β might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1α, MIP-1β, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes. PMID:12651610

Far-red fluorescent probes for canonical and non-canonical nucleic acid structures: current progress and future implications.

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Suseela, Y V; Narayanaswamy, Nagarjun; Pratihar, Sumon; Govindaraju, Thimmaiah

2018-02-05

The structural diversity and functional relevance of nucleic acids (NAs), mainly deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are indispensable for almost all living organisms, with minute aberrations in their structure and function becoming causative factors in numerous human diseases. The standard structures of NAs, termed canonical structures, are supported by Watson-Crick hydrogen bonding. Under special physiological conditions, NAs adopt distinct spatial organisations, giving rise to non-canonical conformations supported by hydrogen bonding other than the Watson-Crick type; such non-canonical structures have a definite function in controlling gene expression and are considered as novel diagnostic and therapeutic targets. Development of molecular probes for these canonical and non-canonical DNA/RNA structures has been an active field of research. Among the numerous probes studied, probes with turn-on fluorescence in the far-red (600-750 nm) region are highly sought-after due to minimal autofluorescence and cellular damage. Far-red fluorescent probes are vital for real-time imaging of NAs in live cells as they provide good resolution and minimal perturbation of the cell under investigation. In this review, we present recent advances in the area of far-red fluorescent probes of DNA/RNA and non-canonical G-quadruplex structures. For the sake of continuity and completeness, we provide a brief overview of visible fluorescent probes. Utmost importance is given to design criteria, characteristic properties and biological applications, including in cellulo imaging, apart from critical discussion on limitations of the far-red fluorescent probes. Finally, we offer current and future prospects in targeting canonical and non-canonical NAs specific to cellular organelles, through sequence- and conformation-specific far-red fluorescent probes. We also cover their implications in chemical and molecular biology, with particular focus on decoding various disease

Congo red and protein aggregation in neurodegenerative diseases.

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Frid, Petrea; Anisimov, Sergey V; Popovic, Natalija

2007-01-01

Congo red is a commonly used histological dye for amyloid detection. The specificity of this staining results from Congo red's affinity for binding to fibril proteins enriched in beta-sheet conformation. Unexpectedly, recent investigations indicate that the dye also possesses the capacity to interfere with processes of protein misfolding and aggregation, stabilizing native protein monomers or partially folded intermediates, while reducing concentration of more toxic protein oligomers. Inhibitory effects of Congo red upon amyloid toxicity may also range from blockade of channel formation and interference with glycosaminoglycans binding or immune functions, to the modulation of gene expression. Particularly, Congo red exhibits ameliorative effect in models of neurodegenerative disorders, such as Alzheimer's, Parkinson's, Huntington's and prion diseases. Another interesting application of Congo red analogues is the development of imaging probes. Based on their small molecular size and penetrability through blood-brain barrier, Congo red congeners can be used for both antemortem and in vivo visualization and quantification of brain amyloids. Therefore, understanding mechanisms involved in dye-amyloidal fibril binding and inhibition of aggregation will provide instructive guides for the design of future compounds, potentially useful for monitoring and treating neurodegenerative diseases.

Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.

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Bryan Sands

Full Text Available Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.

Analysis of reporter proteins GUS and DsRed driven under the control of CaMV35S promoter in syncytia induced by beet cyst nematode Heterodera schachtii in Arabidopsis roots

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Muhammad Amjad Ali

2016-05-01

Full Text Available Background: Cyst nematodes induce specialized feeding structures called syncytia in the plant roots. The expression of CaMV promoter in syncytia has remained topic of debate. The objective of this research was to study the activity of CaMV promoter by using reporter proteins like GUS and DsRed under the control of CaMV35S promoter in syncytia induced by H. schachtii in Arabidopsis roots. Methods: pMAA-Red and pPZP3425 plasmids were used to study expression of GUS and DsRed in syncytia. The plants were grown in 2% Knop medium under sterile conditions in growth chambers at 25°C in long day conditions. GUS activity in syncytia was studied through staining of syncytia using X-gluc solution. Ds-Red fluorescence in syncytia was detected by using an inverse microscope equipped with UV filter. Results: The expression analysis of DsRed protein driven by CaMV promoter demonstrated that this promoter is active in syncytia at all the time points. All the syncytia showed DsRed expression at 5 dpi. At 7 dpi, 10 dpi and 15 dpi over 90%, 80% and 50% of the syncytia showed DsRed fluorescence respectively. There was very high fluorescence in the syncytia as compared to the uninfected root segments due to high expression. CaMV::GUS lines showed GUS expression in 80% of 5dpi syncytia. However, unlike expression of DsRed, the number of GUS stained syncytia decreased quickly to around 50% at 7 dpi and to about 5% in the 15 dpi syncytia. Conclusions: The results conclude that CaMV promoter is more active in younger syncytia as compared to older syncytia but can be used for expression in syncytia. Moreover, DsRed protein could be used as better reporter for evaluation of gene expression in syncytia as compared to GUS.

Development of CRTEIL and CETRIZ, Cre-loxP-Based Systems, Which Allow Change of Expression of Red to Green or Green to Red Fluorescence upon Transfection with a Cre-Expression Vector

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Masato Ohtsuka

2009-01-01

Full Text Available We developed Cre-loxP-based systems, termed CRTEIL and CETRIZ, which allow gene switching in a noninvasive manner. Single transfection with pCRTEIL resulted in predominant expression of red fluorescence. Cotransfection with pCRTEIL and Cre-expression plasmid (pCAG/NCre caused switching from red to green fluorescence. Similarly, cotransfection with pCETRIZ and pCAG/NCre resulted in change of green to red fluorescence. These noninvasive systems will be useful in cell lineage analysis, since descendants of cells exhibiting newly activated gene expression can be continuously monitored in noninvasive fashion.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

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Schneidereit, D; Vass, H; Reischl, B; Allen, R J; Friedrich, O

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa

Science.gov (United States)

Vass, H.; Reischl, B.; Allen, R. J.; Friedrich, O.

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules Δdv¯ is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence. PMID:27764134

Integrated image storage solution for the Cath department.

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Weterings, R A

1998-10-01

Contemporary Image Storage systems for the Catheterization department manage and distribute digital cardiac images according to the "cine-film" paradigm. The images are digital, but the applications have not changed much. This situation will change in the near future. New systems are being developed to store additional (clinical related) data with X-ray Angiographic (XA) Images. Furthermore, the image storage domains are no longer an island in the hospital infrastructure. Efficiency requires the availability of images with other data at the various "point of care" locations. This in turn raises requirements and expectations about the standards in the area of application interoperability, since no single vendor can supply the complete solution. Recent DICOM (Digital Imaging and Communications in Medicine) standardization activities play an important role in extending the current scope of image oriented storage solutions towards a more integrated imaging and information (clinical) folder for the Cath department. The paper will address the following issues: New requirements on "self-contained" Image Storage solutions for the Cath lab. How to deal with the demand for interdepartmental communication using upcoming (new) DICOM standards and HL7 (Health Level Seven) in this area. The increasing influence of computer technology, replacing vendor-specific solutions by general-accepted standards from the Information Technology (IT) world. A step-wise approach to come to an integrated clinical (patient) folder with inherent capabilities for data interchange with other Cardiology departments and the hospitals information infrastructure.

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

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Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-03-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

Changes of the laser-induced blue, green and red fluorescence signatures during greening of etiolated leaves of wheat

International Nuclear Information System (INIS)

Stober, F.; Lichtenthaler, H.K.

1992-01-01

The UV-laser-induced blue, green and red fluorescence-emission spectra were used to characterize the pigment status of etiolated leaves of wheat (Triticum aestivum L.) during a 48 h greening period under white light conditions. Upon UV-light excitation (337 nm) leaves not only show a fluorescence emission in the red spectral region between 650 and 800nm (chlorophyll fluorescence with maxima near 690nm and 735 nm), but also in the blue and green regions between 400 to 570 nm with maxima or shoulders near 450 nm (blue) and 530 nm (green). During greening of etiolated leaves the chlorophyll-fluorescence ratio F690/F735 strongly correlated with the total chlorophyll content and the ratio of the chlorophylls to the carotenoids (a+b/x+c). The ratio of the blue to the green fluorescence F450/F530 was also correlated with the total chlorophyll content and the ratio of chlorophylls to total carotenoids (a+b/x+c). Consequently, there also existed a correlation between the chlorophyll-fluorescence ratio F690/F735 and the ratio of the blue to green fluorescence F450/F530. In contrast, the ratios of the blue to red fluorescences F450/F690 and F450/F735 did not show clear relations to the pigment content of the investigated plants. The particular shape of the UV-laser-induced-fluorescence emission spectra of wheat leaves as well as the dependencies of the fluorescence ratios on the pigment content are due to a partial and differential reabsorption of the emitted fluorescences by the photosynthetic pigments

Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

International Nuclear Information System (INIS)

Nienhaus, Karin; Nienhaus, G Ulrich

2016-01-01

Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments. (topical review)

Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

Science.gov (United States)

Nienhaus, Karin; Nienhaus, G. Ulrich

2016-11-01

Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

Pitfalls in alignment of observation models resolved using PROV as an upper ontology

Science.gov (United States)

Cox, S. J. D.

2015-12-01

A number of models for observation metadata have been developed in the earth and environmental science communities, including OGC's Observations and Measurements (O&M), the ecosystems community's Extensible Observation Ontology (OBOE), the W3C's Semantic Sensor Network Ontology (SSNO), and the CUAHSI/NSF Observations Data Model v2 (ODM2). In order to combine data formalized in the various models, mappings between these must be developed. In some cases this is straightforward: since ODM2 took O&M as its starting point, their terminology is almost completely aligned. In the eco-informatics world observations are almost never made in isolation of other observations, so OBOE pays particular attention to groupings, with multiple atomic 'Measurements' in each oboe:Observation which does not have a result of its own and thus plays a different role to an om:Observation. And while SSN also adopted terminology from O&M, mapping is confounded by the fact that SSN uses DOLCE as its foundation and places ssn:Observations as 'Social Objects' which are explicitly disjoint from 'Events', while O&M is formalized as part of the ISO/TC 211 harmonised (UML) model and sees om:Observations as value assignment activities. Foundational ontologies (such as BFO, GFO, UFO or DOLCE) can provide a framework for alignment, but different upper ontologies can be based in profoundly different worldviews and use of incommensurate frameworks can confound rather than help. A potential resolution is provided by comparing recent studies that align SSN and O&M, respectively, with the PROV-O ontology. PROV-O provides just three base classes: Entity, Activity and Agent. om:Observation is sub-classed from prov:Activity, while ssn:Observation is sub-classed from prov:Entity. This confirms that, despite the same name, om:Observation and ssn:Observation denote different aspects of the observation process: the observation event, and the record of the observation event, respectively. Alignment with the simple

A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging.

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Kinnear, Ekaterina; Caproni, Lisa J; Tregoning, John S

2015-01-01

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy.

Directed evolution of an extremely stable fluorescent protein.

Science.gov (United States)

Kiss, Csaba; Temirov, Jamshid; Chasteen, Leslie; Waldo, Geoffrey S; Bradbury, Andrew R M

2009-05-01

In this paper we describe the evolution of eCGP123, an extremely stable green fluorescent protein based on a previously described fluorescent protein created by consensus engineering (CGP: consensus green protein). eCGP123 could not be denatured by a standard thermal melt, preserved almost full fluorescence after overnight incubation at 80 degrees C and possessed a free energy of denaturation of 12.4 kcal/mol. It was created from CGP by a recursive process involving the sequential introduction of three destabilizing heterologous inserts, evolution to overcome the destabilization and finally 'removal' of the destabilizing insert by gene synthesis. We believe that this approach may be generally applicable to the stabilization of other proteins.

Genetic barcoding with fluorescent proteins for multiplexed applications.

Science.gov (United States)

Smurthwaite, Cameron A; Williams, Wesley; Fetsko, Alexandra; Abbadessa, Darin; Stolp, Zachary D; Reed, Connor W; Dharmawan, Andre; Wolkowicz, Roland

2015-04-14

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded 'indefinitely'. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Diffuse fluorescence tomography of exo- and endogenously labeled tumors

Science.gov (United States)

Balalaeva, Irina V.; Turchin, Ilya V.; Orlova, Anna G.; Plekhanov, Vladimir I.; Shirmanova, Marina V.; Kleshnin, Michail S.; Fiks, Ilya I.; Zagainova, Elena V.; Kamensky, Vladislav A.

2007-06-01

Strong light scattering and absorption limit observation of the internal structure of biological tissue. Only special tools for turbid media imaging, such as optical diffuse tomography, enable noninvasive investigation of the internal biological tissues, including visualization and intravital monitoring of deep tumors. In this work the preliminary results of diffuse fluorescence tomography (DFT) of small animals are presented. Usage of exogenous fluorophores, targeted specifically at tumor cells, and fluorescent proteins expressed endogenously can significantly increase the contrast of obtained images. Fluorescent compounds of different nature, such as sulphonated aluminium phthalocyanine (Photosens), red fluorescing proteins and CdTe/CdSe-core/shell nanocrystals (quantum dots) were applied. We tested diffuse fluorescence tomography method at model media, in post mortem and in vivo experiments. The animal was scanned in transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at wavelength of 532 nm or semiconductor laser at wavelength of 655 nm. Quantum dots or protein DsRed2 in glass capsules (inner diameter 2-3 mm) were placed post mortem inside the esophagus of 7-day-old hairless rats to simulate marked tumors. Photosens was injected intravenously to linear mice with metastazing Lewis lung carcinoma. The reconstruction algorithm, based on Algebraic Reconstruction Technique, was created and tested numerically in model experiments. High contrast images of tumor simulating capsules with DsRed2 concentrations about 10 -6 M and quantum dots about 5x10 -11 M have been obtained. Organ distribution of Photosens and its accumulation in tumors and surrounding tissues of animals has been examined. We have conducted the monitoring of tumors, exogenously labeled by photosensitizer. This work demonstrates potential capabilities of DFT method for intravital detection and monitoring of deep fluorescent

Cath lab costs in patients undergoing percutaneous coronary angioplasty - detailed analysis of consecutive procedures.

Science.gov (United States)

Dziki, Beata; Miechowicz, Izabela; Iwachów, Piotr; Kuzemczak, Michał; Kałmucki, Piotr; Szyszka, Andrzej; Baszko, Artur; Siminiak, Tomasz

2017-01-01

Costs of percutaneous coronary interventions (PCI) have an important impact on health care expenditures. Despite the present stress upon the cost-effectiveness issues in medicine, few comprehensive data exist on costs and resource use in different clinical settings. To assess catheterisation laboratory costs related to use of drugs and single-use devices in patients undergoing PCI due to coronary artery disease. Retrospective analysis of 1500 consecutive PCIs (radial approach, n = 1103; femoral approach, n = 397) performed due to ST segment elevation myocardial infarction (STEMI; n = 345) and non ST-segment elevation myocardial infarction (NSTEMI; n = 426) as well as unstable angina (UA; n = 489) and stable angina (SA; n = 241) was undertaken. Comparative cost analysis was performed and shown in local currency units (PLN). The cath lab costs were higher in STEMI (4295.01 ± 2384.54PLN, p costs were positively correlated with X-ray dose, fluoroscopy, and total procedure times. Patients' age negatively correlated with cath lab costs in STEMI/NSTEMI patients. Cath lab costs were higher in STEMI patients compared to other groups. In STEMI/NSTEMI they were lower in older patients. In all analysed groups costs were related to the level of procedural difficulty. In female patients, the costs of PCI performed via radial approach were higher compared to femoral approach. Despite younger age, male patients underwent more expensive procedures.

Fluorescence Quantum Yield Measurements of Fluorescent Proteins: A Laboratory Experiment for a Biochemistry or Molecular Biophysics Laboratory Course

Science.gov (United States)

Wall, Kathryn P.; Dillon, Rebecca; Knowles, Michelle K.

2015-01-01

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts…

Larval vision contributes to gregarious settlement in barnacles: adult red fluorescence as a possible visual signal

KAUST Repository

Matsumura, K.; Qian, P.-Y.

2014-01-01

surfaces. Moreover, we found that shells of adult B. amphitrite emit red auto-fluorescence and the adult extracts with the fluorescence as a visual signal attracted cyprid larvae to settle around it. We propose that the perception of specific visual signals

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

Science.gov (United States)

Guo, Wenyan; Liu, Xiuli; Liu, Yurong; Gang, Yadong; He, Xiaobin; Jia, Yao; Yin, Fangfang; Li, Pei; Huang, Fei; Zhou, Hongfu; Wang, Xiaojun; Gong, Hui; Luo, Qingming; Xu, Fuqiang; Zeng, Shaoqun

2017-01-01

The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain. PMID:28717566

Fish with red fluorescent eyes forage more efficiently under dim, blue-green light conditions.

Science.gov (United States)

Harant, Ulrike Katharina; Michiels, Nicolaas Karel

2017-04-20

Natural red fluorescence is particularly conspicuous in the eyes of some small, benthic, predatory fishes. Fluorescence also increases in relative efficiency with increasing depth, which has generated speculation about its possible function as a "light organ" to detect cryptic organisms under bluish light. Here we investigate whether foraging success is improved under ambient conditions that make red fluorescence stand out more, using the triplefin Tripterygion delaisi as a model system. We repeatedly presented 10 copepods to individual fish (n = 40) kept under a narrow blue-green spectrum and compared their performance with that under a broad spectrum with the same overall brightness. The experiment was repeated for two levels of brightness, a shaded one representing 0.4% of the light present at the surface and a heavily shaded one with about 0.01% of the surface brightness. Fish were 7% more successful at catching copepods under the narrow, fluorescence-friendly spectrum than under the broad spectrum. However, this effect was significant under the heavily shaded light treatment only. This outcome corroborates previous predictions that fluorescence may be an adaptation to blue-green, heavily shaded environments, which coincides with the opportunistic biology of this species that lives in the transition zone between exposed and heavily shaded microhabitats.

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging.

Science.gov (United States)

Poëa-Guyon, Sandrine; Pasquier, Hélène; Mérola, Fabienne; Morel, Nicolas; Erard, Marie

2013-05-01

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

Fluorescence diffuse tomography for tumor detection and monitoring

Science.gov (United States)

Balalaeva, Irina V.; Orlova, Anna G.; Shirmanova, Marina V.; Kibraeva, Elena A.; Zagainova, Elena V.; Turchin, Ilya V.

2007-05-01

Strong light scattering and absorption limit visualization of the internal structure of biological tissue. Only special tools for turbid media imaging, such as optical diffuse tomography, enable noninvasive investigation of the internal biological tissues, including visualization and intravital monitoring of deep tumors. In this work the preliminary results of fluorescence diffuse tomography (FDT) of small animals are presented. Using of exogenous fluorophores, targeted specifically at tumor cells, and fluorescent proteins expressed endogenously can significantly increase the contrast of obtained images. Fluorescent compounds of different nature, such as sulphonated aluminium phthalocyanine (Photosens), red fluorescing proteins and CdTe/CdSe-core/shell nanocrystals (quantum dots) were applied. The animal was scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the wavelength of 532 nm or semiconductor laser at the wavelength of 655 nm. Photosens was injected intravenously into linear mice with metastazing Lewis lung carcinoma in dose 4 mg/kg. Quantum dots (5x10 -11 M) or protein DsRed2 (1-5x10 -6 M) in glass capsules (inner diameter 2-3 mm) were placed inside the esophagus of 7-day-old hairless rats (18-20 g) to simulate marked tumors. Cells of HEK-293 Phoenix line, transitory transfected with Turbo-RFP protein gene, were injected hypodermically to immunodeficient mice. This work demonstrates potential capabilities of FDT method for detection and monitoring of deep fluorescent-labeled tumors in animal models. Strong advantages of fluorescent proteins and quantum dots over the traditional photosensitizer for FDT imaging are shown.

In vivo cellular imaging using fluorescent proteins - Methods and Protocols

Directory of Open Access Journals (Sweden)

M. Monti

2012-12-01

Full Text Available The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible to the cell often can be made visible with the use of fluorescent proteins. With this words, Robert M. Hoffman introduces In vivo Cellular Imaging Using Fluorescent proteins, the eighteen chapters book dedicated to the description of how fluorescence proteins have changed the way to analyze cellular processes in vivo. Modern researches aim to study new and less invasive methods able to follow the behavior of different cell types in different biological contexts: for example, how cancer cells migrate or how they respond to different therapies. Also, in vivo systems can help researchers to better understand animal embryonic development so as how fluorescence proteins may be used to monitor different processes in living organisms at the molecular and cellular level.

Split green fluorescent protein as a modular binding partner for protein crystallization

International Nuclear Information System (INIS)

Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2013-01-01

A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

Science.gov (United States)

Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

2018-06-01

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

Impact of fluorescent protein fusions on the bacterial flagellar motor.

Science.gov (United States)

Heo, M; Nord, A L; Chamousset, D; van Rijn, E; Beaumont, H J E; Pedaci, F

2017-10-03

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation- new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing

International Nuclear Information System (INIS)

Sakhalkar, H S; Dewhirst, M; Oliver, T; Cao, Y; Oldham, M

2007-01-01

Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing

Energy Technology Data Exchange (ETDEWEB)

Sakhalkar, H S [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, M [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Oliver, T [Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 (United States); Cao, Y [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Oldham, M [Department of Radiation Oncology Physics, and Biomedical Engineering, Duke University Medical Center, Durham, NC 27710 (United States)

2007-04-21

Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate

Unusual expression of red fluorescence at M phase induced by anti-microtubule agents in HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci)

Energy Technology Data Exchange (ETDEWEB)

Honda-Uezono, Asumi [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Kaida, Atsushi [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Michi, Yasuyuki; Harada, Kiyoshi [Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Hayashi, Yoshiki; Hayashi, Yoshio [Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392 (Japan); Miura, Masahiko, E-mail: masa.mdth@tmd.ac.jp [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

2012-11-16

Highlights: Black-Right-Pointing-Pointer Fucci visualizes cell cycle by green and red fluorescence. Black-Right-Pointing-Pointer Plinabulin, induced unusual red fluorescence at M-phase in HeLa-Fucci cells. Black-Right-Pointing-Pointer The unusual pattern was followed by mitotic catastrophe. Black-Right-Pointing-Pointer The unusual pattern may be an early indicator of cell death in HeLa cells. -- Abstract: Plinabulin (NPI-2358) is a novel microtubule-depolymerizing agent. In HeLa cells, plinabulin arrests the cell-cycle at M phase and subsequently induces mitotic catastrophe. To better understand the effects on this compound on the cell-cycle, we used the fluorescent ubiquitination-based cell cycle indicator (Fucci), which normally enables G1 and S/G2/M cells to emit red and green fluorescence, respectively. When HeLa-Fucci cells were treated with 50 nM plinabulin, cells began to fluoresce both green and red in an unusual pattern; most cells exhibited the new pattern after 24 h of treatment. X-irradiation efficiently induced G2 arrest in plinabulin-treated cells and significantly retarded the emergence of the unusual pattern, suggesting that entering M phase is essential for induction of the pattern. By simultaneously visualizing chromosomes with GFP-histone H2B, we established that the pattern emerges after nuclear envelope breakdown but before metaphase. Pedigree assay revealed a significant relationship between the unusual expression and mitotic catastrophe. Nocodazole, KPU-133 (a more potent derivative of plinabulin), and paclitaxel also exerted similar effects. From these data, we conclude that the unusual pattern may be associated with dysregulation of late M phase-specific E3 ligase activity and mitotic catastrophe following treatment with anti-microtubule agents.

Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

DEFF Research Database (Denmark)

Tian, He; Naganathan, Saranga; Kazmi, Manija A

2014-01-01

Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal...

Study of neutral red interaction with DNA by resolution of rank deficient multi-way fluorescence data

DEFF Research Database (Denmark)

Moghaddam, Fatemeh Ghasemi; Kompany Zare, Mohsen; Gholami, Somayeh

2012-01-01

The interaction of neutral red (NR) as an efficient anticancer drug with DNA was studied under physiological pH condition. Three-way data array were recorded by measuring excitation-emission fluorescence during the titration of neutral red with DNA at constant pH. The acid-base equilibrium constant...

Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

Science.gov (United States)

Lei, Ting; Huang, Zheng; Ohno, Nobuhiko; Wu, Bao; Sakoh, Takashi; Saitoh, Yurika; Saiki, Ikuo

2014-01-01

The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes. PMID:24394469

mKikGR, a monomeric photoswitchable fluorescent protein.

Directory of Open Access Journals (Sweden)

Satoshi Habuchi

Full Text Available The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

The Equine Antimicrobial Peptide eCATH1 Is Effective against the Facultative Intracellular Pathogen Rhodococcus equi in Mice

Science.gov (United States)

Schlusselhuber, Margot; Torelli, Riccardo; Martini, Cecilia; Leippe, Matthias; Cattoir, Vincent; Leclercq, Roland; Laugier, Claire; Grötzinger, Joachim; Sanguinetti, Maurizio

2013-01-01

Rhodococcus equi, the causal agent of rhodococcosis, is a major pathogen of foals and is also responsible for severe infections in immunocompromised humans. Of great concern, strains resistant to currently used antibiotics have emerged. As the number of drugs that are efficient in vivo is limited because of the intracellular localization of the bacterium inside macrophages, new active but cell-permeant drugs will be needed in the near future. In the present study, we evaluated, by in vitro and ex vivo experiments, the ability of the alpha-helical equine antimicrobial peptide eCATH1 to kill intracellular bacterial cells. Moreover, the therapeutic potential of the peptide was assessed in experimental rhodococcosis induced in mice, while the in vivo toxicity was evaluated by behavioral and histopathological analysis. The study revealed that eCATH1 significantly reduced the number of bacteria inside macrophages. Furthermore, the bactericidal potential of the peptide was maintained in vivo at doses that appeared to have no visible deleterious effects for the mice even after 7 days of treatment. Indeed, daily subcutaneous injections of 1 mg/kg body weight of eCATH1 led to a significant reduction of the bacterial load in organs comparable to that obtained after treatment with 10 mg/kg body weight of rifampin. Interestingly, the combination of the peptide with rifampin showed a synergistic interaction in both ex vivo and in vivo experiments. These results emphasize the therapeutic potential that eCATH1 represents in the treatment of rhodococcosis. PMID:23817377

Exploiting fluorescence for multiplex immunoassays on protein microarrays

International Nuclear Information System (INIS)

Herbáth, Melinda; Balogh, Andrea; Matkó, János; Papp, Krisztián; Prechl, József

2014-01-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

Impact of fluorescent protein fusions on the bacterial flagellar motor

NARCIS (Netherlands)

Heo, M.; Nord, A. L.; Chamousset, D.; van Rijn, E.; Beaumont, H.J.E.; Pedaci, F.

2017-01-01

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side

Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

Energy Technology Data Exchange (ETDEWEB)

Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

2005-01-01

The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

Spatially Controlled Fabrication of Brightly Fluorescent Nanodiamond-Array with Enhanced Far-Red Si-V Luminescence

Science.gov (United States)

Singh, Sonal; Thomas, Vinoy; Martyshkin, Dmitry; Kozlovskaya, Veronika; Kharlampieva, Eugenia

2014-01-01

We demonstrate a novel approach to precise pattern fluorescent nanodiamond-arrays with enhanced far-red intense photostable luminescence from silicon-vacancy (Si-V) defect centers. The precision-patterned pre-growth seeding of nanodiamonds is achieved by scanning probe “Dip-Pen” nanolithography technique using electrostatically-driven transfer of nanodiamonds from “inked” cantilevers to a UV-treated hydrophilic SiO2 substrate. The enhanced emission from nanodiamond-dots in the far-red is achieved by incorporating Si-V defect centers in subsequent chemical vapor deposition treatment. The development of a suitable nanodiamond ink, mechanism of ink transport, and effect of humidity, dwell time on nanodiamond patterning are investigated. The precision-patterning of as-printed (pre-CVD) arrays with dot diameter and dot height as small as 735 nm ± 27 nm, 61 nm ± 3 nm, respectively and CVD-treated fluorescent ND-arrays with consistently patterned dots having diameter and height as small as 820 nm ± 20 nm, 245 nm ± 23 nm, respectively using 1 s dwell time and 30% RH is successfully achieved. We anticipate that the far-red intense photostable luminescence (~738 nm) observed from Si-V defect centers integrated in spatially arranged nanodiamonds could be beneficial for the development of the next generation fluorescent based devices and applications. PMID:24394286

Mechanical stretching of proteins-a theoretical survey of the Protein Data Bank

International Nuclear Information System (INIS)

Sulkowska, Joanna I; Cieplak, Marek

2007-01-01

The mechanical stretching of single proteins has been studied experimentally for about 50 proteins, yielding a variety of force patterns and peak forces. Here we perform a theoretical survey of proteins of known native structure and map out the landscape of possible dynamical behaviours under stretching at constant speed. We consider 7510 proteins comprising not more than 150 amino acids and 239 longer proteins. The model used is constructed based on the native geometry. It is solved by methods of molecular dynamics and validated by comparing the theoretical predictions to experimental results. We characterize the distribution of peak forces and investigate correlations with the system size and with the structure classification as characterized by the CATH scheme. Despite the presence of such correlations, proteins with the same CATH index may belong to different classes of dynamical behaviour. We identify proteins with the biggest forces and show that they belong to few topology classes. We determine which protein segments act as mechanical clamps and show that, in most cases, they correspond to long stretches of parallel β-strands, but other mechanisms are also possible. (topical review)

In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence.

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Shokoufeh Karimi

Full Text Available Lactobacillus reuteri is a symbiont that inhabits the gastrointestinal (GI tract of mammals, and several strains are used as probiotics. After introduction of probiotic strains in a complex ecosystem like the GI tract, keeping track of them is a challenge. The main objectives of this study were to introduce reporter proteins that would enable in vivo and in vitro detection of L. reuteri and increase knowledge about its interactions with the host. We describe for the first time cloning of codon-optimized reporter genes encoding click beetle red luciferase (CBRluc and red fluorescent protein mCherry in L. reuteri strains ATCC PTA 6475 and R2LC. The plasmid persistence of mCherry-expressing lactobacilli was evaluated by both flow cytometry (FCM and conventional plate count (PC, and the plasmid loss rates measured by FCM were lower overall than those determined by PC. Neutralization of pH and longer induction duration significantly improved the mCherry signal. The persistency, dose-dependent signal intensity and localization of the recombinant bacteria in the GI tract of mice were studied with an in vivo imaging system (IVIS, which allowed us to detect fluorescence from 6475-CBRluc-mCherry given at a dose of 1×1010 CFU and luminescence signals at doses ranging from 1×105 to 1×1010 CFU. Both 6475-CBRluc-mCherry and R2LC-CBRluc were localized in the colon 1 and 2 h after ingestion, but the majority of the latter were still found in the stomach, possibly reflecting niche specificity for R2LC. Finally, an in vitro experiment showed that mCherry-producing R2LC adhered efficiently to the intra cellular junctions of cultured IPEC-J2 cells. In conclusion, the two reporter genes CBRluc and mCherry were shown to be suitable markers for biophotonic imaging (BPI of L. reuteri and may provide useful tools for future studies of in vivo and in vitro interactions between the bacteria and the host.

In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence.

Science.gov (United States)

Karimi, Shokoufeh; Ahl, David; Vågesjö, Evelina; Holm, Lena; Phillipson, Mia; Jonsson, Hans; Roos, Stefan

2016-01-01

Lactobacillus reuteri is a symbiont that inhabits the gastrointestinal (GI) tract of mammals, and several strains are used as probiotics. After introduction of probiotic strains in a complex ecosystem like the GI tract, keeping track of them is a challenge. The main objectives of this study were to introduce reporter proteins that would enable in vivo and in vitro detection of L. reuteri and increase knowledge about its interactions with the host. We describe for the first time cloning of codon-optimized reporter genes encoding click beetle red luciferase (CBRluc) and red fluorescent protein mCherry in L. reuteri strains ATCC PTA 6475 and R2LC. The plasmid persistence of mCherry-expressing lactobacilli was evaluated by both flow cytometry (FCM) and conventional plate count (PC), and the plasmid loss rates measured by FCM were lower overall than those determined by PC. Neutralization of pH and longer induction duration significantly improved the mCherry signal. The persistency, dose-dependent signal intensity and localization of the recombinant bacteria in the GI tract of mice were studied with an in vivo imaging system (IVIS), which allowed us to detect fluorescence from 6475-CBRluc-mCherry given at a dose of 1×1010 CFU and luminescence signals at doses ranging from 1×105 to 1×1010 CFU. Both 6475-CBRluc-mCherry and R2LC-CBRluc were localized in the colon 1 and 2 h after ingestion, but the majority of the latter were still found in the stomach, possibly reflecting niche specificity for R2LC. Finally, an in vitro experiment showed that mCherry-producing R2LC adhered efficiently to the intra cellular junctions of cultured IPEC-J2 cells. In conclusion, the two reporter genes CBRluc and mCherry were shown to be suitable markers for biophotonic imaging (BPI) of L. reuteri and may provide useful tools for future studies of in vivo and in vitro interactions between the bacteria and the host.

Engineering and Characterization of a Superfolder Green Fluorescent Protein

International Nuclear Information System (INIS)

Pedelacq, J.; Cabantous, S.; Tran, T.; Terwilliger, T.; Waldo, G.

2006-01-01

Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP

Highly Selective Fluorescent Sensing of Proteins Based on a Fluorescent Molecularly Imprinted Nanosensor

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Shuo Wang

2013-09-01

Full Text Available A fluorescent molecularly imprinted nanosensor was obtained by grafting imprinted polymer onto the surface of multi-wall carbon nanotubes and post-imprinting treatment with fluorescein isothiocyanate (FITC. The fluorescence of lysozyme-imprinted polymer (Lys-MIP was quenched more strongly by Lys than that of nonimprinted polymer (NIP, which indicated that the Lys-MIP could recognize Lys. The resulted imprinted material has the ability to selectively sense a target protein, and an imprinting factor of 3.34 was achieved. The Lys-MIP also showed selective detection for Lys among other proteins such as cytochrome C (Cyt C, hemoglobin (HB and bovine serum albumin (BSA due to the imprinted sites in the Lys-MIP. This approach combines the high selectivity of surface molecular imprinting technology and fluorescence, and converts binding events into detectable signals by monitoring fluorescence spectra. Therefore, it will have further applications for Lys sensing.

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.

Directory of Open Access Journals (Sweden)

Michael Breen

Full Text Available Influenza A and B viruses (IAV and IBV, respectively cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1 was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer's spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection.

Simultaneous neuron- and astrocyte-specific fluorescent marking

Energy Technology Data Exchange (ETDEWEB)

Schulze, Wiebke [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Hayata-Takano, Atsuko [Molecular Research Center for Children' s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kamo, Toshihiko [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nakazawa, Takanobu, E-mail: takanobunakazawa-tky@umin.ac.jp [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nagayasu, Kazuki [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kasai, Atsushi; Seiriki, Kaoru [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Shintani, Norihito [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ago, Yukio [Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Farfan, Camille [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); and others

2015-03-27

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.

Simultaneous neuron- and astrocyte-specific fluorescent marking

International Nuclear Information System (INIS)

Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille

2015-01-01

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein

Deep-Red Fluorescent Gold Nanoclusters for Nucleoli Staining: Real-Time Monitoring of the Nucleolar Dynamics in Reverse Transformation of Malignant Cells.

Science.gov (United States)

Wang, Xiaojuan; Wang, Yanan; He, Hua; Ma, Xiqi; Chen, Qi; Zhang, Shuai; Ge, Baosheng; Wang, Shengjie; Nau, Werner M; Huang, Fang

2017-05-31

Nucleoli are important subnuclear structures inside cells. We report novel fluorescent gold nanoclusters (K-AuNCs) that are able to stain the nucleoli selectively and make it possible to explore the nucleolar morphology with fluorescence imaging technique. This novel probe is prepared through an easy synthesis method by employing a tripeptide (Lys-Cys-Lys) as the surface ligand. The properties, including deep-red fluorescence emission (680 nm), large Stocks shift, broad excitation band, low cytotoxicity, and good photostability, endow this probe with potential for bioanalytical applications. Because of their small size and their positively charged surface, K-AuNCs are able to accumulate efficiently at the nucleolar regions and provide precise morphological information. K-AuNCs are also used to monitor the nucleolar dynamics along the reverse-transformation process of malignant cells, induced by the agonist of protein A, 8-chloro-cyclic adenosine monophosphate. This gives a novel approach for investigating the working mechanism of antitumor drugs.

On the purported "backbone fluorescence" in protein three-dimensional fluorescence spectra

DEFF Research Database (Denmark)

Bortolotti, Annalisa; Wong, Yin How; Korsholm, Stine S.

2016-01-01

In this study, several proteins (albumin, lysozyme, insulin) and model compounds (Trp, Tyr, homopolypeptides) were used to demonstrate the origin of the fluorescence observed upon their excitation at 220-230 nm. In the last 10 years we have observed a worrying increase in the number of articles...... as any traditional protein emission spectrum. The many papers in reputable journals erroneously reporting this peak assignment, contradicting 5 decades of prior knowledge, have led to the creation of a new dogma, where many authors and reviewers now take the purported backbone fluorescence...... as an established fact. We hope the current paper helps counter this new situation and leads to a reassessment of those papers that make this erroneous claim....

The Use of Virtual Reality Computer Simulation in Learning Port-A Cath Injection

Science.gov (United States)

Tsai, Sing-Ling; Chai, Sin-Kuo; Hsieh, Li-Feng; Lin, Shirling; Taur, Fang-Meei; Sung, Wen-Hsu; Doong, Ji-Liang

2008-01-01

Cost-benefit management trends in Taiwan healthcare settings have led nurses to perform more invasive skills, such as Port-A cath administration of medications. Accordingly, nurses must be well-prepared prior to teaching by the mentor and supervision method. The purpose of the current study was to develop a computer-assisted protocol using virtual…

Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

Science.gov (United States)

Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

2017-01-01

In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

Cardiac magnetic resonance and the need for routine cardiac catheterization in single ventricle patients prior to Fontan: a comparison of 3 groups: pre-Fontan CMR versus cath evaluation.

Science.gov (United States)

Fogel, Mark A; Pawlowski, Thomas W; Whitehead, Kevin K; Harris, Matthew A; Keller, Marc S; Glatz, Andrew C; Zhu, Winnie; Shore, David; Diaz, Laura K; Rome, Jonathan J

2012-09-18

This study investigated whether cardiac magnetic resonance (CMR) and echocardiography (echo) can replace catheterization (cath) for routine evaluation prior to Fontan and under what circumstances CMR and cath are used together. Routine cath prior to Fontan has been utilized for years; noninvasive methods, however, may be sufficient. This study reviews clinical data in 119 consecutive patients investigating 3 groups: those who underwent CMR alone (MR; n = 41), cath alone (C; n = 41), or both cath and CMR (C+M; n = 37) prior to Fontan. No clinically significant differences were noted in patient characteristics, hemodynamics, or clinical status prior to or after surgery between the C and MR groups. CMR added information in 82%. There were no discrepant findings between CMR and cath data in the C+M group. Diagnostic success was ≥95% in all groups. Of those undergoing Fontan completion, the C+M group had similar outcomes to C and MR; C and CMR were utilized in combination to assess aortopulmonary collaterals or the need for an intervention or evaluate its success. Echo could not delineate pulmonary arterial anatomy in 46% to 53% of patients. The C+M and C groups were exposed to 6.8 ± 4.1 mSv of radiation. Single ventricle patients not requiring an intervention can undergo successful Fontan completion with CMR and echo alone with similar short-term outcomes to C, which was used as a control, preventing an invasive test and exposure to radiation. CMR can add information in a significant number of patients. Cath and CMR are utilized together for interventions and assessment of aortopulmonary collaterals. Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Two-photon excited UV fluorescence for protein crystal detection

International Nuclear Information System (INIS)

Madden, Jeremy T.; DeWalt, Emma L.; Simpson, Garth J.

2011-01-01

Complementary measurements using SONICC and TPE-UVF allow the sensitive and selective detection of protein crystals. Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC

Photonic reagents for concentration measurement of flu-orescent proteins with overlapping spectra

Science.gov (United States)

Goun, Alexei; Bondar, Denys I.; Er, Ali O.; Quine, Zachary; Rabitz, Herschel A.

2016-05-01

By exploiting photonic reagents (i.e., coherent control by shaped laser pulses), we employ Optimal Dynamic Discrimination (ODD) as a novel means for quantitatively characterizing mixtures of fluorescent proteins with a large spectral overlap. To illustrate ODD, we simultaneously measured concentrations of in vitro mixtures of Enhanced Blue Fluorescent Protein (EBFP) and Enhanced Cyan Fluorescent Protein (ECFP). Building on this foundational study, the ultimate goal is to exploit the capabilities of ODD for parallel monitoring of genetic and protein circuits by suppressing the spectral cross-talk among multiple fluorescent reporters.

Do the fluorescent red eyes of the marine fish Tripterygion delaisi stand out? In situ and in vivo measurements at two depths.

Science.gov (United States)

Harant, Ulrike K; Santon, Matteo; Bitton, Pierre-Paul; Wehrberger, Florian; Griessler, Thomas; Meadows, Melissa G; Champ, Connor M; Michiels, Nico K

2018-05-01

Since the discovery of red fluorescence in fish, much effort has been invested to elucidate its potential functions, one of them being signaling. This implies that the combination of red fluorescence and reflection should generate a visible contrast against the background. Here, we present in vivo iris radiance measurements of Tripterygion delaisi under natural light conditions at 5 and 20 m depth. We also measured substrate radiance of shaded and exposed foraging sites at those depths. To assess the visual contrast of the red iris against these substrates, we used the receptor noise model for chromatic contrasts and Michelson contrast for achromatic calculations. At 20 m depth, T. delaisi iris radiance generated strong achromatic contrasts against substrate radiance, regardless of exposure, and despite substrate fluorescence. Given that downwelling light above 600 nm is negligible at this depth, we can attribute this effect to iris fluorescence. Contrasts were weaker in 5 m. Yet, the pooled radiance caused by red reflection and fluorescence still exceeded substrate radiance for all substrates under shaded conditions and all but Jania rubens and Padina pavonia under exposed conditions. Due to the negative effects of anesthesia on iris fluorescence, these estimates are conservative. We conclude that the requirements to create visual brightness contrasts are fulfilled for a wide range of conditions in the natural environment of T. delaisi .

Protein recognition by a pattern-generating fluorescent molecular probe

Science.gov (United States)

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M.; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M.; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

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Yuki Horiuchi

2018-01-01

Full Text Available Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

Rhodamine 800 as reference substance for fluorescence quantum yield measurements in deep red emission range

Energy Technology Data Exchange (ETDEWEB)

Alessi, A., E-mail: andrea.alessi@eni.com [Centro Ricerche per le Energie non Convenzionali, Istituto eni Donegani, e.n.i. S.p.A., Via G. Fauser 4, 28100 Novara (Italy); Salvalaggio, M. [Centro Ricerche per le Energie non Convenzionali, Istituto eni Donegani, e.n.i. S.p.A., Via G. Fauser 4, 28100 Novara (Italy); Ruzzon, G. [HORIBA Jobin Yvon Srl, Via Cesare Pavese 35/AB, 20090 Opera Milano (Italy)

2013-02-15

The determination of fluorescence quantum yields ({Phi}{sub f}) of deep red dyes emitting at 635-900 nm is difficult due to lack of suitable standards. In this work, we propose a commercial dye, rhodamine 800 (Rho800), as reference standard which belongs to the family of xanthenes. The quantum yield of rhodamine 800 in absolute ethanol has been studied using a relative method with cresyl violet (CV) and rhodamine 101 (Rho101) as references, and an absolute fluorometric method by integrating sphere measurements. - Highlights: Black-Right-Pointing-Pointer A red emitting dye Rhodamine 800 was electronic spectroscopy characterized. Black-Right-Pointing-Pointer Its fluorescence quantum yield was studied using a relative and an absolute method. Black-Right-Pointing-Pointer The values found are greater than the values currently known in the literature.

Red fluorescent chitosan nanoparticles grafted with poly(2-methacryloyloxyethyl phosphorylcholine) for live cell imaging.

Science.gov (United States)

Wang, Ke; Fan, Xingliang; Zhang, Xiaoyong; Zhang, Xiqi; Chen, Yi; Wei, Yen

2016-08-01

Poly(2-methacryloyloxyethyl phosphorylcholine) conjugated red fluorescent chitosan nanoparticles (GCC-pMPC) were facilely fabricated by "grafting from" method via surface initiated atom transfer radical polymerization (ATRP). Firstly, glutaraldehyde crosslinked red fluorescent chitosan nanoparticles (GCC NPs) with many amino groups and hydroxyl groups on their surface were prepared, which were then reacted with 2-bromoisobutyryl bromide to form GCC-Br; subsequently, poly(MPC) (pMPC) brushes were grafted onto GCC NPs surface using GCC-Br as initiator via ATRP. Compared with PEGylated nanoparticles, zwitterionic polymers modified nanoparticles demonstrated better performance in their cellular uptake. Moreover, the obtained GCC-pMPC demonstrated excellent water-dispersibility, biocompatibility, and photostability, which made them highly potential for long-term tracing applications. Importantly, the successful live cell imaging of GCC-pMPC would remarkably advance the research of their further bioapplications. Copyright © 2016 Elsevier B.V. All rights reserved.

Cross-over between discrete and continuous protein structure space: insights into automatic classification and networks of protein structures.

Directory of Open Access Journals (Sweden)

Alberto Pascual-García

2009-03-01

. As a domain set, we have selected a consensus set of 2,890 domains decomposed very similarly in SCOP and CATH. As an alignment algorithm, we used a global version of MAMMOTH developed in our group, which is both rapid and accurate. As a similarity measure, we used the size-normalized contact overlap, and as a clustering algorithm, we used average linkage. The resulting automatic classification at the cross-over point was more consistent than expert ones with respect to the structure similarity measure, with 86% of the clusters corresponding to subsets of either SCOP or CATH superfamilies and fewer than 5% containing domains in distinct folds according to both SCOP and CATH. Almost 15% of SCOP superfamilies and 10% of CATH superfamilies were split, consistent with the notion of fold change in protein evolution. These results were qualitatively robust for all choices that we tested, although we did not try to use alignment algorithms developed by other groups. Folds defined in SCOP and CATH would be completely joined in the regime of large transitivity violations where clustering is more arbitrary. Consistently, the agreement between SCOP and CATH at fold level was lower than their agreement with the automatic classification obtained using as a clustering algorithm, respectively, average linkage (for SCOP or single linkage (for CATH. The networks representing significant evolutionary and structural relationships between clusters beyond the cross-over point may allow us to perform evolutionary, structural, or functional analyses beyond the limits of classification schemes. These networks and the underlying clusters are available at http://ub.cbm.uam.es/research/ProtNet.php.

Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging

NARCIS (Netherlands)

Ordonez Alvarez, Soledad; Amarullah, Ilham H; Wubbolts, Richard W; Veldhuizen, Edwin J A; Haagsman, Henk P

2014-01-01

Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the

A interpretação de provérbios em função do sexo e da idade

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José Fernando B. Lomonaco

Full Text Available O presente estudo teve o objetivo de investigar a transição do pensamento concreto para o abstrato através da interpretação de provérbios. Pata tal finalidade foram traduzidos e adaptados 15 provérbios constantes do Teste de Provérbios (Gorham, 1956, os quais foram aplicados a uma amostra de sujeitos agrupados em três faixas etárias: 9, 12 e 15 anos. A tarefa dos sujeitos consistia em escolher, dentre quatro alternativas de resposta, aquela que melhor explicasse o significado de cada provérbio. Tais alternativas são consideradas ou classificadas como abstratas quando implicam num afastamento do sentido literal do provérbio ou concretas, quando estão fortemente ligadas a esse tipo dê significado. Verificou-se que: a à medida que os sujeitos se tomavam mais velhos aumentava a emissão de respostas abstratas e diminuía a de concretas e, b essa tendência foi mais acentuada no sexo feminino uma vez que aos 12 anos as meninas emitiram significantemente mais respostas abstratas do que os meninos. Os resultados foram discutidos em termos de diferenças no condicionamento verbal de meninos e meninas.

Effect of Different Light Qualities on Growth, Pigment Content, Chlorophyll Fluorescence, and Antioxidant Enzyme Activity in the Red Alga Pyropia haitanensis (Bangiales, Rhodophyta).

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Wu, Huanyang

2016-01-01

Spectral light changes evoke different morphogenetic and photosynthetic responses that can vary among different algae species. The aim of this study is to investigate the photosynthetic characteristics of the red macroalgae grown under different spectrum environments. In this study, Pyropia haitanensis were cultured under blue, red, and green LED and fluorescent tubes light. The growth rate, photopigment composition, chlorophyll fluorescence, and antioxidative enzymes activities in different light spectrums were investigated. The results revealed that growth rate was significantly higher in the thalli grown under blue, green, and fluorescent tubes light. Contents of Chl a and phycobiliprotein in red light were lower among all the growth conditions. Furthermore, a striking increase in SOD and CAT activity was observed in red light treatment along with the NPQ increase. The results revealed that the photosynthetic efficiency and increased growth rate of P. haitanensis benefitted from light spectrums such as blue, green, and fluorescent tubes light by pigment composition and photochemical efficiency manipulation, whereas red light has disadvantageous effects. Accordingly, the results for improving quality and the economic yield of algae species in some extent and the combination of different wavelengths could allow better economic resource exploitation.

Effect of Different Light Qualities on Growth, Pigment Content, Chlorophyll Fluorescence, and Antioxidant Enzyme Activity in the Red Alga Pyropia haitanensis (Bangiales, Rhodophyta

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Huanyang Wu

2016-01-01

Full Text Available Spectral light changes evoke different morphogenetic and photosynthetic responses that can vary among different algae species. The aim of this study is to investigate the photosynthetic characteristics of the red macroalgae grown under different spectrum environments. In this study, Pyropia haitanensis were cultured under blue, red, and green LED and fluorescent tubes light. The growth rate, photopigment composition, chlorophyll fluorescence, and antioxidative enzymes activities in different light spectrums were investigated. The results revealed that growth rate was significantly higher in the thalli grown under blue, green, and fluorescent tubes light. Contents of Chl a and phycobiliprotein in red light were lower among all the growth conditions. Furthermore, a striking increase in SOD and CAT activity was observed in red light treatment along with the NPQ increase. The results revealed that the photosynthetic efficiency and increased growth rate of P. haitanensis benefitted from light spectrums such as blue, green, and fluorescent tubes light by pigment composition and photochemical efficiency manipulation, whereas red light has disadvantageous effects. Accordingly, the results for improving quality and the economic yield of algae species in some extent and the combination of different wavelengths could allow better economic resource exploitation.

Highly efficient red OLEDs using DCJTB as the dopant and delayed fluorescent exciplex as the host.

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Zhao, Bo; Zhang, Tianyou; Chu, Bei; Li, Wenlian; Su, Zisheng; Wu, Hairuo; Yan, Xingwu; Jin, Fangming; Gao, Yuan; Liu, Chengyuan

2015-05-29

In this manuscript, we demonstrated a highly efficient DCJTB emission with delayed fluorescent exciplex TCTA:3P-T2T as the host. For the 1.0% DCJTB doped concentration, a maximum luminance, current efficiency, power efficiency and EQE of 22,767 cd m(-2), 22.7 cd A(-1), 21.5 lm W(-1) and 10.15% were achieved, respectively. The device performance is the best compared to either red OLEDs with traditional fluorescent emitter or traditional red phosphor of Ir(piq)3 doped into CBP host. The extraction of so high efficiency can be explained as the efficient triplet excitons up-conversion of TCTA:3P-T2T and the energy transfer from exciplex host singlet state to DCJTB singlet state.

Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research.

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Takuya Kobayashi

Full Text Available BACKGROUND: Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel multifunctional green fluorescent protein (mfGFP tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis, streptavidin-binding peptide (SBP, and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM. These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry. CONCLUSIONS AND SIGNIFICANCE: The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

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Whittington, Niteace C; Wray, Susan

2017-10-23

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

The potential of a fluorescent-based approach for bioassay of antifungal agents against chili anthracnose disease in Thailand.

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Chutrakul, Chanikul; Khaokhajorn, Pratoomporn; Auncharoen, Patchanee; Boonruengprapa, Tanapong; Mongkolporn, Orarat

2013-01-01

Severe chili anthracnose disease in Thailand is caused by Colletotrichum gloeosporioides and C. capsici. To discover anti-anthracnose substances we developed an efficient dual-fluorescent labeling bioassay based on a microdilution approach. Indicator strains used in the assay were constructed by integrating synthetic green fluorescent protein (sGFP) and Discosoma sp. red fluorescent protein (DsRedExp) genes into the genomes of C. gloeosporioides or C. capsici respectively. Survival of co-spore cultures in the presence of inhibitors was determined by the expression levels of these fluorescent proteins. This developed assay has high potential for utilization in the investigation of selective inhibition activity to either one of the pathogens as well as the broad-range inhibitory effect against both pathogens. The value of using the dual-fluorescent assay is rapid, reliable, and consistent identification of anti-anthracnose agents. Most of all, the assay enables the identification of specific inhibitors under the co-cultivation condition.

Spatially controlled fabrication of a bright fluorescent nanodiamond-array with enhanced far-red Si-V luminescence.

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Singh, Sonal; Thomas, Vinoy; Martyshkin, Dmitry; Kozlovskaya, Veronika; Kharlampieva, Eugenia; Catledge, Shane A

2014-01-31

We demonstrate a novel approach to precisely pattern fluorescent nanodiamond-arrays with enhanced far-red intense photostable luminescence from silicon-vacancy (Si-V) defect centers. The precision-patterned pre-growth seeding of nanodiamonds is achieved by a scanning probe 'dip-pen' nanolithography technique using electrostatically driven transfer of nanodiamonds from 'inked' cantilevers to a UV-treated hydrophilic SiO2 substrate. The enhanced emission from nanodiamond dots in the far-red is achieved by incorporating Si-V defect centers in a subsequent chemical vapor deposition treatment. The development of a suitable nanodiamond ink and mechanism of ink transport, and the effect of humidity and dwell time on nanodiamond patterning are investigated. The precision patterning of as-printed (pre-CVD) arrays with dot diameter and dot height as small as 735 nm ± 27 nm and 61 nm ± 3 nm, respectively, and CVD-treated fluorescent ND-arrays with consistently patterned dots having diameter and height as small as 820 nm ± 20 nm and, 245 nm ± 23 nm, respectively, using 1 s dwell time and 30% RH is successfully achieved. We anticipate that the far-red intense photostable luminescence (~738 nm) observed from Si-V defect centers integrated in spatially arranged nanodiamonds could be beneficial for the development of next generation fluorescence-based devices and applications.

Control of the blue fluorescent protein with advanced evolutionary pulse shaping

International Nuclear Information System (INIS)

Tkaczyk, Eric R.; Mauring, Koit; Tkaczyk, Alan H.; Krasnenko, Veera; Ye, Jing Yong; Baker, James R.; Norris, Theodore B.

2008-01-01

We demonstrate optical coherent control of the two-photon fluorescence of the blue fluorescent protein (BFP), which is of interest in investigations of protein-protein interactions. In addition to biological relevance, BFP represents an interesting target for coherent control from a chemical perspective due to its many components of highly nonexponential fluorescence decay and low quantum yield resulting from excited state isomerization. Using a genetic algorithm with a multiplicative (rather than ratiometric) fitness parameter, we are able to control the ratio of BFP fluorescence to second-harmonic generation without a considerable drop in the maximized signal. The importance of linear chirp and power-scaling on the discrimination process is investigated in detail

An orange fluorescent protein tagging system for real-time pollen tracking.

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Rice, J Hollis; Millwood, Reginald J; Mundell, Richard E; Chambers, Orlando D; Abercrombie, Laura L; Davies, H Maelor; Stewart, C Neal

2013-09-27

Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum × Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification. We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum 'TN 90' and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression. The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.

Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

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Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

2015-05-15

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

Decay time shortening of fluorescence from donor-acceptor pair proteins using ultrafast time-resolved fluorescence resonance energy transfer spectroscopy

International Nuclear Information System (INIS)

Baba, Motoyoshi; Suzuki, Masayuki; Ganeev, Rashid A.; Kuroda, Hiroto; Ozaki, Tsuneyuki; Hamakubo, Takao; Masuda, Kazuyuki; Hayashi, Masahiro; Sakihama, Toshiko; Kodama, Tatsuhiko; Kozasa, Tohru

2007-01-01

We improved an ultrafast time-resolved fluorescence resonance energy transfer (FRET) spectroscopy system and measured directly the decrease in the fluorescence decay time of the FRET signal, without any entanglement of components in the picosecond time scale from the donor-acceptor protein pairs (such as cameleon protein for calcium ion indicator, and ligand-activated GRIN-Go proteins pair). The drastic decrease in lifetime of the donor protein fluorescence under the FRET condition (e.g. a 47.8% decrease for a GRIN-Go protein pair) proves the deformation dynamics between donor and acceptor fluorescent proteins in an activated state of a mixed donor-acceptor protein pair. This study is the first clear evidence of physical contact of the GRIN-Go proteins pair using time-resolved FRET system. G protein-coupled receptors (GPCRs) are the most important protein family for the recognition of many chemical substances at the cell surface. They are the targets of many drugs. Simultaneously, we were able to observe the time-resolved spectra of luminous proteins at the initial stage under the FRET condition, within 10 ns from excitation. This new FRET system allows us to trace the dynamics of the interaction between proteins at the ligand-induced activated state, molecular structure change and combination or dissociation. It will be a key technology for the development of protein chip technology

Enhanced emission of nile red fluorescent nanoparticles embedded in hybrid sol-gel glasses.

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Ferrer, Maria L; del Monte, Francisco

2005-01-13

Highly fluorescent Nile Red (NR) nanoparticles embedded in a hybrid sol-gel glass are reported. The crystallite growth within the confined system created by the porous hybrid matrix results in NR nanoparticles of averaged dimensions below 36 nm. The preparation process allows for the control of both the conformation adopted by single NR molecules prior to aggregation (e.g., near planar) and the configuration of the aggregates (e.g., oblique with phi architecture which ultimately forms the nanoparticles. The full preservation of the fluorescent configuration of the aggregates in the nanoparticles is confirmed through the application of the exciton theory, and it is responsible for the significant increase of the fluorescence emission intensity (e.g., up to 525- and 70-fold as compared to that obtained for single NR molecules embedded in pure and hybrid silica glasses, respectively).

Glycine Insertion Makes Yellow Fluorescent Protein Sensitive to Hydrostatic Pressure

Science.gov (United States)

Watanabe, Tomonobu M.; Imada, Katsumi; Yoshizawa, Keiko; Nishiyama, Masayoshi; Kato, Chiaki; Abe, Fumiyoshi; Morikawa, Takamitsu J.; Kinoshita, Miki; Fujita, Hideaki; Yanagida, Toshio

2013-01-01

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure. PMID:24014139

Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads

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Dae Hong Jeong

2012-03-01

Full Text Available Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.

Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm.

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Li, ZhaoYang; Zhang, ZhiPing; Bi, LiJun; Cui, ZongQiang; Deng, JiaoYu; Wang, DianBing; Zhang, Xian-En

2016-01-01

GFP-like fluorescent proteins with diverse emission wavelengths have been developed through mutagenesis, offering many possible choices in cellular and tissue imaging, such as multi-targets imaging, deep tissue imaging that require longer emission wavelength. Here, we utilized a combined approach of random mutation and structure-based rational design to develop new NIR fluorescent proteins on the basis of a far-red fluorescent protein, mNeptune (Ex/Em: 600/650 nm). We created a number of new monomeric NIR fluorescent proteins with the emission range of 681-685 nm, which exhibit the largest Stocks shifts (77-80 nm) compared to other fluorescent proteins. Among them, mNeptune681 and mNeptune684 exhibit more than 30 nm redshift in emission relative to mNeptune, owing to the major role of the extensive hydrogen-bond network around the chromophore and contributions of individual mutations to the observed redshift. Furthermore, the two variants still maintain monomeric state in solution, which is a trait crucial for their use as protein tags. In conclusion, our results suggest that there is untapped potential for developing fluorescent proteins with desired properties.

ONIOM Investigation of the Second-Order Nonlinear Optical Responses of Fluorescent Proteins.

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de Wergifosse, Marc; Botek, Edith; De Meulenaere, Evelien; Clays, Koen; Champagne, Benoît

2018-05-17

The first hyperpolarizability (β) of six fluorescent proteins (FPs), namely, enhanced green fluorescent protein, enhanced yellow fluorescent protein, SHardonnay, ZsYellow, DsRed, and mCherry, has been calculated to unravel the structure-property relationships on their second-order nonlinear optical properties, owing to their potential for multidimensional biomedical imaging. The ONIOM scheme has been employed and several of its refinements have been addressed to incorporate efficiently the effects of the microenvironment on the nonlinear optical responses of the FP chromophore that is embedded in a protective β-barrel protein cage. In the ONIOM scheme, the system is decomposed into several layers (here two) treated at different levels of approximation (method1/method2), from the most elaborated method (method1) for its core (called the high layer) to the most approximate one (method2) for the outer surrounding (called the low layer). We observe that a small high layer can already account for the variations of β as a function of the nature of the FP, provided the low layer is treated at an ab initio level to describe properly the effects of key H-bonds. Then, for semiquantitative reproduction of the experimental values obtained from hyper-Rayleigh scattering experiments, it is necessary to incorporate electron correlation as described at the second-order Møller-Plesset perturbation theory (MP2) level as well as implicit solvent effects accounted for using the polarizable continuum model (PCM). This led us to define the MP2/6-31+G(d):HF/6-31+G(d)/IEFPCM scheme as an efficient ONIOM approach and the MP2/6-31+G(d):HF/6-31G(d)/IEFPCM as a better compromise between accuracy and computational needs. Using these methods, we demonstrate that many parameters play a role on the β response of FPs, including the length of the π-conjugated segment, the variation of the bond length alternation, and the presence of π-stacking interactions. Then, noticing the small diversity

A green fluorescent protein with photoswitchable emission from the deep sea.

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Alexander Vogt

Full Text Available A colorful variety of fluorescent proteins (FPs from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that approximately 15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37 degrees C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.

Plasmonic photocatalyst-like fluorescent proteins for generating reactive oxygen species

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Leem, Jung Woo; Kim, Seong-Ryul; Choi, Kwang-Ho; Kim, Young L.

2018-03-01

The recent advances in photocatalysis have opened a variety of new possibilities for energy and biomedical applications. In particular, plasmonic photocatalysis using hybridization of semiconductor materials and metal nanoparticles has recently facilitated the rapid progress in enhancing photocatalytic efficiency under visible or solar light. One critical underlying aspect of photocatalysis is that it generates and releases reactive oxygen species (ROS) as intermediate or final products upon light excitation or activation. Although plasmonic photocatalysis overcomes the limitation of UV irradiation, synthesized metal/semiconductor nanomaterial photocatalysts often bring up biohazardous and environmental issues. In this respect, this review article is centered in identifying natural photosensitizing organic materials that can generate similar types of ROS as those of plasmonic photocatalysis. In particular, we propose the idea of plasmonic photocatalyst-like fluorescent proteins for ROS generation under visible light irradiation. We recapitulate fluorescent proteins that have Type I and Type II photosensitization properties in a comparable manner to plasmonic photocatalysis. Plasmonic photocatalysis and protein photosensitization have not yet been compared systemically in terms of ROS photogeneration under visible light, although the phototoxicity and cytotoxicity of some fluorescent proteins are well recognized. A comprehensive understanding of plasmonic photocatalyst-like fluorescent proteins and their potential advantages will lead us to explore new environmental, biomedical, and defense applications.

Spatially controlled fabrication of a bright fluorescent nanodiamond-array with enhanced far-red Si-V luminescence

International Nuclear Information System (INIS)

Singh, Sonal; Thomas, Vinoy; Kharlampieva, Eugenia; Catledge, Shane A; Martyshkin, Dmitry; Kozlovskaya, Veronika

2014-01-01

We demonstrate a novel approach to precisely pattern fluorescent nanodiamond-arrays with enhanced far-red intense photostable luminescence from silicon-vacancy (Si-V) defect centers. The precision-patterned pre-growth seeding of nanodiamonds is achieved by a scanning probe ‘dip-pen’ nanolithography technique using electrostatically driven transfer of nanodiamonds from ‘inked’ cantilevers to a UV-treated hydrophilic SiO 2 substrate. The enhanced emission from nanodiamond dots in the far-red is achieved by incorporating Si-V defect centers in a subsequent chemical vapor deposition treatment. The development of a suitable nanodiamond ink and mechanism of ink transport, and the effect of humidity and dwell time on nanodiamond patterning are investigated. The precision patterning of as-printed (pre-CVD) arrays with dot diameter and dot height as small as 735 nm ± 27 nm and 61 nm ± 3 nm, respectively, and CVD-treated fluorescent ND-arrays with consistently patterned dots having diameter and height as small as 820 nm ± 20 nm and, 245 nm ± 23 nm, respectively, using 1 s dwell time and 30% RH is successfully achieved. We anticipate that the far-red intense photostable luminescence (∼738 nm) observed from Si-V defect centers integrated in spatially arranged nanodiamonds could be beneficial for the development of next generation fluorescence-based devices and applications. (paper)

New Methods for Retrieval of Chlorophyll Red Fluorescence from Hyperspectral Satellite Instruments: Simulations and Application to GOME-2 and SCIAMACHY

Science.gov (United States)

Joiner, Joanna; Yoshida, Yasuko; Guanter, Luis; Middleton, Elizabeth M.

2016-01-01

Global satellite measurements of solar-induced fluorescence (SIF) from chlorophyll over land and ocean have proven useful for a number of different applications related to physiology, phenology, and productivity of plants and phytoplankton. Terrestrial chlorophyll fluorescence is emitted throughout the red and far-red spectrum, producing two broad peaks near 683 and 736nm. From ocean surfaces, phytoplankton fluorescence emissions are entirely from the red region (683nm peak). Studies using satellite-derived SIF over land have focused almost exclusively on measurements in the far red (wavelengths greater than 712nm), since those are the most easily obtained with existing instrumentation. Here, we examine new ways to use existing hyperspectral satellite data sets to retrieve red SIF (wavelengths less than 712nm) over both land and ocean. Red SIF is thought to provide complementary information to that from the far red for terrestrial vegetation. The satellite instruments that we use were designed to make atmospheric trace-gas measurements and are therefore not optimal for observing SIF; they have coarse spatial resolution and only moderate spectral resolution (0.5nm). Nevertheless, these instruments, the Global Ozone Monitoring Instrument 2 (GOME-2) and the SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY), offer a unique opportunity to compare red and far-red terrestrial SIF at regional spatial scales. Terrestrial SIF has been estimated with ground-, aircraft-, or satellite-based instruments by measuring the filling-in of atmospheric andor solar absorption spectral features by SIF. Our approach makes use of the oxygen (O2) gamma band that is not affected by SIF. The SIF-free O2 gamma band helps to estimate absorption within the spectrally variable O2 B band, which is filled in by red SIF. SIF also fills in the spectrally stable solar Fraunhofer lines (SFLs) at wavelengths both inside and just outside the O2 B band, which further helps

Generation and characterization of a transgenic pig carrying a DsRed-monomer reporter gene.

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Chih-Jen Chou

Full Text Available Pigs are an optimal animal for conducting biomedical research because of their anatomical and physiological resemblance to humans. In contrast to the abundant resources available in the study of mice, few fluorescent protein-harboring porcine models are available for preclinical studies. In this paper, we report the successful generation and characterization of a transgenic DsRed-Monomer porcine model.The transgene comprised a CMV enhancer/chicken-beta actin promoter and DsRed monomeric cDNA. Transgenic pigs were produced by using pronuclear microinjection. PCR and Southern blot analyses were applied for identification of the transgene. Histology, blood examinations and computed tomography were performed to study the health conditions. The pig amniotic fluid progenitor/stem cells were also isolated to examine the existence of red fluorescence and differentiation ability.Transgenic pigs were successfully generated and transmitted to offspring at a germ-line transmission rate of 43.59% (17/39. Ubiquitous expression of red fluorescence was detected in the brain, eye, tongue, heart, lung, liver, pancreas, spleen, stomach, small intestine, large intestine, kidney, testis, and muscle; this was confirmed by histology and western blot analyses. In addition, we confirmed the differentiation potential of amniotic fluid progenitor stem cells isolated from the transgenic pig.This red fluorescent pig can serve as a host for other fluorescent-labeled cells in order to study cell-microenvironment interactions, and can provide optimal red-fluorescent-labeled cells and tissues for research in developmental biology, regenerative medicine, and xenotransplantation.

Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy

NARCIS (Netherlands)

van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

2008-01-01

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH

Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

Science.gov (United States)

Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

1998-01-01

Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

Science.gov (United States)

Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

2016-01-01

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Preparation of Au Nanoclusters-Modified Polylactic Acid Fiber with Bright Red Fluorescence and its Use as Sensing Probe.

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Zhu, Wenli; Li, Huili; Wan, Ajun; Liu, Lanbo

2017-01-01

In present work, the Au nanoclusters-modified polylactic acid fiber (PLA-Au NCs) with bright red fluorescence were fabricated by the encapsulation of Au nanoclusters (Au NCs) in the PLA fiber treated with H 2 O 2 . The Au 25 nanoclusters stabilized by bovine serum albumin (BSA-Au NCs) were prepared via an improved "green" synthetic routine. With pretreatment of the PLA fiber in H 2 O 2 concentration of 12 and 18 %, the as-prepared PLA-Au NCs exhibited brighter red emission with a strong peak centered at ~640 nm than BSA-Au NCs. The fluorescence can be quenched by nitric oxide (NO). A good linear relationship between the relative fluorescence quenching intensity of the as-prepared PLA-Au NCs and the concentration of NO can be obtained in the range of 0.0732 to 0.7320 mM, and the detection limit was 0.0070 mM.

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

Science.gov (United States)

Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia; Munteanu, Emilia L; Feinstein, Paul; Pon, Liza A

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

Larval vision contributes to gregarious settlement in barnacles: adult red fluorescence as a possible visual signal

KAUST Repository

Matsumura, K.

2014-02-26

Gregarious settlement, an essential behavior for many barnacle species that can only reproduce by mating with a nearby barnacle, has long been thought to rely on larval ability to recognize chemical signals from conspecifics during settlement. However, the cyprid, the settlement stage larva in barnacles, has one pair of compound eyes that appear only at the late nauplius VI and cyprid stages, but the function(s) of these eyes remains unknown. Here we show that cyprids of the intertidal barnacle Balanus (=Amphibalanus) amphitrite can locate adult barnacles even in the absence of chemical cues, and prefer to settle around them probably via larval sense of vision. We also show that the cyprids can discriminate color and preferred to settle on red surfaces. Moreover, we found that shells of adult B. amphitrite emit red auto-fluorescence and the adult extracts with the fluorescence as a visual signal attracted cyprid larvae to settle around it. We propose that the perception of specific visual signals can be involved in behavior of zooplankton including marine invertebrate larvae, and that barnacle auto-fluorescence may be a specific signal involved in gregarious larval settlement.

Fluorescent tags of protein function in living cells.

Science.gov (United States)

Whitaker, M

2000-02-01

A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection. BioEssays 22:180-187, 2000. Copyright 2000 John Wiley & Sons, Inc.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

International Nuclear Information System (INIS)

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

2014-01-01

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

Energy Technology Data Exchange (ETDEWEB)

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

2014-02-21

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

Importância dos provérbios no judeu-espanhol

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Aléxia Teles Duchowny

2011-10-01

Full Text Available Divulgação da existência da cultura e língua dos judeus sefarditas orientais, através da verificação da importância dos provérbios escritos em judeu-espanhol.Divulgation de l’existence de la culture et de la langue des juifs sefardis orientaux à travers la vérification de l’importance des dictons écrits en judeo-espagnol.

Effect of tissue scaffold topography on protein structure monitored by fluorescence spectroscopy.

Science.gov (United States)

Portugal, Carla A M; Truckenmüller, Roman; Stamatialis, Dimitrios; Crespo, João G

2014-11-10

The impact of surface topography on the structure of proteins upon adhesion was assessed through non-invasive fluorescence monitoring. This study aimed at obtaining a better understanding about the role of protein structural status on cell-scaffold interactions. The changes induced upon adsorption of two model proteins with different geometries, trypsin (globular conformation) and fibrinogen (rod-shaped conformation) on poly-l-lactic acid (PLLA) scaffolds with different surface topographies, flat, fibrous and surfaces with aligned nanogrooves, were assessed by fluorescence spectroscopy monitoring, using tryptophan as structural probe. Hence, the maximum emission blue shift and the increase of fluorescence anisotropy observed after adsorption of globular and rod-like shaped proteins on surfaces with parallel nanogrooves were ascribed to more intense protein-surface interactions. Furthermore, the decrease of fluorescence anisotropy observed upon adsorption of proteins to scaffolds with fibrous morphology was more significant for rod-shaped proteins. This effect was associated to the ability of these proteins to adjust to curved surfaces. The additional unfolding of proteins induced upon adsorption on scaffolds with a fibrous morphology may be the reason for better cell attachment there, promoting an easier access of cell receptors to initially hidden protein regions (e.g. RGDS sequence), which are known to have a determinant role in cell attaching processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Dynamics of red fluorescent dental plaque during experimental gingivitis--A cohort study.

Science.gov (United States)

van der Veen, Monique H; Volgenant, Catherine M C; Keijser, Bart; Ten Cate, Jacob Bob M; Crielaard, Wim

2016-05-01

The dynamics of red fluorescent plaque (RFP) in comparison to clinical plaque and bleeding scores were studied during an experimental gingivitis protocol in a cohort of healthy participants. Forty-one participants were monitored for RFP before (24h plaque), during 14 days plaque accumulation (days 2, 5, 9, 14) and after 7 days recovery (24h plaque). RFP was assessed on fluorescence photographs of the vestibular aspect of the anterior teeth (cuspid to cuspid) in the upper and lower jaw. Clinical plaque and bleeding were assessed at days -14, 0, 14 and 21. RFP of 24h plaque was reproducible (days -14, 0), then increased during 14 days plaque accumulation and returned to baseline after 7 days recovery. Groups of low, moderate and high RFP formers were statistically significantly different at all times even already at baseline. The individual RFP response during 14 days plaque accumulation correlated well with RFP of 24h plaque (days -14, 0). RFP correlated moderate to well with clinical plaque at days -14, 0, 14 and 21. From day 2 of the gingivitis challenge RFP correlated with bleeding at day 14. RFP provided an objective measure of oral hygiene status. Given the correlation with clinical parameters found, the amount of RFP after 24h plaque accumulation was indicatory for the inflammatory response during a prolonged period of no oral hygiene. This trial was registered at the public trial register ​of the Central Committee on Research Involving Human Subjects (CCMO) under number NL51111.029.14 CLINICAL SIGNIFICANCE: This paper shows the association between RFP after 24h plaque accumulation and inflammatory response after a prolonged period of no oral hygiene. Red plaque fluorescence can be used to identify subjects at risk for developing gingival inflammation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

Science.gov (United States)

Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

2014-05-01

Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of Depilation-Induced Skin Pigmentation and Diet-Induced Fluorescence on In Vivo Fluorescence Imaging

OpenAIRE

Kwon, Sunkuk; Sevick-Muraca, Eva M.

2017-01-01

Near-infrared fluorescence imaging (NIRFI) and far-red fluorescence imaging (FRFI) were used to investigate effects of depilation-induced skin pigmentation and diet-induced background fluorescence on fluorescent signal amplitude and lymphatic contraction frequency in C57BL6 mice. Far-red fluorescent signal amplitude, but not frequency, was affected by diet-induced fluorescence, which was removed by feeding the mice an alfalfa-free diet, and skin pigmentation further impacted the amplitude mea...

Synthesis and characterization of novel 2, 2'-bipyrimidine fluorescent derivative for protein binding

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Padalkar Vikas S

2011-11-01

Full Text Available Abstract Background Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility. Results Novel 4, 4'-(1, 1'-(5-(2-methoxyphenoxy-[2, 2'-bipyrimidine]-4, 6-diylbis(1H-pyrazol-3, 1-diyl dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2-methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375 and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry. Conclusion In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay.

Click strategies for single-molecule protein fluorescence.

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Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

2012-03-21

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

A Fluorescent Probe for Sensitive Detection of Hydrazine and Its Application in Red Wine and Water.

Science.gov (United States)

Wang, Jialin; Wang, Hao; Yang, Shaoxiang; Tian, Hongyu; Liu, Yongguo; Hao, Yanfeng; Zhang, Jie; Sun, Baoguo

2018-01-01

A fluorescent probe, 7-(diethylamino)-2-oxo-2H-chromene-4-carbaldehyde (probe 1), was designed and synthesized for the sensitive detection of hydrazine. The addition of N 2 H 4 caused the fluorescence intensity of probe 1 to decrease. The probe's fluorescence was turn-off after adding N 2 H 4 , which could be observed under UV light at 365 nm. Moreover, once treated with different concentrations N 2 H 4 solutions, the solution color change could be distinguished, which indicates that probe 1 could be used as a visual sensor for hydrazine. Moreover, probe 1 can be used as a signal tool to determine hydrazine levels in solutions, such as red wine and water.

Structural characterization of the photoswitchable fluorescent protein Dronpa-C62S

International Nuclear Information System (INIS)

Nam, Ki-Hyun; Kwon, Oh Yeun; Sugiyama, Kanako; Lee, Won-Ho; Kim, Young Kwan; Song, Hyun Kyu; Kim, Eunice Eunkyung; Park, Sam-Yong; Jeon, Hyesung; Hwang, Kwang Yeon

2007-01-01

The photoswitching behavior of green fluorescent proteins (GFPs) or GFP-like proteins is increasingly recognized as a new technique for optical marking. Recently, Ando and his colleagues developed a new green fluorescent protein Dronpa, which possesses the unique photochromic property of being photoswitchable in a non-destructive manner. To better understand this mechanism, we determined the crystal structures of a new GFP Dronpa and its mutant C62S, at 1.9 A and 1.8 A, respectively. Determination of the structures demonstrates that a unique hydrogen-bonding network and the sulfur atom of the chromophore are critical to the photoswitching property of Dronpa. Reversible photoswitching was lost in cells expressing the Dronpa-C62S upon repetitive irradiation compared to the native protein. Structural and mutational analyses reveal the chemical basis for the functional properties of photoswitchable fluorescent proteins and provide the basis for subsequent coherent engineering of this subfamily of Dronpa homolog's

Highly efficient red fluorescent organic light-emitting diodes by sorbitol-doped PEDOT:PSS

Science.gov (United States)

Zheng, Yan-Qiong; Yu, Jun-Le; Wang, Chao; Yang, Fang; Wei, Bin; Zhang, Jian-Hua; Zeng, Cheng-Hui; Yang, Yang

2018-06-01

This work shows a promising approach to improve device performance by optimizing the electron transport and hole injection layers for tetraphenyldibenzoperiflanthene (DBP):rubrene-based red fluorescent organic light-emitting diodes (OLEDs). We compared the effect of two electron transport layers (ETLs), and found that the rubrene/bathophenanthroline (Bphen) ETL-based OLED showed a much higher external quantum efficiency (EQE) (4.67%) than the Alq3 ETL-based OLED (EQE of 3.08%). The doping ratio of DBP in rubrene was tuned from 1.0 wt% to 4.5 wt%, and the 1.5 wt%-DBP:rubrene-based OLED demonstrated the highest EQE of 5.24% and lowest turn-on voltage of 2.2 V. Atomic force microscopy images indicated that 1.5 wt% DBP-doped rubrene film exhibited a regular strip shape, and this regular surface was favorable to the hole and electron recombination in the emitting layer. Finally, the sorbitol-doped poly(3, 4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) was used to further improve the EQE; doping with 6 wt% sorbitol achieved the highest current efficiency of 7.03 cd A‑1 and an EQE of 7.50%. The significantly enhanced performance implies that the hole injection is a limiting factor for DBP:rubrene-based red fluorescent OLEDs.

Tolerance of a knotted near infrared fluorescent protein to random circular permutation

Science.gov (United States)

Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

2016-01-01

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

Science.gov (United States)

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

Science.gov (United States)

Close, Devin W; Paul, Craig Don; Langan, Patricia S; Wilce, Matthew C J; Traore, Daouda A K; Halfmann, Randal; Rocha, Reginaldo C; Waldo, Geoffery S; Payne, Riley J; Rucker, Joseph B; Prescott, Mark; Bradbury, Andrew R M

2015-07-01

In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. © 2014 Wiley Periodicals, Inc.

Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer: Fluorescence in situ hybridization analysis of a tissue microarray.

Science.gov (United States)

Hu, Xiaoyu; Yang, Zhu; Zeng, Manman; Liu, Y I; Yang, Xiaotao; Li, Yanan; Li, X U; Yu, Qiubo

2016-07-01

The aim of the present study was to investigate the status of speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) gene located on chromosome 17q21 in ovarian cancer (OC). The present study evaluated a tissue microarray, which contained 90 samples of ovarian cancer and 10 samples of normal ovarian tissue, using fluorescence in situ hybridization (FISH). FISH is a method where a SPOP-specific DNA red fluorescence probe was used for the experimental group and a centromere-specific DNA green fluorescence probe for chromosome 17 was used for the control group. The present study demonstrated that a deletion of the SPOP gene was observed in 52.27% (46/88) of the ovarian cancer tissues, but was not identified in normal ovarian tissues. Simultaneously, monosomy 17 was frequently identified in the ovarian cancer tissues, but not in the normal ovarian tissues. Furthermore, the present data revealed that the ovarian cancer histological subtype and grade were significantly associated with a deletion of the SPOP gene, which was assessed by the appearance of monosomy 17 in the ovarian cancer samples; the deletion of the SPOP gene was observed in a large proportion of serous epithelial ovarian cancer (41/61; 67.21%), particularly in grade 3 (31/37; 83.78%). In conclusion, deletion of the SPOP gene on chromosome 17 in ovarian cancer samples, which results from monosomy 17, indicates that the SPOP gene may serve as a tumor suppressor gene in ovarian cancer.

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

Science.gov (United States)

Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

2008-09-01

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

Human cytomegaloviruses expressing yellow fluorescent fusion proteins--characterization and use in antiviral screening.

Directory of Open Access Journals (Sweden)

Sarah Straschewski

Full Text Available Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity. We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP fused with the viral proteins IE-2, ppUL32 (pp150, and ppUL83 (pp65. In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI. The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus-infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.

Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

Energy Technology Data Exchange (ETDEWEB)

Shi Ruina [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Huang Yong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Wang Dan [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Zhao Meiping [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Li Yuanzong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China)]. E-mail: yzli@pku.edu.cn

2006-09-25

The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10{sup -8} to 2.0 x 10{sup -6} M with a detection limit of 1.6 x 10{sup -8} M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.

Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

International Nuclear Information System (INIS)

Shi Ruina; Huang Yong; Wang Dan; Zhao Meiping; Li Yuanzong

2006-01-01

The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10 -8 to 2.0 x 10 -6 M with a detection limit of 1.6 x 10 -8 M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

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Erin Wilson

2018-05-01

Full Text Available A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP. The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD600 and plate counting (colony-forming units (CFUs. While both OD600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.

Intrinsic fluorescence of protein in turbid media using empirical relation based on Monte Carlo lookup table

Science.gov (United States)

Einstein, Gnanatheepam; Udayakumar, Kanniyappan; Aruna, Prakasarao; Ganesan, Singaravelu

2017-03-01

Fluorescence of Protein has been widely used in diagnostic oncology for characterizing cellular metabolism. However, the intensity of fluorescence emission is affected due to the absorbers and scatterers in tissue, which may lead to error in estimating exact protein content in tissue. Extraction of intrinsic fluorescence from measured fluorescence has been achieved by different methods. Among them, Monte Carlo based method yields the highest accuracy for extracting intrinsic fluorescence. In this work, we have attempted to generate a lookup table for Monte Carlo simulation of fluorescence emission by protein. Furthermore, we fitted the generated lookup table using an empirical relation. The empirical relation between measured and intrinsic fluorescence is validated using tissue phantom experiments. The proposed relation can be used for estimating intrinsic fluorescence of protein for real-time diagnostic applications and thereby improving the clinical interpretation of fluorescence spectroscopic data.

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions.

Science.gov (United States)

Žoldák, Gabriel; Jancura, Daniel; Sedlák, Erik

2017-06-01

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady-state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high-throughput drug screening campaigns. © 2017 The Protein Society.

Fluorescent reporter signals, EGFP and DsRed, encoded in HIV-1 facilitate the detection of productively infected cells and cell-associated viral replication levels

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Kazutaka eTerahara

2012-01-01

Full Text Available Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1 strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively-infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

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Meek, Megan E; Van Dolah, Frances M

2016-05-01

Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

Multidimensional fluorescence microscopy of multiple organelles in Arabidopsis seedlings

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Morales Andrea

2008-05-01

Full Text Available Abstract Background The isolation of green fluorescent protein (GFP and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types. Results We generated a transgenic Arabidopsis plant that contains four spectrally different fluorescent proteins. Nuclei, plastids, mitochondria and plasma membranes were genetically tagged with cyan, red, yellow and green fluorescent proteins, respectively. In addition, methods to track nuclei, mitochondria and chloroplasts and quantify the interaction between these organelles at a submicron resolution were developed. These analyzes revealed that N-ethylmaleimide disrupts nuclear-mitochondrial but not nuclear-plastids interactions in root epidermal cells of live Arabidopsis seedlings. Conclusion We developed a tool and associated methods for analyzing the complex dynamic of organelle-organelle interactions in real time in planta. Homozygous transgenic Arabidopsis (Kaleidocell is available through Arabidopsis Biological Resource Center.

Sizing protein-templated gold nanoclusters by time resolved fluorescence anisotropy decay measurements

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Soleilhac, Antonin; Bertorelle, Franck; Antoine, Rodolphe

2018-03-01

Protein-templated gold nanoclusters (AuNCs) are very attractive due to their unique fluorescence properties. A major problem however may arise due to protein structure changes upon the nucleation of an AuNC within the protein for any future use as in vivo probes, for instance. In this work, we propose a simple and reliable fluorescence based technique measuring the hydrodynamic size of protein-templated gold nanoclusters. This technique uses the relation between the time resolved fluorescence anisotropy decay and the hydrodynamic volume, through the rotational correlation time. We determine the molecular size of protein-directed AuNCs, with protein templates of increasing sizes, e.g. insulin, lysozyme, and bovine serum albumin (BSA). The comparison of sizes obtained by other techniques (e.g. dynamic light scattering and small-angle X-ray scattering) between bare and gold clusters containing proteins allows us to address the volume changes induced either by conformational changes (for BSA) or the formation of protein dimers (for insulin and lysozyme) during cluster formation and incorporation.

Sizing protein-templated gold nanoclusters by time resolved fluorescence anisotropy decay measurements.

Science.gov (United States)

Soleilhac, Antonin; Bertorelle, Franck; Antoine, Rodolphe

2018-03-15

Protein-templated gold nanoclusters (AuNCs) are very attractive due to their unique fluorescence properties. A major problem however may arise due to protein structure changes upon the nucleation of an AuNC within the protein for any future use as in vivo probes, for instance. In this work, we propose a simple and reliable fluorescence based technique measuring the hydrodynamic size of protein-templated gold nanoclusters. This technique uses the relation between the time resolved fluorescence anisotropy decay and the hydrodynamic volume, through the rotational correlation time. We determine the molecular size of protein-directed AuNCs, with protein templates of increasing sizes, e.g. insulin, lysozyme, and bovine serum albumin (BSA). The comparison of sizes obtained by other techniques (e.g. dynamic light scattering and small-angle X-ray scattering) between bare and gold clusters containing proteins allows us to address the volume changes induced either by conformational changes (for BSA) or the formation of protein dimers (for insulin and lysozyme) during cluster formation and incorporation. Copyright © 2017 Elsevier B.V. All rights reserved.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

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Pandey, Naresh; Kuypers, Brianna E; Nassif, Barbara; Thomas, Emily E; Alnahhas, Razan N; Segatori, Laura; Silberg, Jonathan J

2016-07-12

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

New fluorescent dipolar pyrazine derivatives for non-doped red organic light-emitting diodes

International Nuclear Information System (INIS)

Gao Baoxiang; Zhou Quanguo; Geng Yanhou; Cheng Yanxiang; Ma Dongge; Xie Zhiyuan; Wang Lixiang; Wang Fosong

2006-01-01

Dipolar fluorescent compounds containing electron-accepting pyrazine-2,3-dicarbonitrile and electron-donating arylamine moiety have been designed and synthesized. The optical and electrochemical properties of these compounds can be adjusted by changing π-bridge length and the donor (D) strength. Organic light-emitting devices based on these compounds are fabricated. Saturated red emission of (0.67, 0.33) and the external quantum efficiency as high as 1.41% have been demonstrated for one of these compounds

Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

Science.gov (United States)

Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

2005-04-01

This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.

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Zhou Han

Full Text Available ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein. Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼ 1%. The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells.

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Frozza, Caroline Olivieri da Silva; Ribeiro, Tanara da Silva; Gambato, Gabriela; Menti, Caroline; Moura, Sidnei; Pinto, Paulo Marcos; Staats, Charley Christian; Padilha, Francine Ferreira; Begnini, Karine Rech; de Leon, Priscila Marques Moura; Borsuk, Sibele; Savegnago, Lucielli; Dellagostin, Odir; Collares, Tiago; Seixas, Fabiana Kömmling; Henriques, João Antonio Pêgas; Roesch-Ely, Mariana

2014-01-01

Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC-MS-MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120 μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6 μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120 μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hybrid white organic light-emitting devices based on phosphorescent iridium–benzotriazole orange–red and fluorescent blue emitters

International Nuclear Information System (INIS)

Xia, Zhen-Yuan; Su, Jian-Hua; Chang, Chi-Sheng; Chen, Chin H.

2013-01-01

We demonstrate that high color purity or efficiency hybrid white organic light-emitting devices (OLEDs) can be generated by integrating a phosphorescent orange–red emitter, bis[4-(2H-benzotriazol-2-yl)-N,N-diphenyl-aniline-N 1 ,C 3 ] iridium acetylacetonate, Ir(TBT) 2 (acac) with fluorescent blue emitters in two different emissive layers. The device based on deep blue fluorescent material diphenyl-[4-(2-[1,1′;4′,1″]terphenyl-4-yl-vinyl)-phenyl]-amine BpSAB and Ir(TBT) 2 (acac) shows pure white color with the Commission Internationale de L'Eclairage (CIE) coordinates of (0.33,0.30). When using sky-blue fluorescent dopant N,N′-(4,4′-(1E,1′E)-2,2′-(1,4-phenylene)bis(ethene-2,1-diyl) bis(4,1-phenylene))bis(2-ethyl-6-methyl-N-phenylaniline) (BUBD-1) and orange–red phosphor with a color-tuning phosphorescent material fac-tris(2-phenylpyridine) iridium (Ir(ppy) 3 ), it exhibits peak luminance yield and power efficiency of 17.4 cd/A and 10.7 lm/W, respectively with yellow-white color and CIE color rendering index (CRI) value of 73. - Highlights: ► An iridium-based orange–red phosphor Ir(TBT) 2 (acac) was applied in hybrid white OLEDs. ► Duel- and tri-emitter WOLEDs were achieved with either high color purity or efficiency performance. ► Peak luminance yield of tri-emitter WOLEDs was 17.4 cd/A with yellow-white color and color rendering index (CRI) value of 73.

Development of a convenient in vivo hepatotoxin assay using a transgenic zebrafish line with liver-specific DsRed expression.

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Xiaoyan Zhang

Full Text Available Previously we have developed a transgenic zebrafish line (LiPan with liver-specific red fluorescent protein (DsRed expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine] caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics and chlorphenamine (pain killer did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry

Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression

Science.gov (United States)

Zhang, Xiaoyan; Li, Caixia; Gong, Zhiyuan

2014-01-01

Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid

Dual-channel red/blue fluorescence dosimetry with broadband reflectance spectroscopic correction measures protoporphyrin IX production during photodynamic therapy of actinic keratosis

Science.gov (United States)

Kanick, Stephen Chad; Davis, Scott C.; Zhao, Yan; Hasan, Tayyaba; Maytin, Edward V.; Pogue, Brian W.; Chapman, M. Shane

2014-07-01

Dosimetry for aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy of actinic keratosis was examined with an optimized fluorescence dosimeter to measure PpIX during treatment. While insufficient PpIX generation may be an indicator of incomplete response, there exists no standardized method to quantitate PpIX production at depths in the skin during clinical treatments. In this study, a spectrometer-based point probe dosimeter system was used to sample PpIX fluorescence from superficial (blue wavelength excitation) and deeper (red wavelength excitation) tissue layers. Broadband white light spectroscopy (WLS) was used to monitor aspects of vascular physiology and inform a correction of fluorescence for the background optical properties. Measurements in tissue phantoms showed accurate recovery of blood volume fraction and reduced scattering coefficient from WLS, and a linear response of PpIX fluorescence versus concentration down to 1.95 and 250 nM for blue and red excitations, respectively. A pilot clinical study of 19 patients receiving 1-h ALA incubation before treatment showed high intrinsic variance in PpIX fluorescence with a standard deviation/mean ratio of >0.9. PpIX fluorescence was significantly higher in patients reporting higher pain levels on a visual analog scale. These pilot data suggest that patient-specific PpIX quantitation may predict outcome response.

Accurate thermometry based on the red and green fluorescence intensity ratio in NaYF4: Yb, Er nanocrystals for bioapplication.

Science.gov (United States)

Liu, Lixin; Qin, Feng; Lv, Tianquan; Zhang, Zhiguo; Cao, Wenwu

2016-10-15

A biological temperature measurement method based on the fluorescence intensity ratio (FIR) was developed to reduce uncertainty. The upconversion luminescence of NaYF4:Yb, Er nanocrystals was studied as a function of temperature around the physiologically relevant range of 300-330 K. We found that the green-green FIR Fe and red-green FIR (I660/I540) varied linearly as temperature increased. The thermometric uncertainties using the two FIRs were discussed and were determined to be almost constant at 0.6 and 0.09 K for green-green and red-green, respectively. The lower thermometric uncertainty comes from the intense signal-to-noise ratio of the measured FIRs owing to their comparable fluorescence intensities.

Development of a Xanthene-Based Red-Emissive Fluorescent Probe for Visualizing H2O2 in Living Cells, Tissues and Animals.

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Zhang, Nan; Dong, Baoli; Kong, Xiuqi; Wang, Chao; Song, Wenhui; Lin, Weiying

2018-04-25

Hydrogen peroxide (H 2 O 2 ) plays important roles in the regulation of many biological processes, and the abnormal level of H 2 O 2 has close relation with the initiation and progression of many diseases. Herein, we describe a novel red-emissive fluorescence probe (RhoB) for the visualization of H 2 O 2 in living cells, tissues and animals. RhoB was constructed on the basis of a xanthene-based red-emissive dye, and displayed nearly no fluorescence. After the treatment with H 2 O 2 , RhoB can exhibit red fluorescence with the emission wavelength at 638 nm. RhoB exhibited highly sensitive and selective response to H 2 O 2 . Density functional theory (DFT) calculations were conducted to shed light on the optical properties of RhoB, and natural bond orbital (NBO) calculations demonstrate that the boron atom shows the highest positive electricity and further support the response mechanism. RhoB was successfully applied for imaging of exogenous and endogenous H 2 O 2 in living cells, and also can be utilized for visualizing H 2 O 2 in living tissues and animals.

Improving the spectral analysis of Fluorescence Resonance Energy Transfer in live cells: application to interferon receptors and Janus kinases.

Science.gov (United States)

Krause, Christopher D; Digioia, Gina; Izotova, Lara S; Pestka, Sidney

2013-10-01

The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

S-ProvFlow: provenance model and tools for scalable and adaptive analysis pipelines in geoscience.

Science.gov (United States)

Spinuso, A.; Mihajlovski, A.; Atkinson, M.; Filgueira, R.; Klampanos, I.; Sanchez, S.

2017-12-01

The reproducibility of scientific findings is essential to improve the quality and application of modern data-driven research. Delivering such reproducibility is challenging in the context of systems handling large data-streams with sophisticated computational methods. Similarly, the SKA (Square Kilometer Array) will collect an unprecedented volume of radio-wave signals that will have to be reduced and transformed into derived products, with impact on space-weather research. This highlights the importance of having cross-disciplines mechanisms at the producer's side that rely on usable lineage data to support validation and traceability of the new artifacts. To be informative, provenance has to describe each methods' abstractions and their implementation as mappings onto distributed platforms and their concurrent execution, capturing relevant internal dependencies at runtime. Producers and intelligent toolsets should be able to exploit the produced provenance, steering real-time monitoring activities and inferring adaptations of methods at runtime.We present a model of provenance (S-PROV) that extends W3C PROV and ProvONE, broadening coverage of provenance to aspects related to distribution, scale-up and steering of stateful streaming operators in analytic pipelines. This is supported by a technical framework for tuneable and actionable lineage, ensuring its relevance to the users' interests, fostering its rapid exploitation to facilitate research practices. By applying concepts such as provenance typing and profiling, users define rules to capture common provenance patterns and activate selective controls based on domain-metadata. The traces are recorded in a document-store with index optimisation and a web API serves advanced interactive tools (S-ProvFlow, https://github.com/KNMI/s-provenance). These allow different classes of consumers to rapidly explore the provenance data. The system, which contributes to the SKA-Link initiative, within technology and

The Struggle of Cath Avery Against Her Introvert Personality on Fangirl by Rainbow Rowell

OpenAIRE

Tarsila, Hersi Intan

2016-01-01

This thesis will analyze the main character of Fangirl by Rainbow Rowell, Cath Avery. The main purpose of this thesis is to analyze the intrinsic and extrinsic aspects of the story. In this thesis, the intrinsic aspects will use characters, characterizations, conflicts and settings and the extrinsic aspect will use Carl Gustav Jung's Analytical Psychology Theory. The methods that are used in this thesis are the library research and psychological approach. The result of the analysis shows that...

Early history, discovery, and expression of Aequorea green fluorescent protein, with a note on an unfinished experiment.

Science.gov (United States)

Tsuji, Frederick I

2010-08-01

The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (lambda(max) = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment. (c) 2010 Wiley-Liss, Inc.

Stabilization of structure in near-infrared fluorescent proteins by binding of biliverdin chromophore

Science.gov (United States)

Stepanenko, Olesya V.; Stepanenko, Olga V.; Bublikov, G. S.; Kuznetsova, I. M.; Verkhusha, V. V.; Turoverov, K. K.

2017-07-01

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes and their mutants with different location of Cys residues, which able to bind a biliverdin chromophore, or without these Cys residues were studied using intrinsic tryptophan fluorescence, NIR fluorescence and circular dichroism. It was shown that a covalent binding of the biliverdin chromophore to a Cys residue via thioether group substantially stabilizes the spatial structure of NIR FPs. The stability of the protein structure and the chromophore association strength strongly depends on the location of Cys residues and decreases in the following order: a protein with Cys residues in both domains, a protein with Cys in PAS domains, and a protein with Cys in GAF domains. NIR FPs without Cys residues capable to covalently attach biliverdin have the lowest stability, comparable to NIR FP apoforms.

Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters

Science.gov (United States)

2016-01-13

affected by the environment of the stabilizing protein, allowing these hybrid systems to act as sensors in many applications.2,9,14–19 This has led...Biosens Bioelectron. 2012;32:297–299. 8. Joseph D, Geckeler KE. Synthesis of highly fluorescent gold nanoclusters using egg white proteins. Colloids Surf...Chang HW, Chien YC, Hsiao JK, Cheng JT, Chou PT. Insulin -directed synthesis of fluorescent gold nanoclusters: preservation of insulin bioactivity and

Planned caesarean in the interventional radiology cath lab to enable immediate uterine artery embolization for the conservative treatment of placenta accreta

International Nuclear Information System (INIS)

Bouvier, A.; Sentilhes, L.; Thouveny, F.; Bouet, P.-E.; Gillard, P.; Willoteaux, S.; Aubé, C.

2012-01-01

Aim: To evaluate the feasibility and efficacy of routine uterine artery embolization (UAE) immediately after planned caesareans performed in the cath lab for conservative treatment of placenta accreta. Materials and methods: A retrospective study included all patients who had a planned caesarean in the cath lab for conservative treatment of placenta accreta at Angers University Hospital, which is a tertiary care centre, from April 2001 to September 2010. Twelve patients underwent UAE immediately after caesarean with the placenta left partially or totally in situ. The success rate of embolization, blood loss, and complications were reported. Results: Diagnosis of abnormal placentation was confirmed by caesarean findings in 14 cases. Four patients had a percreta form with bladder invasion. In seven cases blood loss was insignificant and UAE was prophylactic; no secondary haemorrhage was observed in this group. Postpartum haemorrhage occurred in five cases: control of immediate postpartum bleeding by embolization was successful in three and failed in two leading to hysterectomy. In one case uterine necrosis occurred 6 weeks after embolization, requiring a hysterectomy. Delayed complications resulted in hysterectomy and partial bladder resection 3 months after delivery for one of the patients with placenta percreta. Conclusion: UAE immediately after a caesarean performed in the cath lab is a feasible therapeutic option for conservative treatment of placenta accreta. Advantages include reducing stress and risks associated with transferring women with potentially unstable haemodynamics.

Fluorescence ON–OFF switching using micelle of stimuli-responsive double hydrophilic block copolymers: Nile Red fluorescence in micelles of poly(acrylic acid-b-N-isopropylacrylamide)

Energy Technology Data Exchange (ETDEWEB)

Yee, Min Min; Tsubone, Miyabi; Morita, Takuya [Department of Chemistry, Graduate School of Science & Engineering, Saga University, 1 Honjo, Saga 840-8502 (Japan); Yusa, Shin-ichi [Department of Materials Science and Chemistry, University of Hyogo, 2167 Shosha, Himeji 671-2280 (Japan); Nakashima, Kenichi, E-mail: nakashik@cc.saga-u.ac.jp [Department of Chemistry, Graduate School of Science & Engineering, Saga University, 1 Honjo, Saga 840-8502 (Japan)

2016-08-15

The dual-mode fluorescence ON–OFF switching of Nile Red (NR) by using stimuli-responsive polymeric micelle of poly(acrylic acid-b-N-isopropylacrylamide) (PAA-b-PNIPAM) has been studied. PAA-b-PNIPAM, one of double hydrophilic block copolymers, is known to form PNIPAM-core/PAA-corona micelles in aqueous solutions when the temperature of the solution is elevated up to the lower critical solution temperature (LCST) of PNIPAM block. It also forms PAA-core/PNIPAM-corona micelles when the anionic PAA block is charge-neutralized with cationic cetyltrimethylammonium ion. Fluorescence properties of NR in the micelles are elucidated by observing various fluorescence parameters such as intensity, polarization, and quantum yield. It is found that the fluorescence intensity is negligibly low (OFF-state) when PAA-b-PNIPAM exists as a form of unimer, whereas it is remarkably enhanced (ON-state) when the PNIPAM-core or PAA-core micelles are formed. These results demonstrate that a novel fluorescence ON–OFF switching system can be constructed by using PAA-b-PNIPAM micelles and NR.

Structural and Functional View of Polypharmacology.

Science.gov (United States)

Moya-García, Aurelio; Adeyelu, Tolulope; Kruger, Felix A; Dawson, Natalie L; Lees, Jon G; Overington, John P; Orengo, Christine; Ranea, Juan A G

2017-08-31

Protein domains mediate drug-protein interactions and this principle can guide the design of multi-target drugs i.e. polypharmacology. In this study, we associate multi-target drugs with CATH functional families through the overrepresentation of targets of those drugs in CATH functional families. Thus, we identify CATH functional families that are currently enriched in drugs (druggable CATH functional families) and we use the network properties of these druggable protein families to analyse their association with drug side effects. Analysis of selected druggable CATH functional families, enriched in drug targets, show that relatives exhibit highly conserved drug binding sites. Furthermore, relatives within druggable CATH functional families occupy central positions in a human protein functional network, cluster together forming network neighbourhoods and are less likely to be within proteins associated with drug side effects. Our results demonstrate that CATH functional families can be used to identify drug-target interactions, opening a new research direction in target identification.

Stylophora pistillata in the Red Sea demonstrate higher GFP fluorescence under ocean acidification conditions

Science.gov (United States)

Grinblat, Mila; Fine, Maoz; Tikochinski, Yaron; Loya, Yossi

2018-03-01

Ocean acidification is thought to exert a major impact on calcifying organisms, including corals. While previous studies have reported changes in the physiological response of corals to environmental change, none have described changes in expression of the ubiquitous host pigments—fluorescent proteins (FPs)—to ocean acidification. The function of FPs in corals is controversial, with the most common consideration being that these primarily regulate the light environment in the coral tissue and protect the host from harmful UV radiation. Here, we provide for the first time experimental evidence that increased fluorescence of colonies of the coral Stylophora pistillata is independent of stress and can be regulated by a non-stressful decrease in pH. Stylophora pistillata is the most abundant and among the most resilient coral species in the northern Gulf of Eilat/Aqaba (GoE/A). Fragmented "sub-colonies" ( n = 72) incubated for 33 days under three pH treatments (ambient, 7.9, and 7.6), under ambient light, and running seawater showed no stress or adverse physiological performance, but did display significantly higher fluorescence, with lower pH. Neither the average number of planulae shed from the experimental sub-colonies nor planulae green fluorescent protein (GFP) expression changed significantly among pH treatments. Sub-colonies incubated under the lower-than-ambient pH conditions showed an increase in both total protein and GFP expression. Since extensive protein synthesis requires a high level of transcription, we suggest that GFP constitutes a UV protection mechanism against potential RNA as well as against DNA damage caused by UV exposure. Manipulating the regulation of FPs in adult corals and planulae, under controlled and combined effects of pH, light, and temperature, is crucial if we are to obtain a better understanding of the role played by this group of proteins in cnidarians.

Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.

Science.gov (United States)

Ast, Cindy; Foret, Jessica; Oltrogge, Luke M; De Michele, Roberto; Kleist, Thomas J; Ho, Cheng-Hsun; Frommer, Wolf B

2017-09-05

Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

Fluorescence quenching and the "ring-mode" to "red-mode" transition in alkali inductively coupled plasmas

Science.gov (United States)

Huang, M.; Bazurto, R.; Camparo, J.

2018-01-01

The ring-mode to red-mode transition in alkali metal inductively coupled plasmas (ICPs) (i.e., rf-discharge lamps) is perhaps the most important physical phenomenon affecting these devices as optical pumping light sources for atomic clocks and magnetometers. It sets the limit on useful ICP operating temperature, thereby setting a limit on ICP light output for atomic-clock/magnetometer signal generation, and it is a temperature region of ICP operation associated with discharge instability. Previous work has suggested that the mechanism driving the ring-mode to red-mode transition is associated with radiation trapping, but definitive experimental evidence validating that hypothesis has been lacking. Based on that hypothesis, one would predict that the introduction of an alkali-fluorescence quenching gas (i.e., N2) into the ICP would increase the ring-mode to red-mode transition temperature. Here, we test that prediction, finding direct evidence supporting the radiation-trapping hypothesis.

A Naturally Fluorescent Mgp Transgenic Mouse for Angiogenesis and Glaucoma Longitudinal Studies.

Science.gov (United States)

Asokan, Priyadarsini; Mitra, Rajendra N; Periasamy, Ramesh; Han, Zongchao; Borrás, Teresa

2018-02-01

Our goal was to generate and characterize a new mouse model in which only angiogenesis- and glaucoma-relevant tissues would be naturally fluorescent. The Matrix Gla (MGP) gene is highly expressed in vascular smooth muscle cells (VSMC) and trabecular meshwork (TM). We sought to direct our Mgp-Cre.KI mouse recombinase to VSMC/TM cells to produce their longitudinal fluorescent profiles. Homozygous Mgp-Cre.KI mice were crossed with Ai9 homozygous reporter mice harboring a loxP-flanked STOP cassette preventing transcription of a DsRed fluorescent protein (tdTomato). The F1 double-heterozygous (Mgp-tdTomato) was examined by direct fluorescence, whole mount, histology, and fundus photography. Custom-made filters had 554/23 emission and 609/54 exciter nanometer wavelengths. Proof of concept of the model's usefulness was conducted by inducing guided imaging laser burns. Evaluation of a vessel's leakage and proliferation was followed by noninvasive angiography. The Mgp-tdTomato mouse was viable, fertile, with normal IOP and ERG. Its phenotype exhibited red paws and snout (cartilage expression), which precluded genotyping. A fluorescent red ring was seen at the limbus and confirmed to be TM expression by histology. The entire retinal vasculature was red fluorescent (VSMC) and directly visualized by fundus photography. Laser burns on the Mgp-tdTomato allowed separation of leakiness and neovascularization evaluation parameters. The availability of a transgenic mouse naturally fluorescent in glaucoma-relevant tissues and retinal vasculature brings the unique opportunity to study a wide spectrum of single and combined glaucomatous conditions in vivo. Moreover, the Mgp-tdTomato mouse provides a new tool to study mechanisms and therapeutics of retinal angiogenesis longitudinally.

A Naturally Fluorescent Mgp Transgenic Mouse for Angiogenesis and Glaucoma Longitudinal Studies

Science.gov (United States)

Asokan, Priyadarsini; Mitra, Rajendra N.; Periasamy, Ramesh; Han, Zongchao

2018-01-01

Purpose Our goal was to generate and characterize a new mouse model in which only angiogenesis- and glaucoma-relevant tissues would be naturally fluorescent. The Matrix Gla (MGP) gene is highly expressed in vascular smooth muscle cells (VSMC) and trabecular meshwork (TM). We sought to direct our Mgp-Cre.KI mouse recombinase to VSMC/TM cells to produce their longitudinal fluorescent profiles. Methods Homozygous Mgp-Cre.KI mice were crossed with Ai9 homozygous reporter mice harboring a loxP-flanked STOP cassette preventing transcription of a DsRed fluorescent protein (tdTomato). The F1 double-heterozygous (Mgp-tdTomato) was examined by direct fluorescence, whole mount, histology, and fundus photography. Custom-made filters had 554/23 emission and 609/54 exciter nanometer wavelengths. Proof of concept of the model's usefulness was conducted by inducing guided imaging laser burns. Evaluation of a vessel's leakage and proliferation was followed by noninvasive angiography. Results The Mgp-tdTomato mouse was viable, fertile, with normal IOP and ERG. Its phenotype exhibited red paws and snout (cartilage expression), which precluded genotyping. A fluorescent red ring was seen at the limbus and confirmed to be TM expression by histology. The entire retinal vasculature was red fluorescent (VSMC) and directly visualized by fundus photography. Laser burns on the Mgp-tdTomato allowed separation of leakiness and neovascularization evaluation parameters. Conclusions The availability of a transgenic mouse naturally fluorescent in glaucoma-relevant tissues and retinal vasculature brings the unique opportunity to study a wide spectrum of single and combined glaucomatous conditions in vivo. Moreover, the Mgp-tdTomato mouse provides a new tool to study mechanisms and therapeutics of retinal angiogenesis longitudinally. PMID:29392320

Red and Far-Red Solar-Induced Chlorophyll Fluorescence Observations in the Tropical Rain Forest of Costa Rica

Science.gov (United States)

Stutz, J.; Grossmann, K.; Seibt, U.; Dierick, D.; Magney, T. S.; Frankenberg, C.

2017-12-01

Solar-Induced Chlorophyll Fluorescence (SIF) is a powerful proxy for photosynthetic activity. SIF can be measured using remote sensing from the leaf to the global scale. However, the relationship between SIF, photosynthetic efficiencies, Gross Primary Productivity (GPP), and their response to environmental stress conditions remain poorly constrained. The impact of canopy radiative transfer and viewing geometry at the canopy scale also requires further study. In addition, there is an urgent need for the validation of space-borne SIF measurements, especially above the tropical rain forest where ground observations at the canopy scale are sparse. Here we present observations of SIF in the red and far-red wavelength range, as well various vegetation indices (NDVI, PRI, EVI), made by a novel ground-based spectrometer system, PhotoSpec, at La Selva Biological Station, Costa Rica. Measurements began in March 2017 and have continued ever since. PhotoSpec uses a narrow (0.7 degrees) field-of-view for the simultaneous co-aligned observations of all parameters at a time resolution of 30 seconds. The 2D scanning telescope unit of PhotoSpec was used for regular surveys of around 20 tree species, 2D-raster on canopies of individual trees, as well as elevation survey scans. SIF retrievals were performed using the in-filling of Fraunhofer lines, which allows the accurate observation of SIF under sunny as well as frequent cloudy conditions. The seasonal changes of SIF at La Selva, as well as the red / far-red SIF ratio, for different tree species are presented. 2D-raster scans allow an assessment of the representativeness of narrow field-of-view observations. We will also compare the PhotoSpec data with coincident satellite observations.

Microspectroscopic analysis of green fluorescent proteins infiltrated into mesoporous silica nanochannels

NARCIS (Netherlands)

Ma, Yujie; Rajendran, Prayanka; Blum, Christian; Cesa, Yanina; Gartmann, Nando; Brühwiler, Dominik; Subramaniam, Vinod

2011-01-01

The infiltration of enhanced green fluorescent protein (EGFP) into nanochannels of different diameters in mesoporous silica particles was studied in detail by fluorescence microspectroscopy at room temperature. Silica particles from the MCM-41, ASNCs and SBA-15 families possessing nanometer-sized

Reef-coral proteins as visual, non-destructive reporters for plant transformation.

Science.gov (United States)

Wenck, A; Pugieux, C; Turner, M; Dunn, M; Stacy, C; Tiozzo, A; Dunder, E; van Grinsven, E; Khan, R; Sigareva, M; Wang, W C; Reed, J; Drayton, P; Oliver, D; Trafford, H; Legris, G; Rushton, H; Tayab, S; Launis, K; Chang, Y-F; Chen, D-F; Melchers, L

2003-11-01

Recently, five novel fluorescent proteins have been isolated from non-bioluminescent species of reef-coral organisms and have been made available through ClonTech. They are AmCyan, AsRed, DsRed, ZsGreen and ZsYellow. These proteins are valuable as reporters for transformation because they do not require a substrate or external co-factor to emit fluorescence and can be tested in vivo without destruction of the tissue under study. We have evaluated them in a large range of plants, both monocots and dicots, and our results indicate that they are valuable reporting tools for transformation in a wide variety of crops. We report here their successful expression in wheat, maize, barley, rice, banana, onion, soybean, cotton, tobacco, potato and tomato. Transient expression could be observed as early as 24 h after DNA delivery in some cases, allowing for very clear visualization of individually transformed cells. Stable transgenic events were generated, using mannose, kanamycin or hygromycin selection. Transgenic plants were phenotypically normal, showing a wide range of fluorescence levels, and were fertile. Expression of AmCyan, ZsGreen and AsRed was visible in maize T1 seeds, allowing visual segregation to more than 99% accuracy. The excitation and emission wavelengths of some of these proteins are significantly different; the difference is enough for the simultaneous visualization of cells transformed with more than one of the fluorescent proteins. These proteins will become useful tools for transformation optimization and other studies. The wide variety of plants successfully tested demonstrates that these proteins will potentially find broad use in plant biology.

Luminescent conjugated oligothiophenes for sensitive fluorescent assignment of protein inclusion bodies.

Science.gov (United States)

Klingstedt, Therése; Blechschmidt, Cristiane; Nogalska, Anna; Prokop, Stefan; Häggqvist, Bo; Danielsson, Olof; Engel, W King; Askanas, Valerie; Heppner, Frank L; Nilsson, K Peter R

2013-03-18

Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hybrid white organic light-emitting devices based on phosphorescent iridium-benzotriazole orange-red and fluorescent blue emitters

Energy Technology Data Exchange (ETDEWEB)

Xia, Zhen-Yuan, E-mail: xiazhenyuan@hotmail.com [Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237 (China); Su, Jian-Hua [Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237 (China); Chang, Chi-Sheng; Chen, Chin H. [Display Institute, Microelectronics and Information Systems Research Center, National Chiao Tung University, Hsinchu, Taiwan 300 (China)

2013-03-15

We demonstrate that high color purity or efficiency hybrid white organic light-emitting devices (OLEDs) can be generated by integrating a phosphorescent orange-red emitter, bis[4-(2H-benzotriazol-2-yl)-N,N-diphenyl-aniline-N{sup 1},C{sup 3}] iridium acetylacetonate, Ir(TBT){sub 2}(acac) with fluorescent blue emitters in two different emissive layers. The device based on deep blue fluorescent material diphenyl-[4-(2-[1,1 Prime ;4 Prime ,1 Double-Prime ]terphenyl-4-yl-vinyl)-phenyl]-amine BpSAB and Ir(TBT){sub 2}(acac) shows pure white color with the Commission Internationale de L'Eclairage (CIE) coordinates of (0.33,0.30). When using sky-blue fluorescent dopant N,N Prime -(4,4 Prime -(1E,1 Prime E)-2,2 Prime -(1,4-phenylene)bis(ethene-2,1-diyl) bis(4,1-phenylene))bis(2-ethyl-6-methyl-N-phenylaniline) (BUBD-1) and orange-red phosphor with a color-tuning phosphorescent material fac-tris(2-phenylpyridine) iridium (Ir(ppy){sub 3} ), it exhibits peak luminance yield and power efficiency of 17.4 cd/A and 10.7 lm/W, respectively with yellow-white color and CIE color rendering index (CRI) value of 73. - Highlights: Black-Right-Pointing-Pointer An iridium-based orange-red phosphor Ir(TBT){sub 2}(acac) was applied in hybrid white OLEDs. Black-Right-Pointing-Pointer Duel- and tri-emitter WOLEDs were achieved with either high color purity or efficiency performance. Black-Right-Pointing-Pointer Peak luminance yield of tri-emitter WOLEDs was 17.4 cd/A with yellow-white color and color rendering index (CRI) value of 73.

Enhancing analysis of cells and proteins by fluorescence imaging on silk-based biomaterials: modulating the autofluorescence of silk.

Science.gov (United States)

Neo, Puay Yong; Tan, Daryl Jian-An; Shi, Pujiang; Toh, Siew Lok; Goh, James Cho-Hong

2015-02-01

Silk is a versatile and established biomaterial for various tissue engineering purposes. However, it also exhibits strong autofluorescence signals-thereby hindering fluorescence imaging analysis of cells and proteins on silk-derived biomaterials. Sudan Black B (SB) is a lysochrome dye commonly used to stain lipids in histology. It has also been reported to be able to quench autofluorescence of tissues in histology and has been tested on artificial biomedical polymers in recent years. It was hypothesized that SB would exert similar quenching effects on silk, modulating the autofluorescence signals, and thereby enabling improved imaging analysis of cells and molecules of interests. The quenching effect of SB on the intrinsic fluorescence properties of silk and on commercial fluorescent dyes were first investigated in this study. SB was then incorporated into typical fluorescence-based staining protocols to study its effectiveness in improving fluorescence-based imaging of the cells and proteins residing with the silk-based biomaterials. Silk processed into various forms of biomaterials (e.g., films, sponges, fibers, and electrospun mats) was seeded with cells and cultured in vitro. At sacrificial time points, specimens were harvested, fixed, and prepared for fluorescence staining. SB, available commercially as a powder, was dissolved in 70% ethanol (0.3% [w/v]) to form staining solutions. SB treatment was introduced at the last step of typical immunofluorescence staining protocols for 15-120 min. For actin staining protocols by phalloidin toxin, SB staining solutions were added before and after permeabilization with Triton-X for 15-30 min. Results showed that ideal SB treatment duration is about 15 min. Apart from being able to suppress the autofluorescence of silk, this treatment duration was also not too long to adversely affect the fluorescent labeling probes used. The relative improvement brought about by SB treatment was most evident in the blue and green

Synchronous fluorescence based biosensor for albumin determination by cooperative binding of fluorescence probe in a supra-biomolecular host-protein assembly.

Science.gov (United States)

Patra, Digambara

2010-01-15

A synchronous fluorescence probe based biosensor for estimation of albumin with high sensitivity and selectivity was developed. Unlike conventional fluorescence emission or excitation spectral measurements, synchronous fluorescence measurement offered exclusively a new synchronous fluorescence peak in the shorter wavelength range upon binding of chrysene with protein making it an easy identification tool for albumin determination. The cooperative binding of a fluorescence probe, chrysene, in a supramolecular host-protein assembly during various albumin assessments was investigated. The presence of supramolecular host molecules such as beta-cyclodextrin, curucurbit[6]uril or curucurbit[7]uril have little influence on sensitivity or limit of detection during albumin determination but reduced dramatically interference from various coexisting metal ion quenchers/enhancers. Using the present method the limit of detection for BSA and gamma-Globulin was found to be 0.005 microM which is more sensitive than reported values. Copyright 2009 Elsevier B.V. All rights reserved.

In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

Directory of Open Access Journals (Sweden)

Intekhab Islam

2016-01-01

Full Text Available Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs are potentially an unlimited source of healthy and functional osteoprogenitors (OPs that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs expressing green fluorescent protein (GFP and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

Science.gov (United States)

Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

2011-01-01

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase…

Mass spectrometry based approach for identification and characterisation of fluorescent proteins from marine organisms

DEFF Research Database (Denmark)

Wojdyla, Katarzyna Iwona; Rogowska-Wrzesinska, Adelina; Wrzesinski, Krzysztof

2011-01-01

We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our...

Far-ultraviolet fluorescence of carbon monoxide in the red giant Arcturus

International Nuclear Information System (INIS)

Ayres, T.R.; Moos, H.W.; Linsky, J.L.

1981-01-01

We present evidence that many of the weak features observed with the International Ultraviolet Explorer (IUE) in the far-ultraviolet (1150--2000 A) spectrum of the archetype red giant Arcturus (K2 III) are A--X fourth positive bands of carbon monoxide excited by chromospheric emissions of O I, C I, and H I. The appearance of fluorescent CO bands near the wavelengths of commonly used indicators of high-temperature (T>2 x 10 4 K) plasma, such as C II lambda1335 and C IV lambda1548, introduces a serious ambiguity in diagnosing the presence of hot material in the outer atmospheres of the cool giants by means of low-dispersion IUE spectra

Realizing the Living Paper using the ProvONE Model for Reproducible Research

Science.gov (United States)

Jones, M. B.; Jones, C. S.; Ludäscher, B.; Missier, P.; Walker, L.; Slaughter, P.; Schildhauer, M.; Cuevas-Vicenttín, V.

2015-12-01

Science has advanced through traditional publications that codify research results as a permenant part of the scientific record. But because publications are static and atomic, researchers can only cite and reference a whole work when building on prior work of colleagues. The open source software model has demonstrated a new approach in which strong version control in an open environment can nurture an open ecosystem of software. Developers now commonly fork and extend software giving proper credit, with less repetition, and with confidence in the relationship to original software. Through initiatives like 'Beyond the PDF', an analogous model has been imagined for open science, in which software, data, analyses, and derived products become first class objects within a publishing ecosystem that has evolved to be finer-grained and is realized through a web of linked open data. We have prototyped a Living Paper concept by developing the ProvONE provenance model for scientific workflows, with prototype deployments in DataONE. ProvONE promotes transparency and openness by describing the authenticity, origin, structure, and processing history of research artifacts and by detailing the steps in computational workflows that produce derived products. To realize the Living Paper, we decompose scientific papers into their constituent products and publish these as compound objects in the DataONE federation of archival repositories. Each individual finding and sub-product of a reseach project (such as a derived data table, a workflow or script, a figure, an image, or a finding) can be independently stored, versioned, and cited. ProvONE provenance traces link these fine-grained products within and across versions of a paper, and across related papers that extend an original analysis. This allows for open scientific publishing in which researchers extend and modify findings, creating a dynamic, evolving web of results that collectively represent the scientific enterprise. The

Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

Science.gov (United States)

van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees

2008-01-01

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

Enzymes and membrane proteins of ADSOL-preserved red blood cells

Directory of Open Access Journals (Sweden)

Maria Sueli Soares Leonart

2000-03-01

Full Text Available CONTEXT: The preservative solution ADSOL (adenine, dextrose, sorbitol, sodium chloride and mannitol maintains red cell viability for blood trans-fusion for 6 weeks. It would be useful to know about its preservation qualities over longer periods. OBJECTIVE: To determine some red cell biochemical parameters for peri-ods of up to 14 weeks in order to determine whether the red cell metabo-lism integrity would justify further studies aiming at increasing red cell preservation and viability. DESIGN: Biochemical evaluation designed to study red cell preservation. SETTING: São Paulo University erythrocyte metabolism referral center. SAMPLE: Six normal blood donors from the University Hospital of the Universidade Federal do Paraná, Curitiba, Brazil. MAIN MEASUREMENTS: Weekly assay of erythrocyte adenosine-5´-triphosphate (ATP, 2,3-diphosphoglycerate (2,3DPG, hexokinase (HX, phosphofructokinase (PFK, pyruvate kinase (PK, glucose-6-phosphate dehydrogenase (G-6-PD, 6-phosphogluconic dehydrogenase (6-PGD, glyceraldehyde-3-phosphate dehydrogenase (GAPD, glutathione reduc-tase (GR, glutathione peroxidase (GSHPx, plasma sodium and potas-sium, blood pH, and membrane proteins of red cells preserved in ADSOL were studied during storage for 14 weeks storage. RESULTS: During ADSOL preservation, erythrocyte ATP concentration decreased 60% after 5 weeks, and 90% after 10 weeks; the pH fell from 6.8 to 6.4 by the 14th week. 2,3-DPG concentration was stable during the first week, but fell 90% after 3 weeks and was exhausted after 5 weeks. By the end of the 5th week, an activity decrease of 16-30% for Hx, GAPD, GR, G-6-PD and 6-PGD, 35% for PFK and GSHPx, and 45% for PK were observed. Thereafter, a uniform 10% decay was observed for all enzymes up to the 14th week. The red blood cell membrane pro-teins did not show significant alterations in polyacrylamide gel electro-phoresis (SDS-PAGE during the 14 weeks. CONCLUSION: Although the blood viability was shown to be poor

Chromophore-protein coupling beyond nonpolarizable models: understanding absorption in green fluorescent protein

NARCIS (Netherlands)

Daday, C.; Curutchet, C.; Sinicropi, A.; Mennucci, B.; Filippi, Claudia

2015-01-01

The nature of the coupling of the photoexcited chromophore with the environment in a prototypical system like green fluorescent protein (GFP) is to date not understood, and its description still defies state-of-the-art multiscale approaches. To identify which theoretical framework of the

Dissecting Redox Biology Using Fluorescent Protein Sensors.

Science.gov (United States)

Schwarzländer, Markus; Dick, Tobias P; Meyer, Andreas J; Morgan, Bruce

2016-05-01

Fluorescent protein sensors have revitalized the field of redox biology by revolutionizing the study of redox processes in living cells and organisms. Within one decade, a set of fundamental new insights has been gained, driven by the rapid technical development of in vivo redox sensing. Redox-sensitive yellow and green fluorescent protein variants (rxYFP and roGFPs) have been the central players. Although widely used as an established standard tool, important questions remain surrounding their meaningful use in vivo. We review the growing range of thiol redox sensor variants and their application in different cells, tissues, and organisms. We highlight five key findings where in vivo sensing has been instrumental in changing our understanding of redox biology, critically assess the interpretation of in vivo redox data, and discuss technical and biological limitations of current redox sensors and sensing approaches. We explore how novel sensor variants may further add to the current momentum toward a novel mechanistic and integrated understanding of redox biology in vivo. Antioxid. Redox Signal. 24, 680-712.

Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment

NARCIS (Netherlands)

Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

2008-01-01

We present a new and simple method based on site-directed fluorescence labeling using the BADAN label that allows to examine protein-lipid interactions in great detail. We apply this approach to a membrane-embedded mainly -helical reference protein, the M13 major coat protein, of which in a

Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein.

Science.gov (United States)

Nava, Phillip; Cecchini, Matt; Chirico, Sara; Gordon, Heather; Morley, Samantha; Manor, Danny; Atkinson, Jeffrey

2006-06-01

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein's binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (alpha-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4'R,8'R-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma.

Science.gov (United States)

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong; Li, Ning

2016-12-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

Bodipy-FL-Verapamil: A Fluorescent Probe for the Study of Multidrug Resistance Proteins

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Anna Rosati

2004-01-01

Full Text Available Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV, have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR or MRP (multidrug resistance‐related protein (PANC‐1 and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine or MRP (MK571 and probenecid has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.

Simulation of fluorescence resonance energy transfer experiments: effect of the dyes on protein folding

International Nuclear Information System (INIS)

Allen, Lucy R; Paci, Emanuele

2010-01-01

Fluorescence resonance energy transfer is a powerful technique which is often used to probe the properties of proteins and complex macromolecules. The technique relies on relatively large fluorescent dyes which are engineered into the molecule of interest. In the case of small proteins, these dyes may affect the stability of the protein, and modify the folding kinetics and the folding mechanisms which are being probed. Here we use atomistic simulation to investigate the effect that commonly used fluorescent dyes have on the folding of a four-helix bundle protein. We show that, depending on where the dyes are attached, their effect on the kinetic and thermodynamic properties of the protein may be significant. We find that, while the overall folding mechanism is not affected by the dyes, they can destabilize, or even stabilize, intermediate states.

Cell fusion phenomena detected after in utero transplantation of Ds-red-harboring porcine amniotic fluid stem cells into EGFP transgenic mice.

Science.gov (United States)

Peng, Shao-Yu; Chen, Yu-Hsu; Chou, Chih-Jen; Wang, Yao-Horng; Lee, Hung-Maan; Cheng, Winston Teng-Kui; Shaw, S W Steven; Wu, Shinn-Chih

2014-05-01

Amniotic fluid stem cells (AFSCs) are derived from the amniotic fluid of the developing fetus and can give rise to diverse differentiated cells of ectoderm, mesoderm, and endoderm lineages. Intrauterine transplantation is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant dams. Certain disease such as osteogenesis imperfecta was successfully treated in affected fetal mice using this method. However, the donor cell destiny remains uncertain. The purpose of this study was to investigate the biodistribution and cell fate of Ds-red-harboring porcine AFSCs (Ds-red pAFSCs) after intrauterine transplantation into enhanced green fluorescent protein-harboring fetuses of pregnant mice. Pregnant mice (12.5 days) underwent open laparotomy with intrauterine pAFSC transplantation (5 × 10(4) cells per pup) into fetal peritoneal cavity. Three weeks after birth, the mice were sacrificed. Several samples from different organs were obtained for histological examination and flow cytometric analysis. Ds-red pAFSCs migrated most frequently into the intestines. Furthermore, enhanced green fluorescent protein and red fluorescent protein signals were co-expressed in the intestine and liver cells via immunohistochemistry studies. In utero xenotransplantation of pAFSCs fused with recipient intestinal cells instead of differentiating or maintaining the undifferentiated status in the tissue. © 2014 John Wiley & Sons, Ltd.

CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells.

Science.gov (United States)

Sharma, Arun; Toepfer, Christopher N; Ward, Tarsha; Wasson, Lauren; Agarwal, Radhika; Conner, David A; Hu, Johnny H; Seidman, Christine E

2018-01-24

Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology-directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein can be rapidly created by employing CRISPR/Cas9 genome editing, enhancing HDR efficiency and optimizing homology arm length. These fluorescently tagged hiPSCs can be used to visualize protein function and dynamics in real time as cells proliferate and differentiate. Given that nearly any intracellular protein can be fluorescently tagged, this system serves as a powerful tool to facilitate new discoveries across many biological disciplines. In this unit, we present protocols for the design, generation, and monoclonal expansion of genetically customized hiPSCs encoding fluorescently tagged endogenous proteins. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

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Shingo Sotoma

2016-03-01

Full Text Available The impeccable photostability of fluorescent nanodiamonds (FNDs is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

Magnetic polymer microcapsules loaded with Nile Red fluorescent dye

Science.gov (United States)

Bartel, Marta; Wysocka, Barbara; Krug, Pamela; Kępińska, Daria; Kijewska, Krystyna; Blanchard, Gary J.; Kaczyńska, Katarzyna; Lubelska, Katarzyna; Wiktorska, Katarzyna; Głowala, Paulina; Wilczek, Marcin; Pisarek, Marcin; Szczytko, Jacek; Twardowski, Andrzej; Mazur, Maciej

2018-04-01

Fabrication of multifunctional smart vehicles for drug delivery is a fascinating challenge of multidisciplinary research at the crossroads of materials science, physics and biology. We demonstrate a prototypical microcapsule system that is capable of encapsulating hydrophobic molecules and at the same time reveals magnetic properties. The microcapsules are prepared using a templated synthesis approach where the molecules to be encapsulated (Nile Red) are present in the organic droplets that are suspended in the polymerization solution which also contains magnetic nanoparticles. The polymer (polypyrrole) grows on the surface of organic droplets encapsulating the fluorescent dye in the core of the formed microcapsule which incorporates the nanoparticles into its wall. For characterization of the resulting structures a range of complementary physicochemical methodology is used including optical and electron microscopy, magnetometry, 1H NMR and spectroscopy in the visible and X-ray spectral ranges. Moreover, the microcapsules have been examined in biological environment in in vitro and in vivo studies.

Optical probing of single fluorescent molecules and proteins

NARCIS (Netherlands)

Garcia Parajo, M.F.; Veerman, J.A.; Bouwhuis, R.; Bouwhuis, Rudo; van Hulst, N.F.; Vallée, R.A.L.

2001-01-01

Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis o­n the underlying principles methodology and the application of single-molecule analysis at room temperature. This Minireview is mainly focused o­n the application of confocal and

t-Butyl group-substituted triphenylamine-containing orange-red fluorescent emitters for organic light-emitting diodes

Energy Technology Data Exchange (ETDEWEB)

Lee, Kum Hee; Kim, Chi Sik [Department of Chemistry, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of); Kim, Young Kwan, E-mail: kimyk@hongik.ac.kr [Department of Information Display, Hongik University, Seoul 121-791 (Korea, Republic of); Yoon, Seung Soo, E-mail: ssyoon@skku.edu [Department of Chemistry, Sungkyunkwan University, Suwon, 440-746 (Korea, Republic of)

2012-03-30

Efficient orange-red fluorescent compounds, 4-(dicyanomethylene)-2-adamantyl-6-(4-(N-(4-tert-butylphenyl) -N-(3,5-di-tert-butylphenyl)amino)benzene)vinyl-4H-pyran (DCATP) and 2,6-bis[4-(N-(4-tert-butylphenyl)-N-(3,5-di-tert-butylphenyl)amino)benzene] vinyl-4-(dicyanomethylene)-4H-pyran (BDCTP) containing the tert-butylated triphenylamine in donor moieties, were synthesized and characterized. In these red emitters, bulky groups, such as t-butyl group and adamantane were introduced to increase the steric hindrance between the red emitters. In particular, an efficient orange-red device containing the emitter DCATP as a dopant showed a luminous and power efficiency of 6.87 cd/A and 2.70 lm/W, respectively, at 20 mA/cm{sup 2} with the CIE coordinates of (0.48, 0.50) at 7.0 V. In addition, an efficient red organic light-emitting diode using BDCTP as a dopant exhibited a luminous and power efficiency of 2.30 cd/A and 1.31 lm/W, respectively, at 20 mA/cm{sup 2} and CIE coordinates of (0.61, 0.39). - Highlights: Black-Right-Pointing-Pointer Two orange-red emitters with t-butylated triphenylamine derivatives were studied. Black-Right-Pointing-Pointer We examine changes in electron D-A and electron D-A-D type in the terminal groups. Black-Right-Pointing-Pointer Electron D-A-D type material shows improved color chromaticity.

A SIMPLE FLUORESCENT LABELING METHOD FOR STUDIES OF PROTEIN OXIDATION, PROTEIN MODIFICATION, AND PROTEOLYSIS

Science.gov (United States)

Pickering, Andrew. M.; Davies, Kelvin. J. A.

2014-01-01

Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the Proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve 3H or 14C methylation which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid-precipitation. Alternative labeling methods, have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied, or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, that binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid-precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, 3H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well-suited to study increased proteolytic susceptibility following protein modification, since the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite, and is stable over time and to extremes of pH, temperature (even boiling), freeze-thawing, mercaptoethanol, and methanol. PMID:21988844

FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

Directory of Open Access Journals (Sweden)

Shweta A Raina

Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

Science.gov (United States)

Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2014-07-01

KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

Fluorescence correlation spectroscopy on electron transfer reactions : probing inter- and intramolecular redox processes

NARCIS (Netherlands)

Sen, S.

2016-01-01

We developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing fluorophore ATTO655 was selected for labeling Azurin, a small blue mononuclear copper protein. Single molecule

An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v1; ref status: indexed, http://f1000r.es/2tt

Directory of Open Access Journals (Sweden)

Shin-ichiro Kojima

2014-02-01

Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v2; ref status: indexed, http://f1000r.es/39j

Directory of Open Access Journals (Sweden)

Shin-ichiro Kojima

2014-05-01

Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli

DEFF Research Database (Denmark)

Toddo, Stephen; Soderstrom, Bill; Palombo, Isolde

2012-01-01

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps....../periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli....

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

Science.gov (United States)

Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

2016-01-01

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

Fluorescence detection of a protein-bound 2Fe2S cluster.

Science.gov (United States)

Hoff, Kevin G; Goodlitt, Rochelle; Li, Rui; Smolke, Christina D; Silberg, Jonathan J

2009-03-02

A fluorescent biosensor is described for 2Fe2S clusters that is composed of green fluorescent protein (GFP) fused to glutaredoxin 2 (Grx2), as illustrated here. 2Fe2S detection is based on the reduction of GFP fluorescence upon the 2Fe2S-induced dimerization of GFP-Grx2. This assay is sufficiently sensitive to detect submicromolar changes in 2Fe2S levels, thus making it suitable for high-throughput measurements of metallocluster degradation and synthesis reactions.

Study of protein-probe complexation equilibria and protein-surfactant interaction using charge transfer fluorescence probe methyl ester of N,N-dimethylamino naphthyl acrylic acid

Energy Technology Data Exchange (ETDEWEB)

Mahanta, Subrata; Balia Singh, Rupashree; Bagchi, Arnab [Department of Chemistry University of Calcutta 92, A.P.C. Road, Kolkata 700009 (India); Nath, Debnarayan [Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032 (India); Guchhait, Nikhil, E-mail: nguchhait@yahoo.co [Department of Chemistry University of Calcutta 92, A.P.C. Road, Kolkata 700009 (India)

2010-06-15

In this paper, we demonstrate the interaction between intramolecular charge transfer (ICT) probe-Methyl ester of N,N-dimethylamino naphthyl acrylic acid (MDMANA) with bovine serum albumin (BSA) using absorption and fluorescence emission spectroscopy. The nature of probe protein binding interaction, fluorescence resonance energy transfer from protein to probe and time resolved fluorescence decay measurement predict that the probe molecule binds strongly to the hydrophobic cavity of the protein. Furthermore, the interaction of the anionic surfactant sodium dodecyl sulphate (SDS) with water soluble protein BSA has been investigated using MDMANA as fluorescenece probe. The changes in the spectral characteristics of charge transfer fluorescence probe MDMANA in BSA-SDS environment reflects well the nature of the protein-surfactant binding interaction such as specific binding, non-cooperative binding, cooperative binding and saturation binding.

Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Pletneva, Nadya V.; Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Souslova, Ekaterina; Chudakov, Dmitry M. [Russian Academy of Sciences, Moscow (Russian Federation); Lukyanov, Sergey [Russian Academy of Sciences, Moscow (Russian Federation); Nizhny Novgorod State Medical Academy, Nizhny Novgorod (Russian Federation); Martynov, Vladimir I.; Arhipova, Svetlena; Artemyev, Igor [Russian Academy of Sciences, Moscow (Russian Federation); Wlodawer, Alexander [National Cancer Institute, Frederick, MD 21702 (United States); Dauter, Zbigniew [National Cancer Institute, Argonne, IL 60439 (United States); Pletnev, Sergei [National Cancer Institute, Argonne, IL 60439 (United States); SAIC-Frederick, 9700 South Cass Avenue, Argonne, IL 60439 (United States); Russian Academy of Sciences, Moscow (Russian Federation)

2013-06-01

The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The yellow fluorescent protein phiYFPv (λ{sub em}{sup max} ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

Red fluorescence of dental biofilm as an indicator for assessing the efficacy of antimicrobials

Science.gov (United States)

Lee, Eun-Song; de Josselin de Jong, Elbert; Jung, Hoi-In; Kim, Baek-Il

2018-01-01

The study aimed to determine whether the red fluorescence (RF) of a dental microcosm biofilm as measured with quantitative light-induced fluorescence (QLF) technology is useful for assessing the efficacy of antimicrobials. Dental microcosm biofilms were formed on bovine enamel discs and grown under 0.3% sucrose challenge and treated with chlorhexidine (CHX) solutions at different concentrations (0.05%, 0.1%, and 0.5%) plus a negative control [sterile distilled water (DW)] twice daily for 7 days. The biofilms were photographed using a QLF-digital system to evaluate the RF by calculating the red/green ratio, and pH values of the medium were measured daily. After 7 days, the bacterial viability of the biofilm was assessed by measuring the counts of viable total bacteria and aciduric bacteria, and the percentage surface microhardness changes (%SHC) was evaluated. The RF and cariogenic properties were compared for the different concentrations of CHX, and their correlations were examined. The RF and its increase rate were much lower for CHX-treated biofilms than for DW-treated biofilms. The RF after 7 days of maturation decreased significantly with increasing CHX concentrations (pbiofilms and cariogenic properties, such as the number of total bacteria (r=0.93), number of aciduric bacteria (r=0.97), supernatant pH (r=0.43), and %SHC (r=0.98). In conclusion, the RF of dental biofilms as measured with QLF technology can be used to nondestructively assess and monitor the effect of antimicrobials against biofilm.

Green Fluorescent Protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora ramorum

Science.gov (United States)

Marko Riedel; Gautier Calmin; Lassaad Belbahri; Francois Lefort; Monika Gotz; Stefan Wagner; Sabine. Werres

2009-01-01

Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl2-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen...

Microscopie de fluorescence de protéines autofluorescentes uniques pour la biologie cellulaire

Science.gov (United States)

Cognet, Laurent; Coussen, Françoise; Choquet, Daniel; Lounis, Brahim

In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells. To cite this article: L. Cognet et al., C. R. Physique 3 (2002) 645-656.

Ubiquitous distribution of fluorescent protein in muscles of four ...

Indian Academy of Sciences (India)

In this study, the localization of fluorescent protein (FP) was characterized in the muscles of ... A. mossambica have four exons and three introns, and were common to that of FABP family. ..... organization of the neurons (Rakic 1971; Feng et al.

Les peintures romanes de la cathédrale d’Auxerre. Nouvelles observations 

Directory of Open Access Journals (Sweden)

Alexandre Gordine

2009-09-01

Full Text Available La poursuite des investigations du CEM dans la crypte de la cathédrale Saint-Étienne attire de nouveau l’attention sur ses peintures murales médiévales. Certes, les fresques romanes, notamment la célèbre peinture du Christ à cheval, ont déjà fait l’objet récemment d’une étude très poussée, et à bien des égards exemplaire, par Emmanuelle Cadet et Juliette Rollier-Hanselmann . Néanmoins, les acquisitions de cette étude pourraient être nuancées et même complétées par quelques observations nouvel...

Detection of NT-pro BNP using fluorescent protein modified by streptavidin as a label in immunochromatographic assay

Directory of Open Access Journals (Sweden)

Haixia Li

2016-12-01

Full Text Available A novel fluorescent immunochromatographic assay for the detection of NT-proBNP in human serum has been developed. Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody labeled with fluorescent protein and “sandwiched” by another monoclonal antibody immobilized on the nitrocellulose membrane, the fluorescence and concentration of analytes were measured and then calculated by fluoroanalyzer. The fluorescent protein is a fusion protein and was prepared through the application of Streptavidin gene SA, β subunit cpcB of Phycocyanin, lyase alr0617, and phycoerythrobilin synthetase gene ho1, pebA, pebB for covalent binding. It is characterized with higher stability, good solubility in water and it is not easy to quench fluorescence. Take the advantages of fluorescent protein, the immunochromatographic assay exhibited a wide linear range for NT-proBNP from 200 pg ml−1 to 26,000 pg ml−1, with a detection limit of 47 pg ml−1 under optimal conditions. Compared with chemiluminescence immunoassay (CLIA, 131 human serum samples were analyzed and the correlation coefficient of the developed immunoassay was 0.978. These results demonstrated that fluorescent immunochromatographic assay is a more rapid, sensitive, specific method and could be developed into a platform for more biomarkers determination in clinical practice. Keywords: NT-pro BNP, Fluorescent protein, Immunochromatographic assay

Dual time-resolved temperature-jump fluorescence and infrared spectroscopy for the study of fast protein dynamics.

Science.gov (United States)

Davis, Caitlin M; Reddish, Michael J; Dyer, R Brian

2017-05-05

Time-resolved temperature-jump (T-jump) coupled with fluorescence and infrared (IR) spectroscopy is a powerful technique for monitoring protein dynamics. Although IR spectroscopy of the polypeptide amide I mode is more technically challenging, it offers complementary information because it directly probes changes in the protein backbone, whereas, fluorescence spectroscopy is sensitive to the environment of specific side chains. With the advent of widely tunable quantum cascade lasers (QCL) it is possible to efficiently probe multiple IR frequencies with high sensitivity and reproducibility. Here we describe a dual time-resolved T-jump fluorescence and IR spectrometer and its application to study protein folding dynamics. A Q-switched Ho:YAG laser provides the T-jump source for both time-resolved IR and fluorescence spectroscopy, which are probed by a QCL and Ti:Sapphire laser, respectively. The Ho:YAG laser simultaneously pumps the time-resolved IR and fluorescence spectrometers. The instrument has high sensitivity, with an IR absorbance detection limit of jump induced difference spectrum from 50ns to 0.5ms. This study demonstrates the power of the dual time-resolved T-jump fluorescence and IR spectroscopy to resolve complex folding mechanisms by complementary IR absorbance and fluorescence measurements of protein dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Monitoring protein synthesis by fluorescence recovery after photobleaching (FRAP) in vivo

OpenAIRE

sprotocols

2015-01-01

Currently available methodologies for measuring protein synthesis rates rely on metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides. These approaches are hampered by several limitations and cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a novel method for monitoring protein synthesis in specific cells and tissues of live Caenorhabditis elegans animals. Fluorescent reporter proteins such as...

In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

International Nuclear Information System (INIS)

Hsu, Y.-Y.; Liu, Y.-N.; Wang Wenyen; Kao, Fu-Jen; Kung, S.-H.

2007-01-01

An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP 2 )-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A pro ) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A pro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research

A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

Science.gov (United States)

Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

2018-01-01

In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

Compact whole-body fluorescent imaging of nude mice bearing EGFP expressing tumor

Science.gov (United States)

Chen, Yanping; Xiong, Tao; Chu, Jun; Yu, Li; Zeng, Shaoqun; Luo, Qingming

2005-01-01

Issue of tumor has been a hotspot of current medicine. It is important for tumor research to detect tumors bearing in animal models easily, fast, repetitively and noninvasivly. Many researchers have paid their increasing interests on the detecting. Some contrast agents, such as green fluorescent protein (GFP) and Discosoma red fluorescent protein (Dsred) were applied to enhance image quality. Three main kinds of imaging scheme were adopted to visualize fluorescent protein expressing tumors in vivo. These schemes based on fluorescence stereo microscope, cooled charge-coupled-device (CCD) or camera as imaging set, and laser or mercury lamp as excitation light source. Fluorescence stereo microscope, laser and cooled CCD are expensive to many institutes. The authors set up an inexpensive compact whole-body fluorescent imaging tool, which consisted of a Kodak digital camera (model DC290), fluorescence filters(B and G2;HB Optical, Shenyang, Liaoning, P.R. China) and a mercury 50-W lamp power supply (U-LH50HG;Olympus Optical, Japan) as excitation light source. The EGFP was excited directly by mercury lamp with D455/70 nm band-pass filter and fluorescence was recorded by digital camera with 520nm long-pass filter. By this easy operation tool, the authors imaged, in real time, fluorescent tumors growing in live mice. The imaging system is external and noninvasive. For half a year our experiments suggested the imaging scheme was feasible. Whole-body fluorescence optical imaging for fluorescent expressing tumors in nude mouse is an ideal tool for antitumor, antimetastatic, and antiangiogenesis drug screening.

Biocompatibility study of protein capped and uncapped silver nanoparticles on human hemoglobin

Science.gov (United States)

Bhunia, Amit Kumar; Kanti Samanta, Pijus; Aich, Debasish; Saha, Satyajit; Kamilya, Tapanendu

2015-06-01

The interactions of human hemoglobin with protein capped silver nanoparticles and bare silver nanoparticles were studied to understand fundamental perspectives about the biocompatibility of protein capped silver nanoparticles compared with bare silver nanoparticles. Bare silver (Ag) nanoparticles (NPs) were prepared by the chemical reduction method. High resolution transmission electron microscopy (HRTEM) analysis along with absorption at ~390 nm indicated the formation of bare Ag NPs. Protein coated Ag NPs were prepared by a green synthesis method. Absorption at ~440 nm along with ~280 nm indicated the formation of protein coated Ag NPs. The biocompatibility of the above mentioned Ag NPs was studied by interaction with human hemoglobin (Hb) protein. In presence of bare Ag NPs, the Soret band of Hb was red shifted. This revealed the distortion of iron from the heme pockets of Hb. Also, the fluorescence peak of Hb was quenched and red shifted which indicated that Hb became unfolded in the presence of bare Ag NPs. No red shift of the absorption of Soret, along with no shift and quenching of the fluorescence peak of Hb were observed in the presence of protein coated Ag NPs. A hemolysis assay suggested that protein coated Ag NPs were more biocompatible than bare one.

Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

CERN Document Server

Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

2001-01-01

Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

OpenAIRE

Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

2015-01-01

In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP....

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

DEFF Research Database (Denmark)

Østergaard, H.; Henriksen, A.; Hansen, Flemming G.

2001-01-01

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...... in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivoby non- invasive fluorimetric measurements. The 1.5 Angstrom crystal structure of the oxidized protein revealed a disulfide bond- induced distortion of the beta -barrel, as well...... the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway....

Rotational order–disorder structure of fluorescent protein FP480

International Nuclear Information System (INIS)

Pletnev, Sergei; Morozova, Kateryna S.; Verkhusha, Vladislav V.; Dauter, Zbigniew

2009-01-01

An analysis of the rotational order–disorder structure of fluorescent protein FP480 is presented. In the last decade, advances in instrumentation and software development have made crystallography a powerful tool in structural biology. Using this method, structural information can now be acquired from pathological crystals that would have been abandoned in earlier times. In this paper, the order–disorder (OD) structure of fluorescent protein FP480 is discussed. The structure is composed of tetramers with 222 symmetry incorporated into the lattice in two different ways, namely rotated 90° with respect to each other around the crystal c axis, with tetramer axes coincident with crystallographic twofold axes. The random distribution of alternatively oriented tetramers in the crystal creates a rotational OD structure with statistically averaged I422 symmetry, although the presence of very weak and diffuse additional reflections suggests that the randomness is only approximate

Comparative Study of Lettuce and Radish Grown Under Red and Blue LEDs and White Fluorescent Lamps

Science.gov (United States)

Mickens, Matthew A.; Massa, Gioia; Newsham, Gerard; Wheeler, Raymond; Birmele, Michele

2016-01-01

Growing vegetable crops in space will be an essential part of sustaining astronauts during long-range missions. To drive photosynthesis, red and blue light-emitting diodes (LEDs) have attracted attention because of their efficiency, longevity, small size, and safety. In efforts to optimize crop yield, there is also recent interest in analyzing the subtle effects of additional wavelengths on plant growth. For instance, since plants often look purplish gray under red and blue LEDs, the addition of green light allows easy recognition of disease and the assessment of plant health status. However, it is important to know if wavelengths outside the traditional red and blue wavebands have a direct effect on enhancing or hindering the mechanisms involved in plant growth. In this experiment, a comparative study was performed on two short cycle crops of red romaine lettuce (Lactuca sativa cv. "Outredgeous") and radish (Raphanus sativa cv. 'Cherry Bomb'), which were grown under two light treatments. The first treatment being red (630 nm) and blue (450 nm) LEDs alone, while the second treatment consisted of daylight tri-phosphor fluorescent lamps (CCT approximately 5000 K) at equal photosynthetic photon flux (PPF). The treatment effects were evaluated by measuring the fresh biomass produced, plant morphology and leaf dimensions, leaf chlorophyll content, and adenosine triphosphate (ATP) within plant leaf/storage root tissues.

Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

Science.gov (United States)

Favre, Bertrand; Begré, Nadja; Bouameur, Jamal-Eddine; Borradori, Luca

2016-01-01

Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies. © 2016 Elsevier Inc. All rights reserved.

Colorful packages : fluorescent proteins in complex coacervate core micelles

NARCIS (Netherlands)

Nolles, Antsje

2018-01-01

This thesis explores the encapsulation of fluorescent proteins (FPs) into complex coacervate core micelles (C3Ms) and features the impact of this encapsulation on the biophysical properties of the FPs. In total eight different FPs were investigated originating from two different classes

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

Directory of Open Access Journals (Sweden)

Weatherford Wendy

2005-05-01

Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

Science.gov (United States)

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

Quantization of bovine serum albumin by fluorescence enhancement effects and corresponding binding of macrocyclic host-protein assembly.

Science.gov (United States)

Bardhan, Munmun; Misra, Tapas; Ganguly, Tapan

2012-01-05

The present paper reports the investigations on the spectroscopic behavior of the binary complexes of the dye aurintricarboxylic acid (ATA) with protein bovine serum albumin (BSA) and 18-crown 6 (CW) (ATA·BSA, ATA·CW) and the ternary complex ATA·CW·BSA by using UV-vis steady state and time resolved fluorescence spectroscopy. The primary aim of the work is to determine the protein (BSA) quantization by fluorescence enhancement method and investigate the 'enhancer' activity of crown ether (CW) on it to increase the resolution. Steady state and time resolved fluorescence measurements demonstrated how fluorescence intensity of ATA could be used for the determination of the protein BSA in aqueous solution. The binding of dye (probe/fluorescent medicinal molecule) with protein and the denaturing effect in the polar environment of acetonitrile of the dye protein complex act as drug binding as well as drug release activity. Apart from its basic research point of view, the present study also possesses significant importance and applications in the field of medicinal chemistry. Copyright © 2011 Elsevier B.V. All rights reserved.

Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.

Science.gov (United States)

Huang, Youhua; Huang, Xiaohong; Cai, Jia; Ye, Fuzhou; Guan, Liya; Liu, Hong; Qin, Qiwei

2011-09-01

Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances. Copyright © 2011 Elsevier B.V. All rights reserved.

A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I

Directory of Open Access Journals (Sweden)

Seung-Wan Kim

2016-05-01

Full Text Available The intramolecular fluorescence self-quenching phenomenon is a major drawback in developing high-performance fluorometric biosensors which use common fluorophores as signal generators. We propose two strategies involving liberation of the fluorescent molecules by means of enzymatic fragmentation of protein or dehybridization of double-stranded DNA. In the former, bovine serum albumin (BSA was coupled with the fluorescent BODIPY dye (Red BSA, and then immobilized on a solid surface. When the insolubilized Red BSA was treated with proteinase K (10 units/mL for 30 min, the fluorescent signal was significantly increased (3.5-fold compared to the untreated control. In the second case, fluorophore-tagged DNA probes were linked to gold nanoparticles by hybridization with capture DNA strands densely immobilized on the surface. The quenched fluorescence signal was recovered (3.7-fold by thermal dehybridization, which was induced with light of a specific wavelength (e.g., 530 nm for less than 1 min. We next applied the Red BSA self-quenching relaxation technique employing enzymatic fragmentation to a high-performance immunoassay of cardiac troponin I (cTnI in a microtiter plate format. The detection limit was 0.19 ng/mL cTnI, and the fluorescent signal was enhanced approximately 4.1-fold compared with the conventional method of direct measurement of the fluorescent signal from a non-fragmented fluorophore-labeled antibody.

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

Directory of Open Access Journals (Sweden)

Juan A. González-Vera

2015-11-01

Full Text Available Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

NARCIS (Netherlands)

Mastop, M.; Bindels, D.S.; Shaner, N.C.; Postma, M.; Gadella, T.W.J.; Goedhart, J.

2017-01-01

The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching

Site-specific fluorescent labeling of nascent proteins on the translating ribosome.

Science.gov (United States)

Saraogi, Ishu; Zhang, Dawei; Chandrasekaran, Sandhya; Shan, Shu-ou

2011-09-28

As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.

Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium

International Nuclear Information System (INIS)

Wang Lei; Xu Guang; Shi Zhikun; Jiang Wei; Jin Wenrui

2007-01-01

We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H + L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 x 10 -11 to 8.1 x 10 -10 mol L -1

IR-FEL-induced green fluorescence protein (GFP) gene transfer into plant cell

CERN Document Server

Awazu, K; Tamiya, E

2002-01-01

A Free Electron Laser (FEL) holds potential for various biotechnological applications due to its characteristics such as flexible wavelength tunability, short pulse and high peak power. We could successfully introduce the Green Fluorescent Protein (GFP) gene into tobacco BY2 cells by IR-FEL laser irradiation. The irradiated area of the solution containing BY2 cells and plasmid was about 0.1 mm sup 2. FEL irradiation at a wavelength of 5.75 and 6.1 mu m, targeting absorption by the ester bond of the lipid and the amide I bond of the protein, respectively, was shown to cause the introduction of the fluorescent dye into the cell. On the other hand, transient expression of the GFP fluorescence was only observed after irradiation at 5.75 mu m. The maximum transfer efficiency was about 0.5%.

Provável síndrome de Rasmussen: relato de caso

OpenAIRE

CORAL, LUÍZ CARLOS; HAAS, LEANDRO JOSÉ

1999-01-01

A síndrome de encefalite crônica com epilepsia (síndrome de Rasmussen) ocorre tipicamente em crianças e é caracterizada pelo desenvolvimento de epilepsia focal intratável, hemiparesia progressiva e deterioração intelectual. A etiologia é desconhecida e as anormalidades patológicas variam com a fase da doença. Relatamos o caso de um paciente jovem, com 17 anos de idade, com apresentação clínica da provável síndrome de Rasmussen. Discutimos a etiologia e o tratamento da patologia. The syndro...

Provável síndrome de Rasmussen: relato de caso

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CORAL LUÍZ CARLOS

1999-01-01

Full Text Available A síndrome de encefalite crônica com epilepsia (síndrome de Rasmussen ocorre tipicamente em crianças e é caracterizada pelo desenvolvimento de epilepsia focal intratável, hemiparesia progressiva e deterioração intelectual. A etiologia é desconhecida e as anormalidades patológicas variam com a fase da doença. Relatamos o caso de um paciente jovem, com 17 anos de idade, com apresentação clínica da provável síndrome de Rasmussen. Discutimos a etiologia e o tratamento da patologia.

Effect of pH on the Heat-Induced Denaturation and Renaturation of Green Fluorescent Protein: A Laboratory Experiment

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Flores, Rosa V.; Sola, Hilda M.; Torres, Juan C.; Torres, Rafael E.; Guzman, Ernick E.

2013-01-01

A fluorescence spectroscopy experiment is described where students integrated biochemistry and instrumental analysis, while characterizing the green fluorescent protein excitation and emission spectra in terms of its phenolic and phenolate chromophores. Students studied the combined effect of pH and temperature on the protein's fluorescence,…

Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

Science.gov (United States)

Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

2015-01-01

Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

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Jae Eun Lee

2015-06-01

Full Text Available Two dimensional-fluorescence difference gel electrophoresis (2D DIGE is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

Time-Resolved Fluorescence Immunoassay for C-Reactive Protein Using Colloidal Semiconducting Nanoparticles

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Pekka Hänninen

2011-11-01

Full Text Available Besides the typical short-lived fluorescence with decay times in the nanosecond range, colloidal II/VI semiconductor nanoparticles dispersed in buffer also possess a long-lived fluorescence component with decay times in the microsecond range. Here, the signal intensity of the long-lived luminescence at microsecond range is shown to increase 1,000-fold for CdTe nanoparticles in PBS buffer. This long-lived fluorescence can be conveniently employed for time-gated fluorescence detection, which allows for improved signal-to-noise ratio and thus the use of low concentrations of nanoparticles. The detection principle is demonstrated with a time-resolved fluorescence immunoassay for the detection of C-reactive protein (CRP using CdSe-ZnS nanoparticles and green light excitation.

Effect of tissue scaffold topography on protein structure monitored by fluorescence spectroscopy

NARCIS (Netherlands)

Portugal, C.A.M.; Truckenmüller, R.K.; Stamatialis, Dimitrios; Crespo, J.G.

2014-01-01

The impact of surface topography on the structure of proteins upon adhesion was assessed through non-invasive fluorescence monitoring. This study aimed at obtaining a better understanding about the role of protein structural status on cell–scaffold interactions. The changes induced upon adsorption

Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis

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Bailey Kirra

2010-11-01

Full Text Available Abstract Background Fluorescent proteins are powerful molecular biology tools that have been used to study the subcellular dynamics of proteins within live cells for well over a decade. Two fluorescent proteins commonly used to enable dual protein labelling are GFP (green and mCherry (red. Sporulation in the Gram positive bacterium Bacillus subtilis has been studied for many years as a paradigm for understanding the molecular basis for differential gene expression. As sporulation initiates, cells undergo an asymmetric division leading to differential gene expression in the small prespore and large mother cell compartments. Use of two fluorescent protein reporters permits time resolved examination of differential gene expression either in the same compartments or between compartments. Due to the spectral properties of GFP and mCherry, they are considered an ideal combination for co-localisation and co-expression experiments. They can also be used in combination with fluorescent DNA stains such as DAPI to correlate protein localisation patterns with the developmental stage of sporulation which can be linked to well characterised changes in DNA staining patterns. Findings While observing the recruitment of the transcription machinery into the forespore of sporulating Bacillus subtilis, we noticed the occurrence of stage-specific fluorescence intensity differences between GFP and mCherry. During vegetative growth and the initial stages of sporulation, fluorescence from both GFP and mCherry fusions behaved similarly. During stage II-III of sporulation we found that mCherry fluorescence was considerably diminished, whilst GFP signals remained clearly visible. This fluorescence pattern reversed during the final stage of sporulation with strong mCherry and low GFP fluorescence. These trends were observed in reciprocal tagging experiments indicating a direct effect of sporulation on fluorescent protein fluorophores. Conclusions Great care should be taken

Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

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Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

2014-03-01

Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

Plant-associated fluorescent Pseudomonas from red lateritic soil: Beneficial characteristics and their impact on lettuce growth.

Science.gov (United States)

Maroniche, Guillermo A; Rubio, Esteban J; Consiglio, Adrián; Perticari, Alejandro

2016-11-25

Fluorescent Pseudomonas are ubiquitous soil bacteria that usually establish mutualistic associations with plants, promoting their growth and health by several mechanisms. This makes them interesting candidates for the development of crop bio-inoculants. In this work, we isolated phosphate-solubilizing fluorescent Pseudomonas from the rhizosphere and inner tissues of different plant species growing in red soil from Misiones, Argentina. Seven isolates displaying strong phosphate solubilization were selected for further studies. Molecular identification by rpoD genotyping indicated that they belong to different species within the P. fluorescens and P. putida phylogenetic groups. Screening for in vitro traits such as phosphate solubilization, growth regulators synthesis or degradation, motility and antagonism against phytopathogens or other bacteria, revealed a unique profile of characteristics for each strain. Their plant growth-promoting potential was assayed using lettuce as a model for inoculation under controlled and greenhouse conditions. Five of the strains increased the growth of lettuce plants. Overall, the strongest lettuce growth promoter under both conditions was strain ZME4, isolated from inner tissues of maize. No clear association between lettuce growth promotion and in vitro beneficial traits was detected. In conclusion, several phosphate solubilizing pseudomonads from red soil were isolated that display a rich array of plant growth promotion traits, thus showing a potential for the development of new inoculants.

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

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van Zyl, Winschau F.; Deane, Shelly M.

2015-01-01

Lactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in the in vivo tagging of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423. The mCherry gene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome of L. plantarum 423 by homologous recombination. In the case of E. mundtii ST4SA, the mCherry gene was cloned into the pGKV223D LAB/Escherichia coli expression vector. Expression of the mCherry gene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice. PMID:26116681

The use of yellow fluorescent hybrids to indicate mating in Trypanosoma brucei

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Ferris Vanessa

2008-02-01

Full Text Available Abstract Background Trypanosoma brucei undergoes genetic exchange in its insect vector, the tsetse fly, by an unknown mechanism. The difficulties of working with this experimental system of genetic exchange have hampered investigation, particularly because the trypanosome life cycle stages involved cannot be cultured in vitro and therefore must be examined in the insect. Searching for small numbers of hybrid trypanosomes directly in the fly has become possible through the incorporation of fluorescent reporter genes, and we have previously carried out a successful cross using a reporter-repressor strategy. However, we could not be certain that all fluorescent trypanosomes observed in that cross were hybrids, due to mutations of the repressor leading to spontaneous fluorescence, and we have therefore developed an alternative strategy. Results To visualize the production of hybrids in the fly, parental trypanosome clones were transfected with a gene encoding Green Fluorescent Protein (GFP or Red Fluorescent Protein (RFP. Co-infection of flies with red and green fluorescent parental trypanosomes produced yellow fluorescent hybrids, which were easily visualized in the fly salivary glands. Yellow trypanosomes were not seen in midgut or proventricular samples and first appeared in the glands as epimastigotes as early as 13 days after fly infection. Cloned progeny originating from individual salivary glands had yellow, red, green or no fluorescence and were confirmed as hybrids by microsatellite, molecular karyotype and kinetoplast (mitochondrial DNA analyses. Hybrid clones showed biparental inheritance of both nuclear and kinetoplast genomes. While segregation and reassortment of the reporter genes and microsatellite alleles were consistent with Mendelian inheritance, flow cytometry measurement of DNA content revealed both diploid and polyploid trypanosomes among the hybrid progeny clones. Conclusion The strategy of using production of yellow hybrids

Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

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Tobias Haase

Full Text Available BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC Förster resonance energy transfer (FRET was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP was used as FRET donor and yellow fluorescent protein (YFP as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat, Winger and Marletta (Biochemistry 2005, 44:4083-90 have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a

Isolation, sequencing and expression of RED, a novel human gene encoding an acidic-basic dipeptide repeat.

Science.gov (United States)

Assier, E; Bouzinba-Segard, H; Stolzenberg, M C; Stephens, R; Bardos, J; Freemont, P; Charron, D; Trowsdale, J; Rich, T

1999-04-16

A novel human gene RED, and the murine homologue, MuRED, were cloned. These genes were named after the extensive stretch of alternating arginine (R) and glutamic acid (E) or aspartic acid (D) residues that they contain. We term this the 'RED' repeat. The genes of both species were expressed in a wide range of tissues and we have mapped the human gene to chromosome 5q22-24. MuRED and RED shared 98% sequence identity at the amino acid level. The open reading frame of both genes encodes a 557 amino acid protein. RED fused to a fluorescent tag was expressed in nuclei of transfected cells and localised to nuclear dots. Co-localisation studies showed that these nuclear dots did not contain either PML or Coilin, which are commonly found in the POD or coiled body nuclear compartments. Deletion of the amino terminal 265 amino acids resulted in a failure to sort efficiently to the nucleus, though nuclear dots were formed. Deletion of a further 50 amino acids from the amino terminus generates a protein that can sort to the nucleus but is unable to generate nuclear dots. Neither construct localised to the nucleolus. The characteristics of RED and its nuclear localisation implicate it as a regulatory protein, possibly involved in transcription.

A sulfhydryl-reactive ruthenium (II complex and its conjugation to protein G as a universal reagent for fluorescent immunoassays.

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Jing-Tang Lin

Full Text Available To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2'-dipyridine Ruthenium bis (hexafluorophosphate. The synthesized Ru(II complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. The emission peak wavelength of the Ru(II-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II complex, indicating that Ru(II-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG binding assay was conducted. The result showed that Ru(II-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.

Time variation of fluorescence lifetime in enhanced cyan fluorescence protein

International Nuclear Information System (INIS)

Lee, Soonhyouk; Kim, Soo Yong; Park, Kyoungsook; Jeong, Jinyoung; Chung, Bong Hyun; Kim, Sok Won

2010-01-01

The lifetime variations of enhanced cyan fluorescence protein (ECFP) in relatively short integration time bins were studied via time-correlated single photon counting (TCSPC) measurement. We observed that minimum photon counts are necessary for the lifetime estimation to achieve a certain range of variance. The conditions to decrease the variance of lifetime were investigated and the channel width of the measurement of TCSPC data was found to be another important factor for the variance of lifetime. Though the lifetime of ECFP is best fit by a double exponential, a mono exponential fit for the same integration time is more stable. The results may be useful in the analysis of photophysical dynamics for ensemble molecules in short measurement time windows.

Protein A Detection Based on Quantum Dots-Antibody Bioprobe Using Fluorescence Coupled Capillary Electrophoresis

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Lin Qiu

2014-01-01

Full Text Available In this report, fluorescence detection coupled capillary electrophoresis (CE-FL was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.

Reactivity of polymeric proanthocyanidins toward salivary proteins and their contribution to young red wine astringency.

Science.gov (United States)

Sun, Baoshan; de Sá, Marta; Leandro, Conceição; Caldeira, Ilda; Duarte, Filomena L; Spranger, Isabel

2013-01-30

Recent studies have indicated the presence of significant amount of highly polymerized and soluble proanthocyanidins in red wine and such compounds interacted readily with proteins, suggesting that they might be particularly astringent. Thus, the objective of this work was to verify the astringency of polymeric proanthocyanidins and their contribution to red wine astringency. The precipitation reactions of the purified oligomeric procyanidins (degree of polymerization ranging from 2 to 12-15) and polymeric procyanidins (degree of polymerization ranging from 12-15 to 32-34) with human salivary proteins were studied; salivary proteins composition changes before and after the reaction was verified by SDS-PAGE and procyanidins composition changes by spectrometric, direct HPLC and thiolysis-HPLC methods. The astringency intensity of these two procyanidin fractions was evaluated by a sensory analysis panel. For verifying the correlation between polymeric proanthocyanidins and young red wine astringency, the levels of total oligomeric and total polymeric proanthocyanidins and other phenolic composition in various young red wines were quantified and the astringency intensities of these wines were evaluated by a sensory panel. The results showed that polymeric proanthocyanidins had much higher reactivity toward human salivary proteins and higher astringency intensity than the oligomeric ones. Furthermore, young red wine astringency intensities were highly correlated to levels of polymeric proanthocyanidins, particularly at low concentration range (correlation coefficient r = 0.9840) but not significant correlated to total polyphenols (r = 0.2343) or other individual phenolic compounds (generally r wine astringency and the levels of polymeric polyphenols in red wines may be used as an indicator for its astringency.

Synthesis and bioimaging of biodegradable red fluorescent organic nanoparticles with aggregation-induced emission characteristics.

Science.gov (United States)

Xu, Dazhuang; Zou, Hui; Liu, Meiying; Tian, Jianwen; Huang, Hongye; Wan, Qing; Dai, Yanfeng; Wen, Yuanqing; Zhang, Xiaoyong; Wei, Yen

2017-12-15

Fluorescent organic nanoparticles (FONs) with aggregation-induced emission (AIE) features have recently emerged as promising fluorescent probes for biomedical applications owing to their excellent optical properties, designability and biocompatibility. Significant progress has been made recently for synthesis and biomedical applications of these AIE-active FONs. However, only very limited reports have demonstrated the fabrication of biodegradable AIE-active FONs with red fluorescence emission. In this study, a novel strategy has been developed for the preparation of biodegradable AIE-active polyurethanes (PUs) through a two-step polymerization, in which the diisocyanate-terminated polyethylene glycol (NCO-PEG-NCO) was synthesized and subsequently conjugated with diamine-containing AIE dye (NH 2 -Phe-NH 2 ). The successful synthesis of AIE-active Phe-PEG 2000 PUs is evidenced by a series of characterization techniques. Because of the formation of AIE-active amphiphilic PUs, the final copolymers can self-assemble into spherical nanoparticles, which exhibit strong luminescence and high water dispersion. The biological evaluation results suggest that the AIE-active Phe-PEG 2000 FONs possess low toxicity and desirable cell permeability. Therefore, we anticipate that these AIE-active FONs with biodegradable potential will trigger much research enthusiasm and effort toward the creation of new AIE-active materials with improved properties for various biomedical applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

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Vuento Matti

2006-12-01

Full Text Available Abstract Fluorescence correlation spectroscopy (FCS monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids were fused to the C-terminus of the enhanced green fluorescent protein (EGFP. The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs. The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

Science.gov (United States)

Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

2014-01-01

In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. PMID:25287913

Steady full colour white organic light-emitting devices consisting of an ultrathin red fluorescent layer

International Nuclear Information System (INIS)

Wen Wen; Yu Junsheng; Li Lu; Wang Jun; Jiang Yadong

2009-01-01

White organic light-emitting devices were fabricated using an ultrathin red fluorescent dye of 3-(dicyanomethylene)-5, 5-dimethyl-1-(4-dimethylamino-styryl)cyclohexene inserted in tris(8-quinolinolato) aluminium layer as a red and green emitting layer (EML) and a thin 4, 4'-bis(2, 2'-diphenylvinyl)-1, 1'-diphenyl (DPVBi) layer as blue EML. A maximum power efficiency of 2.4 lm W -1 at 5.5 V and a maximum luminance of 16 690 cd m -2 at 18.5 V were obtained. Pure white emission with a good colour rendering index of 80 was achieved as low as 5 V. The Commission Internationale de l'Eclairage (CIE) coordinates near (0.330, 0.300) show a slight variation of (-0.020, +0.002) in a wide range of voltages. The achievement of full colour white emission at low-operation voltages and high-colour stability is attributed to the confining emission zone function of the thin EML and direct carrier trapping in the ultrathin layer.

Once for All: A Novel Robust System for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants

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Guitao Zhong

2017-06-01

Full Text Available Chimeric fluorescent fusion proteins have been employed as a powerful tool to reveal the subcellular localizations and dynamics of proteins in living cells. Co-expression of a fluorescent fusion protein with well-known organelle markers in the same cell is especially useful in revealing its spatial and temporal functions of the protein in question. However, the conventional methods for co-expressing multiple fluorescent tagged proteins in plants have the drawbacks of low expression efficiency, variations in the expression level and time-consuming genetic crossing. Here, we have developed a novel robust system that allows for high-efficient co-expression of multiple chimeric fluorescent fusion proteins in plants in a time-saving fashion. This system takes advantage of employing a single expression vector which consists of multiple semi-independent expressing cassettes for the protein co-expression thereby overcoming the limitations of using multiple independent expressing plasmids. In addition, it is a highly manipulable DNA assembly system, in which modification and recombination of DNA molecules are easily achieved through an optimized one-step assembly reaction. By employing this effective system, we demonstrated that co-expression of two chimeric fluorescent fusion reporter proteins of vacuolar sorting receptor and secretory carrier membrane protein gave rise to their perspective subcellular localizations in plants via both transient expression and stable transformation. Thus, we believed that this technical advance represents a promising approach for multi-color-protein co-expression in plant cells.

Analytical use of multi-protein Fluorescence Resonance Energy Transfer to demonstrate membrane-facilitated interactions within cytokine receptor complexes.

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Krause, Christopher D; Izotova, Lara S; Pestka, Sidney

2013-10-01

Experiments measuring Fluorescence Resonance Energy Transfer (FRET) between cytokine receptor chains and their associated proteins led to hypotheses describing their organization in intact cells. These interactions occur within a larger protein complex or within a given nano-environment. To illustrate this complexity empirically, we developed a protocol to analyze FRET among more than two fluorescent proteins (multi-FRET). In multi-FRET, we model FRET among more than two fluorophores as the sum of all possible pairwise interactions within the complex. We validated our assumption by demonstrating that FRET among pairs within a fluorescent triplet resembled FRET between each pair measured in the absence of the third fluorophore. FRET between two receptor chains increases with increasing FRET between the ligand-binding chain (e.g., IFN-γR1, IL-10R1 and IFN-λR1) and an acylated fluorescent protein that preferentially resides within subsections of the plasma membrane. The interaction of IL-10R2 with IFN-λR1 or IL-10R1 results in decreased FRET between IL-10R2 and the acylated fluorescent protein. Finally, we analyzed FRET among four fluorescent proteins to demonstrate that as FRET between IFN-γR1 and IFN-γR2 or between IFN-αR1 and IFN-αR2c increases, FRET among other pairs of proteins changes within each complex. Copyright © 2013 Elsevier Ltd. All rights reserved.

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

Science.gov (United States)

Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

2016-04-01

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Energetic costs of protein synthesis do not differ between red- and white-blooded Antarctic notothenioid fishes.

Science.gov (United States)

Lewis, Johanne M; Grove, Theresa J; O'Brien, Kristin M

2015-09-01

Antarctic icefishes (Family Channichthyidae) within the suborder Notothenioidei lack the oxygen-binding protein hemoglobin (Hb), and six of the 16 species of icefishes lack myoglobin (Mb) in heart ventricle. As iron-centered proteins, Hb and Mb can promote the formation of reactive oxygen species (ROS) that damage biological macromolecules. Consistent with this, our previous studies have shown that icefishes have lower levels of oxidized proteins and lipids in oxidative muscle compared to red-blooded notothenioids. Because oxidized proteins are usually degraded by the 20S proteasome and must be resynthesized, we hypothesized that rates of protein synthesis would be lower in icefishes compared to red-blooded notothenioids, thereby reducing the energetic costs of protein synthesis and conferring a benefit to the loss of Hb and Mb. Rates of protein synthesis were quantified in hearts, and the fraction of oxygen consumption devoted to protein synthesis was measured in isolated hepatocytes and cardiomyocytes of notothenioids differing in the expression of Hb and cardiac Mb. Neither rates of protein synthesis nor the energetic costs of protein synthesis differed among species, suggesting that red-blooded species do not degrade and replace oxidatively modified proteins at a higher rate compared to icefishes but rather, persist with higher levels of oxidized proteins. Copyright © 2015 Elsevier Inc. All rights reserved.