14,152 research outputs found
Simultaneous quantitative live cell imaging of multiple FRET-based biosensors.
No full textWe have developed a novel method for multi-color spectral FRET analysis which is used to study a system of three independent FRET-based molecular sensors composed of the combinations of only three fluorescent proteins. This method is made possible by a novel routine for computing the 3-D excitation/emission spectral fingerprint of FRET from reference measurements of the donor and acceptor alone. By unmixing the 3D spectrum of the FRET sample, the total relative concentrations of the fluorophores and their scaled FRET efficiencies are directly measured, from which apparent FRET efficiencies can be computed. If the FRET sample is composed of intramolecular FRET sensors it is possible to determine the total relative concentration of the sensors and then estimate absolute FRET efficiency of each sensor. Using multiple tandem constructs with fixed FRET efficiency as well as FRET-based calcium sensors with novel fluorescent protein combinations we demonstrate that the computed FRET efficiencies are accurate and changes in these quantities occur without crosstalk. We provide an example of this method's potential by demonstrating simultaneous imaging of spatially colocalized changes in [Ca(2+)], [cAMP], and PKA activity
Father Andrew Mullen 1790-1818: a study in early nineteenth century spirituality
No full textThis thesis is laid out in three parts: Part I. The life and death of Andrew Mullen. The life is based, to a large extent, on a long letter to his mother, Catherine Mullen, dated 7 January 1810. The letter gives a definite insight into his spirituality based on his membership of the Archconfraternity of the Blessed Sacrament. There is a hint that he had a premonition of an early death. Part II. The burial of Andrew Mullen and the immediate cult to him This is based on documentary evidence. Part III. Most of this part is a catalogue of testimonies taken from 1993 onwards. Then there is the conclusion on the popular devotion to Andrew Mullen stressing the theological aspect of the subject. In the course of writing the thesis it was decided to separate the documentary evidence from the oral tradition. This was advantageous in developing the thesis, and the documents provided a secure basis for the oral tradition. Two pieces of information were found in March 1997. They are death notices: 2 January 1819, The Leinster Journal and 7 January 1819, The Car low Morning Post. There is a slight discrepancy between the two on the date of his death. Also this discrepancy shows a slight difference from the date of the tombstone
Signal/noise analysis of FRET-based sensors.
No full textAbstractMolecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected
Specific oligomerization of the 5-HT1A receptor in the plasma membrane.
No full textIn the present study we analyze the oligomerization of the 5-HT1A receptor within living cells at the sub-cellular level. Using a 2-excitation Foerster Resonance Energy Transfer (FRET) method combined with spectral microscopy we are able to estimate the efficiency of energy transfer based on donor quenching as well as acceptor sensitization between CFP-and YFP-tagged 5-HT1A receptors at the plasma membrane. Through the analysis of the level of apparent FRET efficiency over the various relative amounts of donor and acceptor, as well as over a range of total surface expressions of the receptor, we verify the specific interaction of these receptors. Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT(1A) receptor mutant. Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane. This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction
Functional analysis of PEX13 mutation in a Zellweger syndrome spectrum patient reveals novel homooligomerization of PEX13 and its role in human peroxisome biogenesis.
No full textIn humans, the concerted action of at least 13 different peroxisomal PEX proteins is needed for proper peroxisome biogenesis. Mutations in any of these PEX genes can lead to lethal neurometabolic disorders of the Zellweger syndrome spectrum (ZSS). Previously, we identified the W313G mutation located within the SH3 domain of the peroxisomal protein, PEX13. As this tryptophan residue is highly conserved in almost all known SH3 proteins, we investigated the pathogenic mechanism of the W313G mutation and its role in PEX13 interactions and functions in peroxisome biogenesis. Here, we report for the first time that human PEX13 interacts with itself in peroxisomes in living cells. We demonstrate that the import of PTS1 (peroxisomal targeting signal 1) proteins is specifically disrupted when homooligomerization of PEX13 is interrupted. Live cell FRET microscopy in living cells as well as co-immunoprecipitation experiments reveal that the highly conserved W313 residue is important for self-association of PEX13 but is not required for interaction with PEX14, a well-established interaction partner at the peroxisomal membrane. Experiments with truncated constructs indicate that although the W313G mutation resides in the C-terminal SH3 domain, the N-terminal half is necessary for peroxisomal localization, which in turn appears to be crucial for homooligomerization. Furthermore, rescue of homooligomerization in the W313G mutant cells through complementation with truncation constructs restores import of peroxisomal matrix proteins. Taken together, the thorough analyses of a ZSS patient mutation unraveled the general cell biological function of PEX13 and its mechanism in the import of peroxisomal matrix PTS1 proteins
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
author-bios-SRD-19-0063.R1 – Supplemental material for The Network Structure of Police Misconduct
No full textSupplemental material, author-bios-SRD-19-0063.R1 for The Network Structure of Police Misconduct by George Wood, Daria Roithmayr and Andrew V. Papachristos in Socius</p
Quantitative Intensity-Based FRET Approaches—A Comparative Snapshot
No full textAbstractFörster resonance energy transfer (FRET) has become an important tool for analyzing different aspects of interactions among biological macromolecules in their native environments. FRET analysis has also been successfully applied to study the spatiotemporal regulation of various cellular processes using genetically encoded FRET-based biosensors. A variety of procedures have been described for measuring FRET efficiency or the relative abundance of donor-acceptor complexes, based on analysis of the donor fluorescence lifetime or the spectrally resolved fluorescence intensity. The latter methods are preferable if one wants to not only quantify the apparent FRET efficiencies but also calculate donor-acceptor stoichiometry and observe fast dynamic changes in the interactions among donor and acceptor molecules in live cells. This review focuses on a comparison of the available intensity-based approaches used to measure FRET. We discuss their strengths and weaknesses in terms of FRET quantification, and provide several examples of biological applications
Dispelling the Myths Behind First-author Citation Counts
No full textWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Andrew Field papers
No full textAndrew Field (1938- ) is a scholar, translator, and author, who has published translations of Russian literature, critical studies, biographies, fiction, essays, and travel articles. He holds degrees from Columbia University as well as a Ph.D. from the University of Queensland, Brisbane, Australia. From 1977 to 1979, he was a professor at Griffith University, Brisbane, Australia. Dr. Field's papers consist of materials relating to the writing of his 1983 study of the life and work of Djuna Barnes, Djuna: the Formidable Miss Barnes (alternately entitled Djuna: The Life and Times of Djuna Barnes). Included in the collection are correspondence, manuscripts, research notes, clippings related to the book's publication and reception, and photographs. Also included is a handwritten manuscript of a poem by Barnes
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