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Sample records for live single red

  1. Single Molecule Detection and Imaging in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Nie, Shuming

    2002-03-01

    Direct observation of single molecules and single molecular events inside living cells could dramatically improve our understanding of basic cellular processes (e.g., signal transduction and gene transcription) as well as improving our knowledge on the intracellular transport and fate of therapeutic agents (e.g., antisense RNA and gene therapy vectors). This talk will focus on using single-molecule fluorescence and luminescent quantum dots to examine the dynamics and spatial distribution of RNA and proteins inside living cells and on the surface membrane surface. These single-molecule studies yield a detailed description of molecular events and cellular structures under physiological conditions.

  2. The Red Queen lives: Epistasis between linked resistance loci.

    PubMed

    Metzger, César M J A; Luijckx, Pepijn; Bento, Gilberto; Mariadassou, Mahendra; Ebert, Dieter

    2016-02-01

    A popular theory explaining the maintenance of genetic recombination (sex) is the Red Queen Theory. This theory revolves around the idea that time-lagged negative frequency-dependent selection by parasites favors rare host genotypes generated through recombination. Although the Red Queen has been studied for decades, one of its key assumptions has remained unsupported. The signature host-parasite specificity underlying the Red Queen, where infection depends on a match between host and parasite genotypes, relies on epistasis between linked resistance loci for which no empirical evidence exists. We performed 13 genetic crosses and tested over 7000 Daphnia magna genotypes for resistance to two strains of the bacterial pathogen Pasteuria ramosa. Results reveal the presence of strong epistasis between three closely linked resistance loci. One locus masks the expression of the other two, while these two interact to produce a single resistance phenotype. Changing a single allele on one of these interacting loci can reverse resistance against the tested parasites. Such a genetic mechanism is consistent with host and parasite specificity assumed by the Red Queen Theory. These results thus provide evidence for a fundamental assumption of this theory and provide a genetic basis for understanding the Red Queen dynamics in the Daphnia-Pasteuria system. PMID:26763092

  3. Long-Lived Bright Red Emitting Azaoxa-Triangulenium Fluorophores

    PubMed Central

    Maliwal, Badri P.; Fudala, Rafal; Raut, Sangram; Kokate, Rutika; Sørensen, Thomas J.; Laursen, Bo W.; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2013-01-01

    The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful. Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36–0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images. ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins

  4. Single-Molecule Studies in Live Cells.

    PubMed

    Yu, Ji

    2016-05-27

    Live-cell single-molecule experiments are now widely used to study complex biological processes such as signal transduction, self-assembly, active trafficking, and gene regulation. These experiments' increased popularity results in part from rapid methodological developments that have significantly lowered the technical barriers to performing them. Another important advance is the development of novel statistical algorithms, which, by modeling the stochastic behaviors of single molecules, can be used to extract systemic parameters describing the in vivo biochemistry or super-resolution localization of biological molecules within their physiological environment. This review discusses recent advances in experimental and computational strategies for live-cell single-molecule studies, as well as a selected subset of biological studies that have utilized these new technologies. PMID:27070321

  5. Single-Molecule Studies in Live Cells

    NASA Astrophysics Data System (ADS)

    Yu, Ji

    2016-05-01

    Live-cell single-molecule experiments are now widely used to study complex biological processes such as signal transduction, self-assembly, active trafficking, and gene regulation. These experiments' increased popularity results in part from rapid methodological developments that have significantly lowered the technical barriers to performing them. Another important advance is the development of novel statistical algorithms, which, by modeling the stochastic behaviors of single molecules, can be used to extract systemic parameters describing the in vivo biochemistry or super-resolution localization of biological molecules within their physiological environment. This review discusses recent advances in experimental and computational strategies for live-cell single-molecule studies, as well as a selected subset of biological studies that have utilized these new technologies.

  6. Trypanosoma cruzi: single cell live imaging inside infected tissues.

    PubMed

    Ferreira, Bianca Lima; Orikaza, Cristina Mary; Cordero, Esteban Mauricio; Mortara, Renato Arruda

    2016-06-01

    Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single-cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed-CL or GFP-G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts. PMID:26639617

  7. Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

    PubMed Central

    Siegel, Amanda P.; Baird, Michelle A.; Davidson, Michael W.; Day, Richard N.

    2013-01-01

    The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy. PMID:24129172

  8. Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells

    PubMed Central

    Francis, Ashwanth C.; Marin, Mariana; Shi, Jiong; Aiken, Christopher; Melikyan, Gregory B.

    2016-01-01

    Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process. PMID:27322072

  9. Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells.

    PubMed

    Francis, Ashwanth C; Marin, Mariana; Shi, Jiong; Aiken, Christopher; Melikyan, Gregory B

    2016-06-01

    Disassembly of the cone-shaped HIV-1 capsid in target cells is a prerequisite for establishing a life-long infection. This step in HIV-1 entry, referred to as uncoating, is critical yet poorly understood. Here we report a novel strategy to visualize HIV-1 uncoating using a fluorescently tagged oligomeric form of a capsid-binding host protein cyclophilin A (CypA-DsRed), which is specifically packaged into virions through the high-avidity binding to capsid (CA). Single virus imaging reveals that CypA-DsRed remains associated with cores after permeabilization/removal of the viral membrane and that CypA-DsRed and CA are lost concomitantly from the cores in vitro and in living cells. The rate of loss is modulated by the core stability and is accelerated upon the initiation of reverse transcription. We show that the majority of single cores lose CypA-DsRed shortly after viral fusion, while a small fraction remains intact for several hours. Single particle tracking at late times post-infection reveals a gradual loss of CypA-DsRed which is dependent on reverse transcription. Uncoating occurs both in the cytoplasm and at the nuclear membrane. Our novel imaging assay thus enables time-resolved visualization of single HIV-1 uncoating in living cells, and reveals the previously unappreciated spatio-temporal features of this incompletely understood process. PMID:27322072

  10. Separation of a single cell by red-laser manipulation

    NASA Astrophysics Data System (ADS)

    Shikano, Shuji; Horio, Koji; Ohtsuka, Yoshihiro; Eto, Yuzuro

    1999-10-01

    A single cell of yeast was separated from a bulk sample of yeast without causing damage to the cell. A focused red-laser light beam was used for trapping and transporting the cell. A specially designed microchannel separator played an essential role in the success of the separation.

  11. Imaging Single Cells in the Living Retina

    PubMed Central

    Williams, David R.

    2011-01-01

    A quarter century ago, we were limited to a macroscopic view of the retina inside the living eye. Since then, new imaging technologies, including confocal scanning laser ophthalmoscopy, optical coherence tomography, and adaptive optics fundus imaging, transformed the eye into a microscope in which individual cells can now be resolved noninvasively. These technologies have enabled a wide range of studies of the retina that were previously impossible. PMID:21596053

  12. Imaging gene expression in single living cells

    PubMed Central

    Shav-Tal, Yaron; Singer, Robert H.; Darzacq, Xavier

    2016-01-01

    Technical advances in the field of live-cell imaging have introduced the cell biologist to a new, dynamic, subcellular world. The static world of molecules in fixed cells has now been extended to the time dimension. This allows the visualization and quantification of gene expression and intracellular trafficking events of the studied molecules and the associated enzymatic processes in individual cells, in real time. PMID:15459666

  13. Creep Function of a Single Living Cell

    PubMed Central

    Desprat, Nicolas; Richert, Alain; Simeon, Jacqueline; Asnacios, Atef

    2005-01-01

    We used a novel uniaxial stretching rheometer to measure the creep function J(t) of an isolated living cell. We show, for the first time at the scale of the whole cell, that J(t) behaves as a power-law J(t) = Atα. For N = 43 mice myoblasts (C2-7), we find α = 0.24 ± 0.01 and A = (2.4 ± 0.3) 10−3 Pa−1 s−α. Using Laplace Transforms, we compare A and α to the parameters G0 and β of the complex modulus G*(ω) = G0ωβ measured by other authors using magnetic twisting cytometry and atomic force microscopy. Excellent agreement between A and G0 on the one hand, and between α and β on the other hand, indicated that the power-law is an intrinsic feature of cell mechanics and not the signature of a particular technique. Moreover, the agreement between measurements at very different size scales, going from a few tens of nanometers to the scale of the whole cell, suggests that self-similarity could be a central feature of cell mechanical structure. Finally, we show that the power-law behavior could explain previous results first interpreted as instantaneous elasticity. Thus, we think that the living cell must definitely be thought of as a material with a large and continuous distribution of relaxation time constants which cannot be described by models with a finite number of springs and dash-pots. PMID:15596508

  14. INTERIOR VIEW OF THE LIVING ROOM. SHOWING THE SINGLE PANEL ...

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

    INTERIOR VIEW OF THE LIVING ROOM. SHOWING THE SINGLE PANEL DOOR TO THE BEDROOM AND THE FRONT ENTRY DOOR WITH VISION PANEL. VIEW FACING SOUTH. - Hickam Field, NCO Housing Type 6, 212 Eleventh Street, Honolulu, Honolulu County, HI

  15. Live single-cell laser tag

    PubMed Central

    Binan, Loïc; Mazzaferri, Javier; Choquet, Karine; Lorenzo, Louis-Etienne; Wang, Yu Chang; Affar, El Bachir; De Koninck, Yves; Ragoussis, Jiannis; Kleinman, Claudia L.; Costantino, Santiago

    2016-01-01

    The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types. PMID:27198043

  16. Live single-cell laser tag.

    PubMed

    Binan, Loïc; Mazzaferri, Javier; Choquet, Karine; Lorenzo, Louis-Etienne; Wang, Yu Chang; Affar, El Bachir; De Koninck, Yves; Ragoussis, Jiannis; Kleinman, Claudia L; Costantino, Santiago

    2016-01-01

    The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types. PMID:27198043

  17. Single Motherhood, Living Arrangements, and Time With Children in Japan.

    PubMed

    Raymo, James M; Park, Hyunjoon; Iwasawa, Miho; Zhou, Yanfei

    2014-08-01

    The authors examined relationships between single parenthood and mothers' time with children in Japan. Using data from the 2011 National Survey of Households with Children (N = 1,926), they first demonstrate that time spent with children and the frequency of shared dinners are significantly lower for single mothers than for their married counterparts. For single mothers living alone, less time with children reflects long work hours and work-related stress. Single mothers coresiding with parents spend less time with children and eat dinner together less frequently than either married mothers or their unmarried counterparts not living with parents, net of (grand)parental support, work hours, income, and stress. The findings suggest that rising divorce rates and associated growth in single-mother families may have a detrimental impact on parents' time with children in Japan and that the relatively high prevalence of intergenerational coresidence among single mothers may do little to temper this impact. PMID:25125704

  18. Single-cell measurement of red blood cell oxygen affinity

    PubMed Central

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability. PMID:26216973

  19. SiR–Hoechst is a far-red DNA stain for live-cell nanoscopy

    PubMed Central

    Lukinavičius, Gražvydas; Blaukopf, Claudia; Pershagen, Elias; Schena, Alberto; Reymond, Luc; Derivery, Emmanuel; Gonzalez-Gaitan, Marcos; D'Este, Elisa; Hell, Stefan W.; Wolfram Gerlich, Daniel; Johnsson, Kai

    2015-01-01

    Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy, thereby limiting their utility. Here we describe a far-red DNA stain, SiR–Hoechst, which displays minimal toxicity, is applicable in different cell types and tissues, and is compatible with super-resolution microscopy. The combination of these properties makes this probe a powerful tool for live-cell imaging. PMID:26423723

  20. SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy.

    PubMed

    Lukinavičius, Gražvydas; Blaukopf, Claudia; Pershagen, Elias; Schena, Alberto; Reymond, Luc; Derivery, Emmanuel; Gonzalez-Gaitan, Marcos; D'Este, Elisa; Hell, Stefan W; Gerlich, Daniel Wolfram; Johnsson, Kai

    2015-01-01

    Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible with super-resolution microscopy, thereby limiting their utility. Here we describe a far-red DNA stain, SiR-Hoechst, which displays minimal toxicity, is applicable in different cell types and tissues, and is compatible with super-resolution microscopy. The combination of these properties makes this probe a powerful tool for live-cell imaging. PMID:26423723

  1. Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging

    PubMed Central

    Hense, Anika; Prunsche, Benedikt; Gao, Peng; Ishitsuka, Yuji; Nienhaus, Karin; Ulrich Nienhaus, G.

    2015-01-01

    The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M−1cm−1, mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths. PMID:26648024

  2. Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging.

    PubMed

    Hense, Anika; Prunsche, Benedikt; Gao, Peng; Ishitsuka, Yuji; Nienhaus, Karin; Nienhaus, G Ulrich

    2015-01-01

    The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M(-1)cm(-1), mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths. PMID:26648024

  3. Single mRNA Tracking in Live Cells

    PubMed Central

    Park, Hye Yoon; Buxbaum, Adina R.; Singer, Robert H.

    2011-01-01

    Asymmetric distribution of mRNA is a prevalent phenomenon observed in diverse cell types. The posttranscriptional movement and localization of mRNA provides an important mechanism to target certain proteins to specific cytoplasmic regions of their function. Recent technical advances have enabled real-time visualization of single mRNA molecules in living cells. Studies analyzing the motion of individual mRNAs have shed light on the complex RNA transport system. This chapter presents an overview of general approaches for single particle tracking and some methodologies that are used for single mRNA detection. PMID:20580973

  4. The Red Hat Society: Exploring the role of play, liminality, and communitas in older women's lives.

    PubMed

    Mackay Yarnal, Careen

    2006-01-01

    There is an extensive literature on play. Yet, the role of play in older adults' lives has received limited attention. Strikingly absent is research on play and older women. Missing from the literature is how older women use play as a liminal context for social interaction and communitas. This is odd because by 2030 one in four American women will be over the age of sixty-five. The primary purpose of this study is to explore the roles of play, liminality, and communitas in older women's lives. The focus is the Red Hat Society, a social group for women over age 50 that fosters play and fun. Using qualitative interviews with focus groups and participant observation of a regional Red Hat Society event, the study highlights some of the strengths and weaknesses of current conceptualizations of play, liminality, and communitas. PMID:17000619

  5. Single-Molecule Ion Channel Conformational Dynamics in Living Cells

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter

    2014-03-01

    Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels that are crucial for cell functions, neuronal signaling, and brain functions. Such complexity makes it difficult, if not impossible, to characterize ion channel dynamics using conventional electrical recording alone since that the measurement does not specifically interrogate the associated conformational changes but rather the consequences of the conformational changes. Recently, new technology developments on single-molecule spectroscopy, and especially, the combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging have provided us the capability of probing ion channel conformational changes simultaneously with the electrical single channel recording. By combining real-time single-molecule fluorescence imaging measurements with real-time single-channel electric current measurements in artificial lipid bilayers and in living cell membranes, we were able to probe single ion-channel-protein conformational changes simultaneously, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level. The function-regulating and site-specific conformational changes of ion channels are now measurable under physiological conditions in real-time, one molecule at a time. We will focus our discussion on the new development and results of real-time imaging of the dynamics of gramicidin, colicin, and NMDA receptor ion channels in lipid bilayers and living cells. Our results shed light on new perspectives of the intrinsic interplay of lipid membrane dynamics, solvation dynamics, and the ion channel functions.

  6. Single-Molecule Tracking in Living Cells Using Single Quantum Dot Applications

    PubMed Central

    Baba, Koichi; Nishida, Kohji

    2012-01-01

    Revealing the behavior of single molecules in living cells is very useful for understanding cellular events. Quantum dot probes are particularly promising tools for revealing how biological events occur at the single molecule level both in vitro and in vivo. In this review, we will introduce how single quantum dot applications are used for single molecule tracking. We will discuss how single quantum dot tracking has been used in several examples of complex biological processes, including membrane dynamics, neuronal function, selective transport mechanisms of the nuclear pore complex, and in vivo real-time observation. We also briefly discuss the prospects for single molecule tracking using advanced probes. PMID:22896768

  7. Single-photon ultrashort-lived radionuclides: symposium proceedings

    SciTech Connect

    Paras, P.; Thiessen, J.W.

    1985-01-01

    The purpose was to define the current role and state-of-the-art regarding the development, clinical applications, and usefulness of generator-produced single-photon ultrashort-lived radionuclides (SPUSLR's) and to predict their future impact on medicine. Special emphasis was placed on the generator production of iridium-191, gold-195, and krypton-81. This report contains expanded summaries of the included papers. (ACR)

  8. Exploring dynamics in living cells by tracking single particles.

    PubMed

    Levi, Valeria; Gratton, Enrico

    2007-01-01

    In the last years, significant advances in microscopy techniques and the introduction of a novel technology to label living cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biology. Our understanding on cell dynamics built from snapshots on fixed specimens has evolved thanks to our actual capability to monitor in real time the evolution of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components ranging from organelles to protein molecules by following their way through the cell. In this review, we introduce the single particle tracking technology to new users. We briefly describe the instrumentation and explain some of the algorithms commonly used to locate and track particles. Also, we present some common tools used to analyze trajectories and illustrate with some examples the applications of single particle tracking to study dynamics in living cells. PMID:17703064

  9. Automatic Detection of Single Fluorophores in Live Cells

    PubMed Central

    Mashanov, G. I.; Molloy, J. E.

    2007-01-01

    Recent developments in light microscopy enable individual fluorophores to be observed in aqueous conditions. Biological molecules, labeled with a single fluorophore, can be localized as isolated spots of light when viewed by optical microscopy. Total internal reflection fluorescence microscopy greatly reduces background fluorescence and allows single fluorophores to be observed inside living cells. This advance in live-cell imaging means that the spatial and temporal dynamics of individual molecules can be measured directly. Because of the stochastic nature of single molecule behavior a statistically meaningful number of individual molecules must be detected and their separate trajectories in space and time stored and analyzed. Here, we describe digital image processing methods that we have devised for automatic detection and tracking of hundreds of molecules, observed simultaneously, in vitro and within living cells. Using this technique we have measured the diffusive behavior of pleckstrin homology domains bound to phosphoinositide phospholipids at the plasma membrane of live cultured mammalian cells. We found that mobility of these membrane-bound protein domains is dominated by mobility of the lipid molecule to which they are attached and is highly temperature dependent. Movement of PH domains isolated from the tail region of myosin-10 is consistent with a simple random walk, whereas, diffusion of intact PLC-δ1 shows behavior inconsistent with a simple random walk. Movement is rapid over short timescales but much slower at longer timescales. This anomalous behavior can be explained by movement being restricted to membrane regions of 0.7 μm diameter. PMID:17208981

  10. Tracking single mRNA molecules in live cells

    NASA Astrophysics Data System (ADS)

    Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

    2016-06-01

    mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

  11. Single molecule spectroscopic characterization of a far-red fluorescent protein (HcRed) from the Anthozoa coral Heteractis crispa

    NASA Astrophysics Data System (ADS)

    Cotlet, Mircea; Habuchi, Satoshi; Whitier, Jennifer E.; Werner, James H.; De Schryver, Frans C.; Hofkens, Johan; Goodwin, Peter M.

    2006-02-01

    We report on the photophysical properties of a far-red intrinsic fluorescent protein by means of single molecule and ensemble spectroscopic methods. The green fluorescent protein (GFP) from Aequorea victoria is a popular fluorescent marker with genetically encoded fluorescence and which can be fused to any biological structure without affecting its function. GFP and its variants provide emission colors from blue to yellowish green. Red intrinsic fluorescent proteins from Anthozoa species represent a recent addition to the emission color palette provided by GFPs. Red intrinsic fluorescent markers are on high demand in protein-protein interaction studies based on fluorescence-resonance energy transfer or in multicolor tracking studies or in cellular investigations where autofluorescence possesses a problem. Here we address the photophysical properties of a far-red fluorescent protein (HcRed), a mutant engineered from a chromoprotein cloned from the sea anemone Heteractis crispa, by using a combination of ensemble and single molecule spectroscopic methods. We show evidence for the presence of HcRed protein as an oligomer and for incomplete maturation of its chromophore. Incomplete maturation results in the presence of an immature (yellow) species absorbing/fluorescing at 490/530-nm. This yellow chromophore is involved in a fast resonance-energy transfer with the mature (purple) chromophore. The mature chromophore of HcRed is found to adopt two conformations, a Transoriented form absorbing and 565-nm and non-fluorescent in solution and a Cis-oriented form absorbing at 590-nm and emitting at 645-nm. These two forms co-exist in solution in thermal equilibrium. Excitation-power dependence fluorescence correlation spectroscopy of HcRed shows evidence for singlet-triplet transitions in the microseconds time scale and for cis-trans isomerization occurring in a time scale of tens of microseconds. Single molecule fluorescence data recorded from immobilized HcRed proteins, all

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

    PubMed

    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. PMID:27088188

  13. Determinants of Pair-Living in Red-Tailed Sportive Lemurs (Lepilemur ruficaudatus)

    PubMed Central

    Hilgartner, Roland; Fichtel, Claudia; Kappeler, Peter M; Zinner, Dietmar

    2012-01-01

    Pair-living and a monogamous mating strategy are rare and theoretically unexpected among mammals. Nevertheless, about 10% of primate species exhibit such a social system, which is difficult to explain in the absence of paternal care. In this study, we investigated the two major hypotheses proposed to explain the evolution of monogamy in mammals, the female defence hypothesis (FDH) and the resource defence hypothesis (RDH), in red-tailed sportive lemurs (Lepilemur ruficaudatus), a nocturnal primate from Madagascar. We analysed behavioural data from eight male–female pairs collected during a 24-mo field study to illuminate the determinants of pair-living in this species. Male and female L. ruficaudatus were found to live in dispersed pairs, which are characterised by low cohesion and low encounter rates within a common home range. Social interactions between pair partners were mainly agonistic and characterised by a complete absence of affiliative interactions – body contact was only observed during mating. During the short annual mating season, males exhibited elevated levels of aggression towards mates, as well as extensive mate guarding and increased locomotor activity. In addition, males were exclusively responsible for the maintenance of proximity between pair partners during this period, and they defended their territories against neighbouring males but not against females. Together, these results point towards the importance of female defence in explaining pair-living in L. ruficaudatus. We discuss the spatial and temporal distribution of receptive females in relation to the female defence strategies of males and suggest possible costs that prevent male red-tailed sportive lemurs from defending more than one female. PMID:23144523

  14. Quantifying the transcriptional output of single alleles in single living mammalian cells

    PubMed Central

    Yunger, Sharon; Rosenfeld, Liat; Garini, Yuval; Shav-Tal, Yaron

    2013-01-01

    Transcription kinetics of actively transcribing genes in vivo have generally been measured using tandem gene arrays. However, tandem arrays do not reflect the endogenous state of genome organization where genes appear as single alleles. We present here a robust technique for the quantification of mRNA synthesis from a single allele in real-time, in single living mammalian cells. The protocol describes how to generate cell clones harboring a tagged allele and how to detect in vivo transcription from this tagged allele at high spatial and temporal resolution throughout the cell cycle. Quantification of nascent mRNAs produced from the single tagged allele is performed using RNA fluorescence in situ hybridization (FISH) and live-cell imaging. Subsequent analyses and data modeling detailed in the protocol include measurements of: transcription rates of RNA polymerase II; determining the number of polymerases recruited to the tagged allele; and measuring the spacing between polymerases. Generating the cells containing the single tagged alleles should take up to a month; RNA FISH or live-cell imaging will require an additional week. PMID:23424748

  15. Bioluminescence microscopy: application to ATP measurements in single living cells

    NASA Astrophysics Data System (ADS)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  16. Single Electron Transfer Living Radical Polymerization via a New Initiator

    NASA Astrophysics Data System (ADS)

    Bai, Xiongxiong; Hu, Ying; Zhang, Xu; Ai, Lingling; Cheng, Chuanjie

    2014-08-01

    Research and development of novel initiating system such as single electron transfer living radical polymerization (SET-LRP) is of high importance in polymer chemistry. A new SET-LRP initiator was synthesized and applied to prepare end-functionalized poly(methyl methacrylate) (PMMA) in this study. α-Trichloromethyl benzyl alcohol was firstly synthesized, followed by preparation of PMMA under SET-LRP conditions. Conversion of MMA was 81.9%, and the molecular weight of PMMA was about 2.5 kDa at 60 °C for 1 h. Consistency of the number-average molecular weight of PMMA from NMR, GPC and theoretical calculation indicated that the polymerization featured controllable property. Broad molecular weight distribution (MWD) may be ascribed to branched polymers formed by initiation and chain transfer.

  17. Monitoring protein synthesis in single live cancer cells.

    PubMed

    Tu, Chengyi; Santo, Loredana; Mishima, Yuko; Raje, Noopur; Smilansky, Zeev; Zoldan, Janet

    2016-05-16

    Protein synthesis is generally under sophisticated and dynamic regulation to meet the ever-changing demands of a cell. Global up or down-regulation of protein synthesis and the shift of protein synthesis location (as shown, for example, during cellular stress or viral infection) are recognized as cellular responses to environmental changes such as nutrient/oxygen deprivation or to alterations such as pathological mutations in cancer cells. Monitoring protein synthesis in single live cells can be a powerful tool for cancer research. Here we employed a microfluidic platform to perform high throughput delivery of fluorescent labeled tRNAs into multiple myeloma cells with high transfection efficiency (∼45%) and high viability (>80%). We show that the delivered tRNAs were actively recruited to the ER for protein synthesis and that treatment with puromycin effectively disrupted this process. Interestingly, we observed the scattered distribution of tRNAs in cells undergoing mitosis, which has not been previously reported. Fluorescence lifetime analysis detected extensive FRET signals generated from tRNAs labeled as FRET pairs, further confirming that the delivered tRNAs were used by active ribosomes for protein translation. Our work demonstrates that the microfluidic delivery of FRET labeled tRNAs into living cancer cells can provide new insights into basic cancer metabolism and has the potential to serve as a platform for drug screening, diagnostics, or personalized medication. PMID:26956582

  18. Direct Visualization of De novo Lipogenesis in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Li, Junjie; Cheng, Ji-Xin

    2014-10-01

    Increased de novo lipogenesis is being increasingly recognized as a hallmark of cancer. Despite recent advances in fluorescence microscopy, autoradiography and mass spectrometry, direct observation of de novo lipogenesis in living systems remains to be challenging. Here, by coupling stimulated Raman scattering (SRS) microscopy with isotope labeled glucose, we were able to trace the dynamic metabolism of glucose in single living cells with high spatial-temporal resolution. As the first direct visualization, we observed that glucose was largely utilized for lipid synthesis in pancreatic cancer cells, which occurs at a much lower rate in immortalized normal pancreatic epithelial cells. By inhibition of glycolysis and fatty acid synthase (FAS), the key enzyme for fatty acid synthesis, we confirmed the deuterium labeled lipids in cancer cells were from de novo lipid synthesis. Interestingly, we also found that prostate cancer cells exhibit relatively lower level of de novo lipogenesis, but higher fatty acid uptake compared to pancreatic cancer cells. Together, our results demonstrate a valuable tool to study dynamic lipid metabolism in cancer and other disorders.

  19. Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

    PubMed Central

    Huang, Lingyan; Behlke, Mark A.; Tsourkas, Andrew

    2014-01-01

    The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3’-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts. PMID:25146531

  20. Block-Cell-Printing for live single-cell printing

    PubMed Central

    Zhang, Kai; Chou, Chao-Kai; Xia, Xiaofeng; Hung, Mien-Chie; Qin, Lidong

    2014-01-01

    A unique live-cell printing technique, termed “Block-Cell-Printing” (BloC-Printing), allows for convenient, precise, multiplexed, and high-throughput printing of functional single-cell arrays. Adapted from woodblock printing techniques, the approach employs microfluidic arrays of hook-shaped traps to hold cells at designated positions and directly transfer the anchored cells onto various substrates. BloC-Printing has a minimum turnaround time of 0.5 h, a maximum resolution of 5 µm, close to 100% cell viability, the ability to handle multiple cell types, and efficiently construct protrusion-connected single-cell arrays. The approach enables the large-scale formation of heterotypic cell pairs with controlled morphology and allows for material transport through gap junction intercellular communication. When six types of breast cancer cells are allowed to extend membrane protrusions in the BloC-Printing device for 3 h, multiple biophysical characteristics of cells—including the protrusion percentage, extension rate, and cell length—are easily quantified and found to correlate well with their migration levels. In light of this discovery, BloC-Printing may serve as a rapid and high-throughput cell protrusion characterization tool to measure the invasion and migration capability of cancer cells. Furthermore, primary neurons are also compatible with BloC-Printing. PMID:24516129

  1. Red, green, and blue lasing enabled by single-exciton gain in colloidal quantum dot films

    DOEpatents

    Nurmikko, Arto V.; Dang, Cuong

    2016-06-21

    The methods and materials described herein contemplate the use films of colloidal quantum dots as a gain medium in a vertical-cavity surface-emitting laser. The present disclosure demonstrates a laser with single-exciton gain in the red, green, and blue wavelengths. Leveraging this nanocomposite gain, the results realize a significant step toward full-color single-material lasers.

  2. The Racial Residential Segregation of Black Single Adults Living Alone

    PubMed Central

    Marsh, Kris; Iceland, John

    2014-01-01

    While many studies have examined the intersection of race and class with residential segregation and residential preferences, very little is known about the role played by household composition in shaping residential patterns. This paper focuses on the residential patterns of a particular kind of household: those consisting of persons single and living alone (SALA). We compare the residential segregation of black SALA households—an important subset of non-family households and a rapidly growing segment of the population—from white SALA households and both white and black married-couple households. We examine how group and metropolitan characteristics influence segregation levels for these household types. Using data from the 2000 census, we find that black SALA households are less segregated from white SALA households than from white married-couple households. Multivariate analyses show that smaller income differences across SALA households account for these segregation patterns, indicating the importance of economic resources in influencing residential patterns. Nevertheless, race continues to play an important role, as black SALA household segregation from both kinds of white households is high in absolute terms and relative to their segregation from black married-couple households. PMID:25558183

  3. Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

    PubMed Central

    Piotrowska-Nitsche, Karolina; Caspary, Tamara

    2013-01-01

    We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time. PMID:23666396

  4. Grapevine red blotch-associated virus is Present in Free-Living Vitis spp. Proximal to Cultivated Grapevines.

    PubMed

    Perry, Keith L; McLane, Heather; Hyder, Muhammad Z; Dangl, Gerald S; Thompson, Jeremy R; Fuchs, Marc F

    2016-06-01

    Red blotch is an emerging disease of grapevine associated with grapevine red blotch-associated virus (GRBaV). The virus spreads with infected planting stocks but no vector of epidemiological significance has been conclusively identified. A vineyard block of red-blotch-affected Vitis vinifera 'Cabernet franc' clone 214 was observed in California, with a clustering of infected, symptomatic vines focused along one edge of the field proximal to a riparian habitat with free-living Vitis spp. No genetic heterogeneity was observed in a 587-nucleotide region of the GRBaV genome in a population of 44 Cabernet franc clone 214 isolates. By contrast, genetic differences were observed in isolates from other cultivars and clones growing in adjacent blocks. GRBaV was confirmed infecting four free-living vines, two of which were shown to be V. californica × V. vinifera hybrids. The genomes of three free-living GRBaV vine isolates and seven from V. vinifera cultivars were compared; free-living vine isolates were shown to be more similar to each other and a 'Merlot' isolate than to the other cultivated vine isolates. The finding that GRBaV is present in free-living Vitis spp. indicates the virus can be spread by natural (nonhuman-mediated) means, and we hypothesize that in-field spread of GRBaV is occurring. PMID:26960112

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

    PubMed Central

    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. PMID:26840497

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

    PubMed

    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. PMID:26840497

  7. Ultrasonic Scattering Measurements of a Live Single Cell at 86 MHz

    PubMed Central

    Lee, Changyang; Jung, Hayong; Lam, Kwok Ho; Yoon, Changhan; Shung, K. Kirk

    2016-01-01

    Cell separation and sorting techniques have been employed biomedical applications such as cancer diagnosis and cell gene expression analysis. The capability to accurately measure ultrasonic scattering properties from cells is crucial in making an ultrasonic cell sorter a reality if ultrasound scattering is to be used as the sensing mechanism as well. To assess the performance of sensing and identifying live single cells with high-frequency ultrasound, an 86-MHz lithium niobate press-focused single-element acoustic transducer was used in a high-frequency ultrasound scattering measurement system that was custom designed and developed for minimizing noise and allowing better mobility. Peak-to-peak echo amplitude, integrated backscatter (IB) coefficient, spectral parameters including spectral slope and intercept, and midband fit from spectral analysis of the backscattered echoes were measured and calculated from a live single cell of two different types on an agar surface: leukemia cells (K562 cells) and red blood cells (RBCs). The amplitudes of echo signals from K562 cells and RBCs were 48.25 ± 11.98 mVpp and 56.97 ± 7.53 mVpp, respectively. The IB coefficient was −89.39 ± 2.44 dB for K562 cells and −89.00 ± 1.19 dB for RBCs. The spectral slope and intercept were 0.30 ± 0.19 dB/MHz and −56.07 ± 17.17 dB, respectively, for K562 cells and 0.78 ± 0.092 dB/MHz and −98.18 ± 8.80 dB, respectively, for RBCs. Midband fits of K562 cells and RBCs were −31.02 ± 3.04 dB and −33.51 ± 1.55 dB, respectively. Acoustic cellular discrimination via these parameters was tested by Student’s t-test. Their values, except for the IB value, showed statistically significant difference (p < 0.001). This paper reports for the first time that ultrasonic scattering measurements can be made on a live single cell with a highly focused high-frequency ultrasound microbeam at 86 MHz. These results also suggest the feasibility of ultrasonic scattering as a sensing mechanism in

  8. Correlates of Living Alone among Single Elderly Chinese Immigrants in Canada

    ERIC Educational Resources Information Center

    Lai, Daniel W. L.; Leonenko, Wendy L.

    2007-01-01

    According to traditional Chinese culture, families will care for their elderly. Therefore, it appears to be uncommon for elderly Chinese to live alone. This study examines the correlates for single elderly Chinese immigrants in Canada to live alone. Using a probability sample of single elderly Chinese immigrants (N = 660) in seven urban centers,…

  9. Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.

    PubMed

    Kreder, Rémy; Pyrshev, Kyrylo A; Darwich, Zeinab; Kucherak, Oleksandr A; Mély, Yves; Klymchenko, Andrey S

    2015-06-19

    Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research. PMID:25710589

  10. Red fluorescent turn-on ligands for imaging and quantifying G protein-coupled receptors in living cells.

    PubMed

    Karpenko, Iuliia A; Kreder, Rémy; Valencia, Christel; Villa, Pascal; Mendre, Christiane; Mouillac, Bernard; Mély, Yves; Hibert, Marcel; Bonnet, Dominique; Klymchenko, Andrey S

    2014-02-10

    Classical fluorescence-based approaches to monitor ligand-protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn-on probes for a G protein-coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor-specific turn-on response. The new ligand was successfully applied for background-free imaging and quantification of oxytocin receptors in living cells. PMID:24449564

  11. Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping.

    PubMed

    Fritsch, Isabel; Luyven, Gabriele; Köhler, Heike; Lutz, Walburga; Möbius, Petra

    2012-02-01

    Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable

  12. Suspicion of Mycobacterium avium subsp. paratuberculosis Transmission between Cattle and Wild-Living Red Deer (Cervus elaphus) by Multitarget Genotyping

    PubMed Central

    Fritsch, Isabel; Luyven, Gabriele; Köhler, Heike; Lutz, Walburga

    2012-01-01

    Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit–variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable

  13. Nd(3+)-Sensitized Ho(3+) Single-Band Red Upconversion Luminescence in Core-Shell Nanoarchitecture.

    PubMed

    Chen, Daqin; Liu, Lu; Huang, Ping; Ding, Mingye; Zhong, Jiasong; Ji, Zhenguo

    2015-07-16

    A strategy to achieve 808 nm excited single-band red upconversion luminescence of Ho(3+) via the core-shell nanoarchitecture design was provided. Specifically, the synthesized Yb/Ho/Ce: NaGdF4@Yb/Nd: NaYF4 active-core@active-shell nanoparticles were evidenced to enable high-content doping of Nd(3+) (∼10 mol %) in the shell layer and, thus, markedly enhance red upconversion emission from Ho(3+) activators in the core with the assistance of spatially confined doping of Nd(3+) ions and efficient energy transfer of Nd(3+) → Yb(3+)(shell) → Yb(3+)(core) → Ho(3+). Importantly, introducing Ce(3+) into the core was beneficial to the competition of radiation transitions from the two intermediate excited states of Ho(3+): (5)S2,(5)F4 (green-emitting) and Ho(3+): (5)F5 (red-emitting), which induced great enhancement in the red to green intensity ratio and ultimately intense single-band red upconversion emission. We believe that this preliminary study will provide an important advance in developing luminescent markers suitable for biolabeling applications. PMID:26266869

  14. Structural relaxation of acridine orange dimer in bulk water and inside a single live lung cell

    NASA Astrophysics Data System (ADS)

    Chowdhury, Rajdeep; Nandi, Somen; Halder, Ritaban; Jana, Biman; Bhattacharyya, Kankan

    2016-02-01

    Structural relaxation of the acridine orange (AO) dimer in bulk water and inside a single live lung cell is studied using time resolved confocal microscopy and molecular dynamics (MD) simulations. The emission maxima ( λem max ˜ 630 nm) of AO in a lung cancer cell (A549) and a non-cancer lung fibroblast cell (WI38) suggest that AO exists as a dimer inside the cell. Time-dependent red shift in emission maximum indicates dynamic relaxation of the AO dimer (in the excited state) with a time constant of 500-600 ps, both in bulk water and inside the cell. We have calculated the equilibrium relaxation dynamics of the AO dimer in the ground state using MD simulations and found a slow component of time scale ˜350 ps. The intra- and inter-molecular components of the total relaxation dynamics of the AO dimer reveal the presence of a slow component of the order of a few hundred picoseconds. Upon restricting intra-molecular dye dynamics by harmonic constraint between AO monomers, the slow component vanishes. Combining the experimental observations and MD simulation results, we ascribe the slow component of the dynamic relaxation of the AO dimer to the structural relaxation, namely, fluctuations in the distance between the two monomers and associated fluctuation in the number of water molecules.

  15. The Living with a Red Dwarf Program: Observing the Decline in dM Star FUV Emissions With Age

    NASA Astrophysics Data System (ADS)

    Engle, Scott G.; Guinan, Edward F.; Mizusawa, Trisha

    2009-05-01

    Red Dwarf (dM) stars are overwhelmingly the most numerous stars in our Galaxy. These cool, faint and low mass stars make up >80% of all stars. Also dM stars have extremely long life times (>50-100 Gyr). Determining the number of red dwarfs with planets and assessing planetary habitability (a planet's potential to develop and sustain life) is critically important because such studies would indicate how common life is in the universe. Our program-``Living with a Red Dwarf''-addresses these questions by investigating the long-term nuclear evolution and the coronal and chromospheric properties of red dwarf stars with widely different ages (~50 Myr-12 Gyr). One major focus of the program is to study the magnetic-dynamo generated coronal and chromospheric X-ray-FUV/UV emissions and flare properties of a sample of dM0-5 stars. Observations carried out by FUSE of a number of young to old dM stars provide important data for understanding transition region heating in these stars with deep convective zones as well as providing measures of FUV irradiances. Also studied are the effects of X-ray-FUV emissions on possible hosted planets and impacts of this radiation on their habitability. Using these data we are constructing irradiance tables (X-UV irradiances) that can be used to model the effects of XUV radiation on planetary atmospheres and possible life on planetary surfaces. The initial results of this program are discussed.

  16. DETECTING SINGLE WHEAT KERNELS CONTAINING LIVE OR DEAD INSECTS USING NEAR-INFRARED REFLECTANCE SPECTROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An automated NIR system was used over a two-month storage period to detect single wheat kernels that contained live or dead internal rice weevils at various stages of growth. Correct classification of sound kernels and kernels containing live pupae, large larvae, medium-sized larvae, and small larv...

  17. Do Children in Single-Parent Households Fare Better Living with Same-Sex Parents?

    ERIC Educational Resources Information Center

    Downey, Douglas B.; Powell, Brian

    1993-01-01

    Used data from National Educational Longitudinal Study (with 3,483 and 409 eighth graders living in mother-only and father-only homes, respectively) to test whether children in single-parent homes fare better living with same-sex parent. Of 35 social psychological and educational outcomes studied, found none in which both males and females…

  18. Red emission fluorescent probes for visualization of monoamine oxidase in living cells.

    PubMed

    Li, Ling-Ling; Li, Kun; Liu, Yan-Hong; Xu, Hao-Ran; Yu, Xiao-Qi

    2016-01-01

    Here we report two novel red emission fluorescent probes for the highly sensitive and selective detection of monoamine oxidase (MAO) with large Stokes shift (227 nm). Both of the probes possess solid state fluorescence and can accomplish the identification of MAO on test papers. The probe MAO-Red-1 exhibited a detection limit down to 1.2 μg mL(-1) towards MAO-B. Moreover, the cleavage product was unequivocally conformedby HPLC and LCMS and the result was in accordance with the proposed oxidative deamination mechanism. The excellent photostability of MAO-Red-1 was proved both in vitro and in vivo through fluorescent kinetic experiment and laser exposure experiment of confocal microscopy, respectively. Intracellular experiments also confirmed the low cytotoxity and exceptional cell imaging abilities of MAO-Red-1. It was validated both in HeLa and HepG2 cells that MAO-Red-1 was capable of reporting MAO activity through the variation of fluorescence intensity. PMID:27499031

  19. Red emission fluorescent probes for visualization of monoamine oxidase in living cells

    PubMed Central

    Li, Ling-Ling; Li, Kun; Liu, Yan-Hong; Xu, Hao-Ran; Yu, Xiao-Qi

    2016-01-01

    Here we report two novel red emission fluorescent probes for the highly sensitive and selective detection of monoamine oxidase (MAO) with large Stokes shift (227 nm). Both of the probes possess solid state fluorescence and can accomplish the identification of MAO on test papers. The probe MAO-Red-1 exhibited a detection limit down to 1.2 μg mL−1 towards MAO-B. Moreover, the cleavage product was unequivocally conformedby HPLC and LCMS and the result was in accordance with the proposed oxidative deamination mechanism. The excellent photostability of MAO-Red-1 was proved both in vitro and in vivo through fluorescent kinetic experiment and laser exposure experiment of confocal microscopy, respectively. Intracellular experiments also confirmed the low cytotoxity and exceptional cell imaging abilities of MAO-Red-1. It was validated both in HeLa and HepG2 cells that MAO-Red-1 was capable of reporting MAO activity through the variation of fluorescence intensity. PMID:27499031

  20. Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

    PubMed Central

    Bosch, Peter J.; Corrêa, Ivan R.; Sonntag, Michael H.; Ibach, Jenny; Brunsveld, Luc; Kanger, Johannes S.; Subramaniam, Vinod

    2014-01-01

    Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines. PMID:25140415

  1. Quantitative Probing of Cu(2+) Ions Naturally Present in Single Living Cells.

    PubMed

    Lee, Junho; Lee, Hwa-Rim; Pyo, Jaeyeon; Jung, Youngseob; Seo, Ji-Young; Ryu, Hye Guk; Kim, Kyong-Tai; Je, Jung Ho

    2016-06-01

    Quantitative probing of Cu(2+) ions naturally present in single living cells is realized by developing a quantum-dot-embedded nanowire-waveguide probe. The intracellular Cu(2+) ion concentration is quantified by direct monitoring of photoluminescence quenching during the insertion of the nanowire in a living neuron. The measured intracellular Cu(2+) ion concentration is 3.34 ± 1.04 × 10(-6) m (mean ± s.e.m.) in single hippocampal neurons. PMID:27027298

  2. τ-SPAD: a new red sensitive single-photon counting module

    NASA Astrophysics Data System (ADS)

    Kell, Gerald; Bülter, Andreas; Wahl, Michael; Erdmann, Rainer

    2011-05-01

    Single Photon Avalanche Diodes (SPADs) are valuable detectors in numerous photon counting applications in the fields of quantum physics, quantum communication, astronomy, metrology and biomedical analytics. They typically feature a much higher photon detection efficiency than photomultiplier tubes, most importantly in the red to near-infrared range of the spectrum. Very often SPADs are combined with Time-Correlated Single Photon Counting (TCSPC) electronics for time-resolved data acquisition and the temporal resolution ("jitter") of a SPAD is therefore one of the key parameters for selecting a detector. We show technical data and first application results from a new type of red sensitive single photon counting module ("τ-SPAD"), which is targeted at timing applications, most prominently in the area of Single Molecule Spectroscopy (SMS). The τ-SPAD photon counting module combines Laser Components' ultra-low noise VLoK silicon avalanche photodiode with specially developed quenching and readout electronics from PicoQuant. It features an extremely high photon detection efficiency of 75% at 670 nm and can be used to detect single photons over the 400 nm to 1100 nm wavelength range. The timing jitter of the output of the τ-SPAD can be as low as 350 ps, making it suitable for time-resolved fluorescence detection applications. First photon coincidence correlation measurements also show that the typical breakdown flash of SPADs is of comparably low intensity for these new SPADs.

  3. New Red-Emitting Tetrazine-Phenoxazine Fluorogenic Labels for Live-Cell Intracellular Bioorthogonal Labeling Schemes.

    PubMed

    Knorr, Gergely; Kozma, Eszter; Herner, András; Lemke, Edward A; Kele, Péter

    2016-06-20

    The synthesis of a set of tetrazine-bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through-bond energy-transfer-based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse-electron-demand Diels-Alder reaction with proteins modified genetically with strained trans-cyclooctenes. PMID:27218228

  4. "Living My Native Life Deadly": Red Lake, Ward Churchill, and the Discourses of Competing Genocides

    ERIC Educational Resources Information Center

    Byrd, Jodi A.

    2007-01-01

    In an attempt to understand how rival narratives of genocide compete even at the cost of disavowing other historical experiences, this article considers how the U.S. national media represented and framed Red Lake in the wake of Ward Churchill's emergence on the national radar. The first section of this article examines how nineteenth-century…

  5. Prevalence and genetic diversity of simian immunodeficiency virus infection in wild-living red colobus monkeys (Piliocolobus badius badius) from the Taï forest, Côte d'Ivoire SIVwrc in wild-living western red colobus monkeys.

    PubMed

    Locatelli, Sabrina; Liegeois, Florian; Lafay, Bénédicte; Roeder, Amy D; Bruford, Michael W; Formenty, Pierre; Noë, Ronald; Delaporte, Eric; Peeters, Martine

    2008-01-01

    Numerous African primates are infected with simian immunodeficiency viruses (SIVs). It is now well established that the clade of SIVs infecting west-central African chimpanzees (Pan troglodytes troglodytes) and western gorillas (Gorilla gorilla gorilla) represent the progenitors of human immunodeficiency virus type 1 (HIV-1), whereas HIV-2 results from different cross-species transmissions of SIVsmm from sooty mangabeys (Cercocebus atys atys). We present here the first molecular epidemiological survey of simian immunodeficiency virus (SIVwrc) in wild-living western red colobus monkeys (Piliocolobus badius badius) which are frequently hunted by the human population and represent a favourite prey of western chimpanzees (Pan troglodytes verus). We collected faecal samples (n=88) and we assessed individual discrimination by microsatellite analyses and visual observation. We tested the inferred 53 adult individuals belonging to two neighbouring habituated groups for presence of SIVwrc infection by viral RNA (vRNA) detection. We amplified viral polymerase (pol) (650 bp) and/or envelope (env) (570 bp) sequences in 14 individuals, resulting in a minimal prevalence of 26% among the individuals sampled, possibly reaching 50% when considering the relatively low sensitivity of viral RNA detection in faecal samples. With a few exceptions, phylogenetic analysis of pol and env sequences revealed a low degree of intragroup genetic diversity and a general viral clustering related to the social group of origin. However, we found a higher intergroup diversity. Behavioural and demographic data collected previously from these communities indicate that red colobus monkeys live in promiscuous multi-male societies, where females leave their natal group at the sub-adult stage of their lives and where extra-group copulations or male immigration have been rarely observed. The phylogenetic data we obtained seem to reflect these behavioural characteristics. Overall, our results indicate that

  6. Role Demands in the Lives of Employed Single Mothers with Preschoolers.

    ERIC Educational Resources Information Center

    Goldberg, Wendy A.; And Others

    1992-01-01

    Examined single mothers' well-being and perceptions of their preschoolers' behavior. Findings from 76 single, employed mothers indicated that variables reflecting interface between work and family roles were important for well-being and perceptions of children's behavior. Depression appeared allied with stability and resources in mothers' lives;…

  7. Translation dynamics of single mRNAs in live cells and neurons

    PubMed Central

    Wu, Bin; Eliscovich, Carolina; Yoon, Young J.; Singer, Robert H.

    2016-01-01

    Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single-molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display “bursting” translation. This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells. PMID:27313041

  8. Translation dynamics of single mRNAs in live cells and neurons.

    PubMed

    Wu, Bin; Eliscovich, Carolina; Yoon, Young J; Singer, Robert H

    2016-06-17

    Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single-molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display "bursting" translation. This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells. PMID:27313041

  9. Return to Being Black, Living in the Red: a race gap in wealth that goes beyond social origins.

    PubMed

    Killewald, Alexandra

    2013-08-01

    In the United States, racial disparities in wealth are vast, yet their causes are only partially understood. In Being Black, Living in the Red, Conley (1999) argued that the sociodemographic traits of young blacks and their parents, particularly parental wealth, wholly explain their wealth disadvantage. Using data from the 1980-2009 waves of the Panel Study of Income Dynamics, I show that this conclusion hinges on the specific sample considered and the treatment of debtors in the sample. I further document that prior research has paid insufficient attention to the possibility of variation in the association between wealth and race at different points of the net worth distribution. Among wealth holders, blacks remain significantly disadvantaged in assets compared with otherwise similar whites. Among debtors, however, young whites hold more debt than otherwise similar blacks. The results suggest that, among young adults, debt may reflect increased access to credit, not simply the absence of assets. The asset disadvantage for black net wealth holders also indicates that research and policy attention should not be focused only on young blacks "living in the red." PMID:23658068

  10. Measurement of UV absorption of single living cell for cell manipulation using NIR femtosecond laser

    NASA Astrophysics Data System (ADS)

    Cho, Sung-Hak; Chang, Won-Seok; Kim, Kwang-Ryul; Hong, Jong Wook

    2009-02-01

    Optical UV absorption of single human living cells ranging from 200 nm to 360 nm was measured in situ for the study of cell manipulation using the near-infrared (NIR) femtosecond laser . Human breast living cells of MCF-10A, MCF-7, and MDA-MB-231 were used in this experiment. The selective photo-disruptions of single living cell and its sub-organelle (nucleus) were also demonstrated using the tightly focused 790 nm wavelength femtosecond laser with pulse duration of 110 fs. It was found that each living cell has its own absorption spectrum in UV wavelength ranges. It was also inferred that intrinsic absorption spectrum is attributed to the amount of DNA and protein of living cell. For the study of photo-disruption of single cell using the multi-photon absorption excited by the NIR femtosecond laser pulse, the origin UV absorption spectrum of targeted living cell is important and fundamental information to understand nonlinear interaction between NIR ultrashort, high-intensity laser light and transparent living cell.

  11. A Comparison of the Self-Esteem between Boys Living with Single-Parent Mothers and Single-Parent Fathers.

    ERIC Educational Resources Information Center

    Lowenstein, Joyce S.; Koopmen, Elizabeth J.

    1978-01-01

    Investigates the self-esteem of boys between the ages of 9 and 14 as related to sex and adjustment of custodial parent, frequency of visitation of noncustodial parent, length of time child lived in a single-parent home, and quality of the relationship between parents. Results revealed few significant differences. (Author)

  12. Red-emitting π-conjugated oligomers infused single-wall carbon nanotube sheets

    NASA Astrophysics Data System (ADS)

    Fujimori, Toshihiko; Urita, Koki

    2016-04-01

    We demonstrate the one-step thermal fusion and infusion of pyrene molecules inside single-wall carbon nanotubes (SWCNTs). Despite the presence of metallic-SWCNTs, which behave as a quencher due to gapless electronic states, the nanohybrids consisting of pyrene and/or azupyrene oligomers infused SWCNT sheets exhibit red fluorescence by the ultraviolet, blue, and green light excitations. The wavelength-independent light-emitting behavior is explained by (1) infused PAH oligomers inside semiconducting-SWCNTs and (2) the peculiar π-π interaction through mixed π-conjugated state between the π-conjugated oligomers and non-armchair metallic-SWCNTs.

  13. Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution.

    PubMed

    Doura, Tomohiro; Kamiya, Mako; Obata, Fumiaki; Yamaguchi, Yoshifumi; Hiyama, Takeshi Y; Matsuda, Takashi; Fukamizu, Akiyoshi; Noda, Masaharu; Miura, Masayuki; Urano, Yasuteru

    2016-08-01

    The LacZ gene, which encodes Escherichia coli β-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms. PMID:27400827

  14. INTERSEEDED RED CLOVER AND KURA CLOVER LIVING-MULCH SYSTEMS FOR CORN SILAGE PRODUCTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In addition to reducing nitrate and soil losses from cropland, greater use of companion crops (cover crops and living mulches) could enhance yields of corn-silage based production systems. In a replicated multi-year study in southern Wisconsin, Roundup-Ready corn was grown for silage with Kura-clove...

  15. Living with a Red Dwarf: Rotation and X-Ray and Ultraviolet Properties of the Halo Population Kapteyn's Star

    NASA Astrophysics Data System (ADS)

    Guinan, Edward F.; Engle, Scott G.; Durbin, Allyn

    2016-04-01

    As part of Villanova's Living with a Red Dwarf program, we have obtained UV, X-ray, and optical data of the Population II red dwarf -- Kapteyn's Star. Kapteyn's Star is noteworthy for its large proper motions and high radial velocity of ∼+245 km s-1. As the nearest Pop II red dwarf, it serves as an old age anchor for calibrating activity/irradiance-rotation-age relations, and an important test bed for stellar dynamos and the resulting X-ray-UV emissions of slowly rotating, near-fully convective red dwarf stars. Adding to the notoriety, Kapteyn's Star has recently been reported to host two super-Earth candidates, one of which (Kapteyn b) is orbiting within the habitable zone. However, Robertson et al. questioned the planet's existence since its orbital period may be an artifact of activity, related to the star's rotation period. Because of its large Doppler-shift, measures of the important, chromospheric H i Lyα 1215.67 Å emission line can be reliably made, because it is mostly displaced from ISM and geo-coronal sources. Lyα emission dominates the FUV region of cool stars. Our measures can help determine the X-ray-UV effects on planets hosted by Kapteyn's Star, and planets hosted by other old red dwarfs. Stellar X-ray and Lyα emissions have strong influences on the heating and ionization of upper planetary atmospheres and can (with stellar winds and flares) erode or even eliminate planetary atmospheres. Using our program stars, we have reconstructed the past exposures of Kapteyn's Star's planets to coronal - chromospheric XUV emissions over time. Based on observations made with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS 5-26555. These observations are associated with program #13020. This work is also based on observations obtained with the Chandra X-ray Observatory, a NASA science mission, program #13200633.

  16. Light sheet microscopy for tracking single molecules on the apical surface of living cells.

    PubMed

    Li, Yu; Hu, Ying; Cang, Hu

    2013-12-12

    Single particle tracking is a powerful tool to study single molecule dynamics in living biological samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single molecules on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF molcules on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temperature, the average diffusion coefficient of EGF on A549 cells was measured to be 0.13 μm(2)/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coefficients and an increase of the average diffusion coefficient at room temperature. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole "lifetime" of a particle from binding till photobleaching or internalization. PMID:23895420

  17. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGESBeta

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; et al

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  18. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  19. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    PubMed Central

    Liu, Daniel S.; Nivón, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-01-01

    Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. PMID:25313043

  20. Label-Free Detection of Single Living Bacteria via Electrochemical Collision Event.

    PubMed

    Lee, Ji Young; Kim, Byung-Kwon; Kang, Mijeong; Park, Jun Hui

    2016-01-01

    We detected single living bacterial cells on ultramicroelectrode (UME) using a single-particle collision method and optical microscopic methods. The number of collision events involving the bacterial cells indicated in current-time (i-t) curves corresponds to the number of bacterial cells (i.e., Escherichia coli) on the UME surface, as observed visually. Simulations were performed to determine the theoretical current response (75 pA) and frequency (0.47 pM(-1) s(-1)) of single Escherichia coli collisions. The experimental current response (83 pA) and frequency (0.26 pM(-1) s(-1)) were on the same order of magnitude as the theoretical values. This single-particle collision approach facilitates detecting living bacteria and determining their concentration in solution and could be widely applied to studying other bacteria and biomolecules. PMID:27435527

  1. Live cell single-molecule and superresolution imaging of proteins in bacteria

    NASA Astrophysics Data System (ADS)

    Biteen, Julie S.; Moerner, W. E.

    2011-03-01

    By providing spatial localization on the nanometer scale, eliminating the need for ensemble averaging, and permitting non-invasive intracellular investigations, single-molecule imaging has brought much insight to biophysics. A particularly enticing application for single-molecule imaging is the capability to investigate live cells and to examine structure and dynamics in the natural environment. To obtain true superresolution, control of the emission of the single molecules provides a way to maintain a sparse concentration of emitters for any frame so that sequential imaging leads to a final reconstruction with information beyond the optical diffraction limit. In this paper, we discuss several single-molecule- based fluorescence methods that are possible, and indeed often enabled, by having live cell specimens.

  2. Label-Free Detection of Single Living Bacteria via Electrochemical Collision Event

    PubMed Central

    Lee, Ji Young; Kim, Byung-Kwon; Kang, Mijeong; Park, Jun Hui

    2016-01-01

    We detected single living bacterial cells on ultramicroelectrode (UME) using a single-particle collision method and optical microscopic methods. The number of collision events involving the bacterial cells indicated in current-time (i-t) curves corresponds to the number of bacterial cells (i.e., Escherichia coli) on the UME surface, as observed visually. Simulations were performed to determine the theoretical current response (75 pA) and frequency (0.47 pM−1 s−1) of single Escherichia coli collisions. The experimental current response (83 pA) and frequency (0.26 pM−1 s−1) were on the same order of magnitude as the theoretical values. This single-particle collision approach facilitates detecting living bacteria and determining their concentration in solution and could be widely applied to studying other bacteria and biomolecules. PMID:27435527

  3. Label-Free Detection of Single Living Bacteria via Electrochemical Collision Event

    NASA Astrophysics Data System (ADS)

    Lee, Ji Young; Kim, Byung-Kwon; Kang, Mijeong; Park, Jun Hui

    2016-07-01

    We detected single living bacterial cells on ultramicroelectrode (UME) using a single-particle collision method and optical microscopic methods. The number of collision events involving the bacterial cells indicated in current-time (i-t) curves corresponds to the number of bacterial cells (i.e., Escherichia coli) on the UME surface, as observed visually. Simulations were performed to determine the theoretical current response (75 pA) and frequency (0.47 pM‑1 s‑1) of single Escherichia coli collisions. The experimental current response (83 pA) and frequency (0.26 pM‑1 s‑1) were on the same order of magnitude as the theoretical values. This single-particle collision approach facilitates detecting living bacteria and determining their concentration in solution and could be widely applied to studying other bacteria and biomolecules.

  4. Efficient two-photon fluorescent probe with red emission for imaging of thiophenols in living cells and tissues.

    PubMed

    Liu, Hong-Wen; Zhang, Xiao-Bing; Zhang, Jing; Wang, Qian-Qian; Hu, Xiao-Xiao; Wang, Peng; Tan, Weihong

    2015-09-01

    Thiophenols, a class of highly toxic and pollutant compounds, are widely used in industrial production. Some aliphatic thiols play important roles in living organisms. Therefore, the development of efficient methods to discriminate thiophenols from aliphatic thiols is of great importance. Although several one-photon fluorescent probes have been reported for thiophenols, two-photon fluorescent probes are more favorable for biological imaging due to its low background fluorescence, deep penetration depth, and so on. In this work, a two-photon fluorescent probe for thiophenols, termed NpRb1, has been developed for the first time by employing 2,4-dinitrobenzene-sulfonate (DNBS) as a recognition unit (also a fluorescence quencher) and a naphthalene-BODIPY-based through-bond energy transfer (TBET) cassette as a fluorescent reporter. The TBET system consists of a D-π-A structured two-photon naphthalene fluorophore and a red-emitting BODIPY. It displayed highly energy transfer efficiency (93.5%), large pseudo-Stokes shifts upon one-photon excitation, and red fluorescence emission (λem = 586 nm), which is highly desirable for bioimaging applications. The probe exhibited a 163-fold thiophenol-triggered two-photon excited fluorescence enhancement at 586 nm. It showed a high selectivity and excellent sensitivity to thiophenols, with a detection limit of 4.9 nM. Moreover, it was successfully applied for practical detection of thiophenol in water samples with a good recovery, two-photon imaging of thiophenol in living cells, and tissues with tissue-imaging depths of 90-220 μm, demonstrating its practical application in environmental samples and biological systems. PMID:26228351

  5. Quantification of red blood cell fragmentation by the automated hematology analyzer XE-2100 in patients with living donor liver transplantation.

    PubMed

    Banno, S; Ito, Y; Tanaka, C; Hori, T; Fujimoto, K; Suzuki, T; Hashimoto, T; Ueda, R; Mizokami, M

    2005-10-01

    The fragmented red cell (FRC) is a useful index for diagnosing and determining the severity of thrombotic thrombocytopenic purpura (TTP), thrombotic microangiopathy (TMA) and other similar conditions, as it is found in peripheral blood in patients with these diseases. The FRC expression rate has conventionally been determined by manual methods using smear samples. However, it is difficult to attain accurate quantification by such methods as they are time consuming and prone to a great margin of error. With cases of living donor liver transplantation, the current study examined the possibility of using a multi-parameter automated hematology analyzer, the XE-2100 (Sysmex Corporation) for FRC quantification. While there was a notable correlation between the manual and automated measurements, the manual measurement resulted in higher values. This suggested remarkable variations in judgment by individuals. The FRC values had a significant correlation with the reticulocyte count, red blood cell distribution width (RDW), fibrin/fibrinogen degradation products (P-FDP) and lactate dehydrogenase (LDH) among the test parameters, and this finding was consistent with the clinical progression in patients. The automated method can offer precise measurements in a short time without inter-observer differences, meeting the requirement for standardization. The determination of FRC count (%) by the XE-2100 that enables early diagnoses and monitoring of TTP or TMA will be useful in the clinical field. PMID:16178907

  6. The pharmacokinetics of a single intramuscular dose of amikacin in red-tailed hawks (Buteo jamaicensis).

    PubMed

    Bloomfield, R B; Brooks, D; Vulliet, R

    1997-03-01

    The pharmacokinetic parameters of amikacin were determined in red-tailed hawks (Buteo jamaicensis) following the i.m. administration of a single 20 mg/kg dose. After a rapid absorption phase, mean amikacin serum concentrations peaked at 65 +/- 12 micrograms/ML 30-45 min following injection. The serum amikacin concentrations decreased to 2.3 +/- 2 micrograms/ml at 12 hr postinjection. Amikacin was eliminated with first-order kinetics characteristic of a single-compartment model with a half-life of 2.02 +/- 0.63 hr. The volume of distribution was estimated to be 0.28 +/- 0.03 L/kg. Forty-two isolates of gram-negative bacteria and coagulase-positive Staphylococcus species were cultured from birds of prey presented to the Veterinary Medical Teaching Hospital at the University of California-Davis. The minimum inhibitory concentration (MICs) of amikacin ranged from 0.5 to 8.0 micrograms/ml (mean = 2.5 micrograms/ml). The 20 mg/kg dose used in this study resulted in serum concentrations at or above the MICs for > 12 hr for most of the isolates examined. The heaviest birds had the lowest peak serum amikacin concentrations, and the lightest birds had the highest, despite exact volume replacement for each sample drawn. This observation suggests that doses should be based on factors other than weight alone. Amikacin administered at 15-20 mg/kg/day, either as a single dose or divided into two or three doses, is effective in treating sensitive pathogens of the red-tailed hawk. PMID:9226617

  7. Localized electroporation and molecular delivery into single living cells by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Nawarathna, D.; Unal, K.; Wickramasinghe, H. Kumar

    2008-10-01

    We present an efficient and fast method for selective and localized electroporation of a single living cell from a population of millions to tens of cells using the modified tip of an atomic force microscope. Electroporation was observed in real time using an inverted microscope. This technique is proposed as a tool for efficient and controlled delivery of biomolecules, proteins, drugs, and genes.

  8. Quantitative Imaging of Single mRNA Splice Variants in Living Cells

    PubMed Central

    Lee, Kyuwan; Cui, Yi

    2015-01-01

    Alternative mRNA splicing is a fundamental process of gene regulation via the precise control of the post-transcriptional step that occurs before mRNA translation. Errors in RNA splicing have been known to correlate with different diseases; however, a key limitation is the lack of technologies for live cell monitoring and quantification to understand the process of alternative splicing. Here, we report a spectroscopic strategy for quantitative imaging of mRNA splice variants in living cells, using nanoplasmonic dimer antennas. The spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1 were monitored at single copy resolution by measuring the hybridization dynamics of nanoplasmonic antennas targeting complementary mRNA sequences in live cells. Our study provides valuable insights on RNA and its transport in living cells, which has the potential to enhance our understanding of cellular protein complex, pharmacogenomics, genetic diagnosis, and gene therapies. PMID:24747838

  9. Living on the edge: Space use of Eurasian red squirrels in marginal high-elevation habitat

    NASA Astrophysics Data System (ADS)

    Romeo, Claudia; Wauters, Lucas A.; Preatoni, Damiano; Tosi, Guido; Martinoli, Adriano

    2010-11-01

    In marginal habitats located at the edge of a species' range, environmental conditions are frequently extreme and individuals may be subject to different selective pressures compared to central populations. These so-called edge or marginal populations tend to have lower densities and reproductive rates than populations located in more suitable habitats, but little is known about local adaptations in spacing behavior. We studied space use and social organization in a population of Eurasian red squirrels ( Sciurus vulgaris) in a high-elevation marginal habitat of dwarf mountain pine ( Pinus mugo) and compared it with spacing patterns in high-quality Scots pine ( Pinus sylvestris) forest at lower-elevation. Home ranges and core areas were larger in the marginal habitat. In both habitats, males used larger home ranges than females, but sex differences in core area size were significant only in the edge population. Patterns of core area overlap were similar in both habitats with intra-sexual territoriality among adult females and higher degrees of inter-sexual overlap, typical for the species throughout its range. However, low densities in the edge population resulted in higher female by males overlap in spring-summer, suggesting males increased home ranges and core areas during mating season to augment access to estrus females. Thus, in the marginal habitat, with low food abundance and low population densities, linked with extreme winter conditions, squirrels, especially males, used large home ranges. Finally, squirrels responded more strongly to variation in food availability (inverse relation between home range size and seed abundance), and even to fluctuations in density (inverse relation between core area size and density of animals of the same sex), in the marginal than in the high-quality habitat, suggesting high behavioral plasticity to respond to the ecological constraints in marginal habitats.

  10. Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision

    PubMed Central

    Lenn, Tchern; Leake, Mark C.

    2012-01-01

    In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences. PMID:22773951

  11. Optical trapping and surgery of living yeast cells using a single laser

    NASA Astrophysics Data System (ADS)

    Ando, Jun; Bautista, Godofredo; Smith, Nicholas; Fujita, Katsumasa; Daria, Vincent Ricardo

    2008-10-01

    We present optical trapping and surgery of living yeast cells using two operational modes of a single laser. We used a focused laser beam operating in continuous-wave mode for noninvasive optical trapping and manipulation of single yeast cell. We verified that such operational mode of the laser does not cause any destructive effect on yeast cell wall. By changing the operation of the laser to femtosecond-pulsed mode, we show that a tightly focused beam dissects the yeast cell walls via nonlinear absorption. Lastly, using the combined technique of optical microsurgery and trapping, we demonstrate intracellular organelle extraction and manipulation from a yeast cell. The technique established here will be useful as an efficient method for both surgery and manipulation of living cells using a single laser beam.

  12. Shape quantification of single red blood cells based on their scattering patterns from microscopic images

    NASA Astrophysics Data System (ADS)

    Schneider, Gert; Artmann, Gerhard

    1995-02-01

    The differentiation between discocytic and stomatocytic red blood cell (RBC) shape using conventional microscopic imaging and image analysis tools is still on a very poor level. A procedure to differentiate the degree of stomatocytic shape changes was developed. We obtained multiple microscopic images of the same RBCs settled on a human albumin coated cover slip. The images were acquired when the microscope objective was subsequently focused through the cell layer. At equidistant horizontal planes (z-axis) below, within, and above the microscopic focal plane the light intensity distribution was considered. Using a model based on light refraction, we calculated the intensity distribution of the planes which are out of focus. Using this tool we are able to differentiate RBC shapes precisely. On the other hand, using this model from and the light intensity distributions of different focal planes, we are able to reconstruct the shape of one single RBC located in the optical axis of the microscope.

  13. Single-shot slightly-off-axis interferometry based Hilbert phase microscopy of red blood cells

    PubMed Central

    Xue, Liang; Lai, Jiancheng; Wang, Shouyu; Li, Zhenhua

    2011-01-01

    A slightly-off-axis interferometry based Hilbert phase microscopy (HPM) method is developed to quantitatively obtain the phase distribution. Owing to its single-shot nature and details detection ability, HPM can be used to investigate rapid phenomena that take place in transparent structures such as biological cells. Moreover, the slightly-off-axis interferometry owns higher effective bandwidth and more sensitivity than traditional off-axis interferometry. The proposed method takes advantages of the above techniques to obtain the phase image of the red blood cells and compared with the traditional off-axis interferometry and phase retrieval algorithm based on the FFT. The experimental results show that the proposed method owns fine spatial details and real-time imaging ability. We are sure that the proposed method provides a breakthrough for real-time observing and quantitative analyzing of cells in vivo. PMID:21483620

  14. A model for oxygen-dependent backscattering spectroscopic contrast from single red blood cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Liu, Rongrong; Yi, Ji; Chen, Siyu; Zhang, Hao F.; Backman, Vadim

    2016-03-01

    The oxygen-dependent absorption of hemoglobin provides the fundamental contrast for all label-free techniques measuring blood oxygenation. When hemoglobin is packaged into red blood cells (RBCs), the structure of the cells creates light scattering which also depends on the absorption based on the Kramers-Kronig relationship. Thus a proper characterization of the optical behaviors of blood has been a key to any accurate measurement of blood oxygenation, particularly at the capillary level where RBCs are dispersed individually in contrast to a densely packed whole blood. Here we provided a theoretical model under Born Approximation to characterize the oxygen dependent backscattering spectroscopic contrast from single RBCs. Using this theoretical model, we conducted simulations on both oxygenated and deoxygenated single RBCs with different sizes for standard and possible deformed cell geometries in blood flow, all which suggested similar backscattering spectroscopic contrast and were confirmed by Mie Theory and experiments using visible Optical Coherence Tomography (visOCT). As long as the cell size satisfies Gaussian distribution with a coefficient variance (C.V.) large enough, there is clear absorption contrast between the backscattering spectra of oxygenated and deoxygenated single RBCs calculated by this model, so oxygen saturation can then be characterized. Thus, this theoretical model can be extended to extract absorption features of other scattering particles as long as they satisfy Born Approximation.

  15. An automated tool for 3D tracking of single molecules in living cells

    NASA Astrophysics Data System (ADS)

    Gardini, L.; Capitanio, M.; Pavone, F. S.

    2015-03-01

    Since the behaviour of proteins and biological molecules is tightly related to cell's environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution. Since protein dynamics inside a cell involve all three dimensions, we developed an automated routine for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.

  16. Styrylpyridine salts-based red emissive two-photon turn-on probe for imaging the plasma membrane in living cells and tissues.

    PubMed

    Guo, Lifang; Zhang, Ruoyao; Sun, Yuming; Tian, Minggang; Zhang, Ge; Feng, Ruiqing; Li, Xuechen; Yu, Xiaoqiang; He, Xiuquan

    2016-05-23

    Based on styrylpyridine salts, a small-molecule red emitting membrane probe with large two-photon absorption cross-section has been synthesized. As a molecular rotor, it enables exclusive lighting up of the plasma membrane in live cells and particular tissues. This probe has the potential to be a powerful tool for bioimaging. PMID:27160329

  17. High efficient white organic light-emitting diodes with single emissive layer using phosphorescent red, green, and blue dopants

    NASA Astrophysics Data System (ADS)

    Kim, You-Hyun; Wai Cheah, Kok; Young Kim, Woo

    2013-07-01

    Phosphorescent white organic light-emitting diodes (PHWOLEDs) with single emissive layer were fabricated by co-doping phosphorescent blue, green, and red emitters with different concentrations. WOLEDs using Ir(piq)3 and Ir(ppy)3 as red and green dopants along with 8% of Firpic as blue dopant with host materials of 4CzPBP in the emissive layer were compared under various doping ratio between Ir(piq)3 and Ir(ppy)3. Triplet-triplet Dexter energy transfer in single emissive PHWOLEDs including three primary colors was saturated from higher triplet energy levels to lower triplet energy levels directly.

  18. Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells.

    PubMed

    Wang, Xiaohua; Li, Xiaojuan; Deng, Xin; Luu, Doan-Trung; Maurel, Christophe; Lin, Jinxing

    2015-12-01

    Measuring the mobility and interactions of proteins is key to understanding cellular signaling mechanisms; however, quantitative analysis of protein dynamics in living plant cells remains a major challenge. Here we describe an automated, single-molecule protocol based on total internal reflection fluorescence microscopy (TIRFM) imaging that allows protein tracking and subunit counting in living plant cells. This protocol uses TIRFM to image transgenic plant tissues expressing fluorescently tagged proteins that are localized to the plasma membrane. Next, a tracking algorithm quantifies dynamic changes in fluorescent protein motion types, temporary particle displacement and protein photobleaching steps. This protocol allows researchers to study the kinetic characteristics of heterogeneously distributed proteins. The approach has potential applications for studies of protein dynamics and subunit stoichiometry for a wide variety of plasma membrane and intracellular proteins in living plant cells and other biological specimens visualized by TIRFM or other fluorescence imaging techniques. The whole protocol can be completed in 5-6 h. PMID:26584445

  19. The motion of a single red blood cell in a capillary

    NASA Astrophysics Data System (ADS)

    Savin, Thierry; Mahadevan, L.

    2009-11-01

    The collective vaso-occlusive event in sickle cell disease induced by multiple red blood cells (RBC's) has recently been evoked and controlled in vitro using a microfluidic platform [1]. The increase in the cells' stiffness in this disease is believed to be a predominant factor at the onset of the occlusion. We report here the motion of a single swollen RBC in a capillary. We use a tapered glass capillary with inner diameter as low as 3 microns, and track the squeezed cell driven by a controlled pressure drop. This allows us to simultaneously measure the variations of the RBC velocity as a function of the pressure gradient and of the local capillary diameter in a single experiment. We show that under certain regimes of confinement, the velocity increases with the pressure head with a characteristic power-law. We analyze our findings in terms of a elasto-hydrodynamical model for soft lubrication.[4pt] [1] Higgins et al., Proc. Natl. Acad. Sci. U.S.A. 104: 20496 (2007).

  20. Yb-fiber laser pumped high-power, broadly tunable, single-frequency red source based on a singly resonant optical parametric oscillator.

    PubMed

    Shukla, Mukesh Kumar; Maji, Partha Sona; Das, Ritwick

    2016-07-01

    We present an efficient and tunable source generating multi-watt single-frequency red radiation by intra-cavity frequency doubling of the signal in a MgO-doped periodically poled LiNbO3 (MgO:PPLN)-based singly resonant optical parametric oscillator (SRO). By optimally designing the SRO cavity in a six-mirror configuration, we generate ≈276  nm tunable idler radiation in mid-infrared with a maximum power of Pi=2.05  W at a pump power of Pp=14.0  W. The resonant signal is frequency doubled using a 10 mm-long BiB3O6 (BiBO) crystal which resulted in tunability of a red beam from ≈753 to 780 nm band with maximum power Pr≈4.0  W recorded at λr≈756  nm. The deployment of a six-mirror SRO ensures single-frequency generation of red across the entire tuning range by inducing additional losses to Raman modes of LiNbO3 and, thus, inhibiting their oscillation. Using a scanning Fabry-Perot interferometer (FPI), nominal linewidth of the red beam is measured to ≈3  MHz which changes marginally over the entire tuning range. Long-term (over 1 h) peak-to-peak frequency fluctuation of the generated red beam is estimated to be about 3.3 GHz under free-running conditions at Pp=14.0  W. The generated red beam is delivered in a TEM00 mode profile with M2≤1.32 at maximum power in a red beam. PMID:27367094

  1. An automated tool for 3D tracking of single molecules in living cells

    NASA Astrophysics Data System (ADS)

    Gardini, L.; Capitanio, M.; Pavone, F. S.

    2015-07-01

    Recently, tremendous improvements have been achieved in the precision of localization of single fluorescent molecules, allowing localization and tracking of biomolecules at the nm level. Since the behaviour of proteins and biological molecules is tightly influenced by the cell's environment, a growing number of microscopy techniques are moving from in vitro to live cell experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). To satisfy these requirements we developed an automated routine that allow 3D tracking of single fluorescent molecules in living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.

  2. Protein Structure-Function Correlation in Living Human Red Blood Cells Probed by Isotope Exchange-based Mass Spectrometry.

    PubMed

    Narayanan, Sreekala; Mitra, Gopa; Muralidharan, Monita; Mathew, Boby; Mandal, Amit K

    2015-12-01

    To gain insight into the underlying mechanisms of various biological events, it is important to study the structure-function correlation of proteins within cells. Structural probes used in spectroscopic tools to investigate protein conformation are similar across all proteins. Therefore, structural studies are restricted to purified proteins in vitro and these findings are extrapolated in cells to correlate their functions in vivo. However, due to cellular complexity, in vivo and in vitro environments are radically different. Here, we show a novel way to monitor the structural transition of human hemoglobin upon oxygen binding in living red blood cells (RBCs), using hydrogen/deuterium exchange-based mass spectrometry (H/DX-MS). Exploiting permeability of D2O across cell membrane, the isotope exchange of polypeptide backbone amide hydrogens of hemoglobin was carried out inside RBCs and monitored using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To explore the conformational transition associated with oxygenation of hemoglobin in vivo, the isotope exchange kinetics was simplified using the method of initial rates. RBC might be considered as an in vivo system of pure hemoglobin. Thus, as a proof-of-concept, the observed results were correlated with structural transition of hemoglobin associated with its function established in vitro. This is the first report on structural changes of a protein upon ligand binding in its endogenous environment. The proposed method might be applicable to proteins in their native state, irrespective of location, concentration, and size. The present in-cell approach opens a new avenue to unravel a plethora of biological processes like ligand binding, folding, and post-translational modification of proteins in living cells. PMID:26531244

  3. A highly selective ratiometric visual and red-emitting fluorescent dual-channel probe for imaging fluoride anions in living cells.

    PubMed

    Zhu, Baocun; Kan, He; Liu, Jingkai; Liu, Hanqing; Wei, Qin; Du, Bin

    2014-02-15

    Recently, growing attention has been paid to the accurate determination of fluoride anion (F(-)) in the environment and living systems for its toxicity and biological function investigation. In this paper, we developed a ratiometric visual and red-emitting fluorescent dual-channel probe (1) employed Si-O bond as a highly selective recognition receptor for imaging F(-) in living cells. Probe 1 possesses a potential internal charge transfer (ICT) structure, and displays a large (158 nm) red-shifted absorption spectrum and the color changes from yellow to blue upon addition of F(-) in the aqueous solution. In addition, probe 1 can be used to detect F(-) quantitatively by the ratiometric absorption and turn-on fluorescence spectroscopy methods with excellent sensitivity. Finally, the results of its application to bioimaging of F(-) in living cells show that probe 1 would be of great benefit to biomedical researchers for investigating the effects of fluoride in biological systems. PMID:24080208

  4. Real-time visualization of intracellular hydrodynamics in single living cells.

    PubMed

    Potma, E; de Boeij, W P; van Haastert, P J; Wiersma, D A

    2001-02-13

    Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high spatial resolution. Studies of the hydrodynamics in the microorganism Dictyostelium discoideum indicated the presence of a microscopic region near the plasma membrane where the mobility of water molecules is severely restricted. Modeling the transient hydrodynamics eventuated in the determination of cell-specific cytosolic diffusion and plasma membrane permeability constants. Our experiments demonstrate that CARS microscopy offers an invaluable tool for probing single-cell water dynamics. PMID:11171993

  5. Fiber optic SERS-based plasmonics nanobiosensing in single living cells

    NASA Astrophysics Data System (ADS)

    Scaffidi, Jonathan P.; Gregas, Molly K.; Seewaldt, Victoria; Vo-Dinh, Tuan

    2009-05-01

    We describe the development of small molecule-sensitive plasmonics-active fiber-optic nanoprobes suitable for intracellular bioanalysis in single living human cells using surface-enhanced Raman scattering (SERS) detection. The practical utility of SERS-based fiber-optic nanoprobes is illustrated by measurements of intracellular pH in HMEC- 15/hTERT immortalized "normal" human mammary epithelial cells and PC-3 human prostate cancer cells. The results indicate that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor biologically-relevant small molecules at the single cell level.

  6. Real-time visualization of intracellular hydrodynamics in single living cells

    PubMed Central

    Potma, Eric O.; de Boeij, Wim P.; van Haastert, Peter J. M.; Wiersma, Douwe A.

    2001-01-01

    Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high spatial resolution. Studies of the hydrodynamics in the microorganism Dictyostelium discoideum indicated the presence of a microscopic region near the plasma membrane where the mobility of water molecules is severely restricted. Modeling the transient hydrodynamics eventuated in the determination of cell-specific cytosolic diffusion and plasma membrane permeability constants. Our experiments demonstrate that CARS microscopy offers an invaluable tool for probing single-cell water dynamics. PMID:11171993

  7. A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells

    PubMed Central

    2014-01-01

    Background Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation. Results We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR. Conclusions Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses. PMID:24834016

  8. Single-Molecule Studies of Integrins by AFM-Based Force Spectroscopy on Living Cells

    NASA Astrophysics Data System (ADS)

    Eibl, Robert H.

    The characterization of cell adhesion between two living cells at the single-molecule level, i.e., between one adhesion receptor and its counter-receptor, appears to be an experimental challenge. Atomic force microscopy (AFM) can be used in its force spectroscopy mode to determine unbinding forces of a single pair of adhesion receptors, even with a living cell as a probe. This chapter provides an overview of AFM force measurements of the integrin family of cell adhesion receptors and their ligands. A focus is given to major integrins expressed on leukocytes, such as lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4). These receptors are crucial for leukocyte trafficking in health and disease. LFA-1 and VLA-1 can be activated within the bloodstream from a low-affinity to a high-affinity receptor by chemokines in order to adhere strongly to the vessel wall before the receptor-bearing leukocytes extravasate. The experimental considerations needed to provide near-physiological conditions for a living cell and to be able to measure adequate forces at the single-molecule level are discussed in detail. AFM technology has been developed into a modern and extremely sensitive tool in biomedical research. It appears now that AFM force spectroscopy could enter, within a few years, medical applications in diagnosis and therapy of cancer and autoimmune diseases.

  9. Nile Blue-Based Nanosized pH Sensors for Simultaneous Far-Red and Near-Infrared Live Bioimaging

    PubMed Central

    2013-01-01

    Diblock copolymer vesicles are tagged with pH-responsive Nile Blue-based labels and used as a new type of pH-responsive colorimetric/fluorescent biosensor for far-red and near-infrared imaging of live cells. The diblock copolymer vesicles described herein are based on poly(2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate) [PMPC-PDPA]: the biomimetic PMPC block is known to facilitate rapid cell uptake for a wide range of cell lines, while the PDPA block constitutes the pH-responsive component that enables facile vesicle self-assembly in aqueous solution. These biocompatible vesicles can be utilized to detect interstitial hypoxic/acidic regions in a tumor model via a pH-dependent colorimetric shift. In addition, they are also useful for selective intracellular staining of lysosomes and early endosomes via subtle changes in fluorescence emission. Such nanoparticles combine efficient cellular uptake with a pH-responsive Nile Blue dye label to produce a highly versatile dual capability probe. This is in marked contrast to small molecule dyes, which are usually poorly uptaken by cells, frequently exhibit cytotoxicity, and are characterized by intracellular distributions invariably dictated by their hydrophilic/hydrophobic balance. PMID:24001153

  10. Nile Blue-based nanosized pH sensors for simultaneous far-red and near-infrared live bioimaging.

    PubMed

    Madsen, Jeppe; Canton, Irene; Warren, Nicholas J; Themistou, Efrosyni; Blanazs, Adam; Ustbas, Burcin; Tian, Xiaohe; Pearson, Russell; Battaglia, Giuseppe; Lewis, Andrew L; Armes, Steven P

    2013-10-01

    Diblock copolymer vesicles are tagged with pH-responsive Nile Blue-based labels and used as a new type of pH-responsive colorimetric/fluorescent biosensor for far-red and near-infrared imaging of live cells. The diblock copolymer vesicles described herein are based on poly(2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate) [PMPC-PDPA]: the biomimetic PMPC block is known to facilitate rapid cell uptake for a wide range of cell lines, while the PDPA block constitutes the pH-responsive component that enables facile vesicle self-assembly in aqueous solution. These biocompatible vesicles can be utilized to detect interstitial hypoxic/acidic regions in a tumor model via a pH-dependent colorimetric shift. In addition, they are also useful for selective intracellular staining of lysosomes and early endosomes via subtle changes in fluorescence emission. Such nanoparticles combine efficient cellular uptake with a pH-responsive Nile Blue dye label to produce a highly versatile dual capability probe. This is in marked contrast to small molecule dyes, which are usually poorly uptaken by cells, frequently exhibit cytotoxicity, and are characterized by intracellular distributions invariably dictated by their hydrophilic/hydrophobic balance. PMID:24001153

  11. Deformation of a single red blood cell in bounded Poiseuille flows

    NASA Astrophysics Data System (ADS)

    Shi, Lingling; Pan, Tsorng-Whay; Glowinski, Roland

    2012-01-01

    Deformation of a red blood cell (RBC) in bounded two-dimensional Poiseuille flows is studied by using an immersed boundary method (IBM). An elastic spring model is applied to simulate the skeleton structure of a RBC membrane. As a benchmarking test, the dynamical behavior of a single RBC under a simple shear flow has been validated. Then we focus on investigating the motion and the deformation of a single RBC in Poiseuille flows by varying the swelling ratio (s*), the initial angle of the long axis of the cell at the centerline (ϕ), the maximum velocity at the centerline of fluid flow (umax), the membrane bending stiffness of a RBC (kb), and the height of the microchannel (H). Two motions of oscillation and vacillating breathing (swing) of a RBC are observed in both narrow and wide channels. The strength of the vacillating-breathing motion depends on the degree of confinement and the value of umax. A RBC exhibits a strong vacillating-breathing motion as the degree of confinement is larger or the value of umax is higher. For the same degree of confinement, the vacillating-breathing motion appears to be relatively weaker but persists longer as the value of umax is lower. The continuation of shape change from the slippery to the parachute by varying the value of umax is obtained for the biconcave shape cell in a narrower channel. In particular, parachute shape and bulletlike shape, depending on the angle ϕ, coexist for the elliptic shape cell given initially with lower umax in a narrower channel.

  12. Life situation and identity among single older home-living people: A phenomenological–hermeneutic study

    PubMed Central

    Söderhamn, Ulrika; Söderhamn, Olle

    2012-01-01

    Being able to continue living in their own home as long as possible is the general preference for many older people, and this is also in line with the public policy in the Nordic countries. The aim of this study was to elucidate the meaning of self-care and health for perception of life situation and identity among single-living older individuals in rural areas in southern Norway. Eleven older persons with a mean age of 78 years were interviewed and encouraged to narrate their self-care and health experiences. The interviews were audio taped, transcribed verbatim and analysed using a phenomenological–hermeneutic method inspired by the philosophy of Ricoeur. The findings are presented as a naïve reading, an inductive structural analysis characterized by two main themes; i.e., “being able to do” and “being able to be”, and a comprehensive interpretation. The life situation of the interviewed single-living older individuals in rural areas in southern Norway was interpreted as inevitable, appropriate and meaningful. Their identity was constituted by their freedom and self-chosen actions in their personal contexts. The overall impression was that independence and the ability to control and govern their own life in accordance with needs and preferences were ultimate goals for the study participants. PMID:22848230

  13. Two-Color STED Microscopy of Living Synapses Using A Single Laser-Beam Pair

    PubMed Central

    Tønnesen, Jan; Nadrigny, Fabien; Willig, Katrin I.; Wedlich-Söldner, Roland; Nägerl, U. Valentin

    2011-01-01

    The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices. PMID:22098754

  14. Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells

    NASA Astrophysics Data System (ADS)

    Chai, Liuying; Zhang, Jianwei; Zhang, Lili; Chen, Tongsheng

    2015-03-01

    Spectral measurement of fluorescence resonance energy transfer (FRET), spFRET, is a widely used FRET quantification method in living cells today. We set up a spectrometer-microscope platform that consists of a miniature fiber optic spectrometer and a widefield fluorescence microscope for the spectral measurement of absolute FRET efficiency (E) and acceptor-to-donor concentration ratio (RC) in single living cells. The microscope was used for guiding cells and the spectra were simultaneously detected by the miniature fiber optic spectrometer. Moreover, our platform has independent excitation and emission controllers, so different excitations can share the same emission channel. In addition, we developed a modified spectral FRET quantification method (mlux-FRET) for the multiple donors and multiple acceptors FRET construct (mD˜nA) sample, and we also developed a spectra-based 2-channel acceptor-sensitized FRET quantification method (spE-FRET). We implemented these modified FRET quantification methods on our platform to measure the absolute E and RC values of tandem constructs with different acceptor/donor stoichiometries in single living Huh-7 cells.

  15. Life situation and identity among single older home-living people: a phenomenological-hermeneutic study.

    PubMed

    Dale, Bjørg; Söderhamn, Ulrika; Söderhamn, Olle

    2012-01-01

    Being able to continue living in their own home as long as possible is the general preference for many older people, and this is also in line with the public policy in the Nordic countries. The aim of this study was to elucidate the meaning of self-care and health for perception of life situation and identity among single-living older individuals in rural areas in southern Norway. Eleven older persons with a mean age of 78 years were interviewed and encouraged to narrate their self-care and health experiences. The interviews were audio taped, transcribed verbatim and analysed using a phenomenological-hermeneutic method inspired by the philosophy of Ricoeur. The findings are presented as a naïve reading, an inductive structural analysis characterized by two main themes; i.e., "being able to do" and "being able to be", and a comprehensive interpretation. The life situation of the interviewed single-living older individuals in rural areas in southern Norway was interpreted as inevitable, appropriate and meaningful. Their identity was constituted by their freedom and self-chosen actions in their personal contexts. The overall impression was that independence and the ability to control and govern their own life in accordance with needs and preferences were ultimate goals for the study participants. PMID:22848230

  16. Evolution of anterior segment reconstruction after live donor adult liver transplantation: a single-center experience.

    PubMed

    Pomposelli, James J; Akoad, Mohamed; Khwaja, Khalid; Lewis, W D; Cheah, Yee L; Verbesey, Jennifer; Jenkins, Roger L; Pomfret, Elizabeth A

    2012-01-01

    Controversy exists regarding the best method for venous outflow reconstruction after live donor liver transplantation using right lobe grafts. Some authors advocate routine inclusion of the middle hepatic vein with the graft, whereas others favor a more selective approach. In this report, we examine the evolution of our decision making and technique of selective anterior venous segment reconstruction during live donor adult liver transplantation performed in 226 recipients. We have developed a simplified back-bench procedure using sequential-composite anastomosis using various vascular conduits with syndactylization to the right hepatic vein creating a single large-outflow anastomosis in the recipient. Conduits used include iliac artery or vein allograft, recanalized umbilical vein, cryopreserved iliac artery allograft, and 6-mm synthetic expanded polytetrafluoroethylene vascular graft. This technique can be performed quickly, safely, and under cold storage conditions and results in excellent outcome while minimizing donor risk. PMID:21980936

  17. Nature of red luminescence band in research-grade ZnO single crystals: A "self-activated" configurational transition

    NASA Astrophysics Data System (ADS)

    Chen, Y. N.; Xu, S. J.; Zheng, C. C.; Ning, J. Q.; Ling, F. C. C.; Anwand, W.; Brauer, G.; Skorupa, W.

    2014-07-01

    By implanting Zn+ ions into research-grade intentionally undoped ZnO single crystal for facilitating Zn interstitials (Zni) and O vacancies (VO) which is revealed by precise X-Ray diffraction rocking curves, we observe an apparent broad red luminescence band with a nearly perfect Gaussian lineshape. This red luminescence band has the zero phonon line at ˜2.4 eV and shows distinctive lattice temperature dependence which is well interpreted with the configurational coordinate model. It also shows a low "kick out" thermal energy and small thermal quenching energy. A "self-activated" optical transition between a shallow donor and the defect center of Zni-VO complex or VZnVO di-vacancies is proposed to be responsible for the red luminescence band. Accompanied with the optical transition, large lattice relaxation simultaneously occurs around the center, as indicated by the generation of multiphonons.

  18. Cell compressibility studies utilizing noncontact hydrostatic pressure measurements on single living cells in a microchamber

    NASA Astrophysics Data System (ADS)

    Lin, L. A. G.; Liu, A. Q.; Yu, Y. F.; Zhang, C.; Lim, C. S.; Ng, S. H.; Yap, P. H.; Gao, H. J.

    2008-06-01

    A micro-optical-fluidic system (MOFS), which integrates a force generating device and an optical detector, is designed to measure the bulk modulus of a single living cell in real time under a controlled hydrostatic pressure. In this design, the accuracy of the bulk modulus measurement is improved because neither the force generating device nor the optical detector needs to be in contact with the cells. The MOFS device has been used to investigate the mechanotransduction of THP-1 human acute monocytic leukemia cells and the effects of the toxin lipopolysaccharide and colchicine on various properties of these cells.

  19. Surface enhanced Raman scattering analyses of individual silver nanoaggregates on living single yeast cell wall

    NASA Astrophysics Data System (ADS)

    Sujith, Athiyanathil; Itoh, Tamitake; Abe, Hiroko; Anas, Abdul Aziz; Yoshida, Kenichi; Biju, Vasudevanpillai; Ishikawa, Mitsuru

    2008-03-01

    We labeled the living yeast cell surface (Saccharomyces cerevisiae strain W303-1A) by silver nanoparticles which can form nanoaggregates and found to show surface enhanced Raman scattering (SERS) activity. Blinking of SERS and its polarization dependence reveal that SERS signals are from amplified electromagnetic field at nanometric Ag nanoparticles gaps with single or a few molecules sensitivity. We tentatively assigned SERS spectra from a yeast cell wall to mannoproteins. Nanoaggregate-by-nanoaggregate variations and temporal fluctuations of SERS spectra are discussed in terms of inhomogeneous mannoprotein distribution on a cell wall and possible ways of Ag nanoaggregate adsorption, respectively.

  20. A red fluorescence 'off-on' molecular switch for selective detection of Al3+, Fe3+ and Cr3+: experimental and theoretical studies along with living cell imaging.

    PubMed

    Goswami, Shymaprosad; Aich, Krishnendu; Das, Sangita; Das, Avijit Kumar; Sarkar, Deblina; Panja, Sukanya; Mondal, Tapan Kumar; Mukhopadhyay, Subhrakanti

    2013-11-25

    A spirobenzopyran-quinoline (SBPQ) based sensor was synthesized which selectively detects trivalent ions viz. Al(3+), Fe(3+) and Cr(3+) through a fluorescence turn on signal in the red region (~675 nm) with the detection limit in the order of 10(-8) M. The potentiality of the probe was confirmed by employing it for fluorescence bio-imaging with Al(3+) in three different types of live-cells. PMID:24104701

  1. Single stance stability and proprioceptive control in older adults living at home: gender and age differences.

    PubMed

    Riva, Dario; Mamo, Carlo; Fanì, Mara; Saccavino, Patrizia; Rocca, Flavio; Momenté, Manuel; Fratta, Marianna

    2013-01-01

    In developed countries, falls in older people represent a rising problem. As effective prevention should start before the risk becomes evident, an early predictor is needed. Single stance instability would appear as a major risk factor. Aims of the study were to describe single stance stability, its sensory components, and their correlation with age and gender. A random sample of 597 older adults (319 men, 278 women) living at home, aged 65-84, was studied. Stability tests were performed with an electronic postural station. The single stance test showed the impairment of single stance stability in older individuals (75-84 yrs). The significant decline of stability in the older subjects may be explained by the impairment of proprioceptive control together with the decrease in compensatory visual stabilization and emergency responses. Younger subjects (65-74 yrs) exhibited better, but still inadequate, proprioceptive control with compensatory visual stabilization. Gender differences appeared in older subjects: women were significantly less stable than men. The measurement of the sensory components of single stance stability could aid in the early detection of a decay in antigravity movements many years before the risk of falling becomes evident. Adequate proprioceptive control could mitigate the effects of all other risks of falling. PMID:23984068

  2. Live Single-Cell Plant Hormone Analysis by Video-Mass Spectrometry.

    PubMed

    Shimizu, Takafumi; Miyakawa, Shinya; Esaki, Tsuyoshi; Mizuno, Hajime; Masujima, Tsutomu; Koshiba, Tomokazu; Seo, Mitsunori

    2015-07-01

    Studies have indicated that endogenous concentrations of plant hormones are regulated very locally within plants. To understand the mechanisms underlying hormone-mediated physiological processes, it is indispensable to know the exact hormone concentrations at cellular levels. In the present study, we established a system to determine levels of ABA and jasmonoyl-isoleucine (JA-Ile) from single cells. Samples taken from a cell of Vicia faba leaves using nano-electrospray ionization (ESI) tips under a microscope were directly introduced into mass spectrometers by infusion and subjected to tandem mass spectrometry (MS/MS) analysis. Stable isotope-labeled [D(6)]ABA or [(13)C(6)]JA-Ile was used as an internal standard to compensate ionization efficiencies, which determine the amount of ions introduced into mass spectrometers. We detected ABA and JA-Ile from single cells of water- and wound-stressed leaves, whereas they were almost undetectable in non-stressed single cells. The levels of ABA and JA-Ile found in the single-cell analysis were compared with levels found by analysis of purified extracts with liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that stress-induced accumulation of ABA and JA-Ile could be monitored from living single cells. PMID:25759328

  3. Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light

    PubMed Central

    Niino, Yusuke; Hotta, Kohji; Oka, Kotaro

    2009-01-01

    Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility. PMID:19551140

  4. A single cyanobacterial ribotype is associated with both red and black bands on diseased corals from Palau.

    PubMed

    Sussman, Meir; Bourne, David G; Willis, Bette L

    2006-03-23

    Filamentous cyanobacteria forming red and black bands (black band disease, BBD) on 3 scleractinian corals from Palau were molecularly identified as belonging to a single ribotype. Red cyanobacterial mats sampled from infections on Pachyseris speciosa and a massive Porites sp. yielded red strains RMS1 and RMS2 respectively; the black cyanobacterial mat sampled from an infection on Montipora sp. yielded black strain BMS1. Following trials of a range of specialized media and culture conditions, 2 media, Grund and ASN-III, were identified as the best for successful isolation and culturing. Cultured cyanobacteria were examined under a light microscope to establish purity, color and morphological appearance. DNA extraction and partial sequencing of the 16S rDNA gene of both red and black cyanobacterial isolates demonstrated 100% sequence identity. These isolated strains were also found to have 99% sequence identity with an uncultured cyanobacterial strain previously identified by molecular techniques as belonging to a cyanobacterial ribotype associated with BBD-infected corals in the Caribbean. This is the first report of the successful isolation and culture of cyanobacterial strains derived from both red bands and BBD. Based on these findings, it is suggested that the classification of these 2 syndromes as separate coral diseases be postponed until further evidence is collected. PMID:16703773

  5. Dielectric dependence of single-molecule photoluminescence intermittency: nile red in poly(vinylidene fluoride).

    PubMed

    Hess, Chelsea M; Riley, Erin A; Reid, Philip J

    2014-07-24

    The dependence of single-molecule photoluminescence intermittency (PI) or "blinking" on the local dielectric constant (ε) is examined for nile red (NR) in thin films of poly(vinylidene fluoride) (PVDF). In previous studies, variation of the local dielectric constant was accomplished by studying luminophores in chemically and structurally different hosts. In contrast, the NR/PVDF guest-host pair allows for the investigation of PI as a function of ε while keeping the chemical composition of both the luminophore and host unchanged. The solvatochromic properties of NR are used to measure the local ε, while fluctuations in NR emission intensity over time provide a measure of the PI. PVDF is an ideal host for this study because it provides submicron-sized dielectric domains that vary from nonpolar (ε ≈ 2) to very polar (ε ≈ 70). The results presented here demonstrate that the local dielectric environment can have a pronounced effect on PI. We find that the NR emissive events increase 5-fold with an increase in ε from 2.2 to 74. A complex dependence on ε is also observed for NR nonemissive event durations, initially increasing as ε increases from 2.2 to 3.4 but decreasing in duration with further increase in ε. The variation in emissive event durations with ε is reproduced using a photoinduced electron-transfer model involving electron transfer from NR to PVDF. In addition, an increase in NR photostability with an increase in ε is observed, suggesting that the dielectric environment plays an important role in defining the photostability of NR in PVDF. PMID:24995904

  6. Aptamer-based single-molecule imaging of insulin receptors in living cells

    NASA Astrophysics Data System (ADS)

    Chang, Minhyeok; Kwon, Mijin; Kim, Sooran; Yunn, Na-Oh; Kim, Daehyung; Ryu, Sung Ho; Lee, Jong-Bong

    2014-05-01

    We present a single-molecule imaging platform that quantitatively explores the spatiotemporal dynamics of individual insulin receptors in living cells. Modified DNA aptamers that specifically recognize insulin receptors (IRs) with a high affinity were selected through the SELEX process. Using quantum dot-labeled aptamers, we successfully imaged and analyzed the diffusive motions of individual IRs in the plasma membranes of a variety of cell lines (HIR, HEK293, HepG2). We further explored the cholesterol-dependent movement of IRs to address whether cholesterol depletion interferes with IRs and found that cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin reduces the mobility of IRs. The aptamer-based single-molecule imaging of IRs will provide better understanding of insulin signal transduction through the dynamics study of IRs in the plasma membrane.

  7. Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells.

    PubMed

    König, Iwo; Zarrine-Afsar, Arash; Aznauryan, Mikayel; Soranno, Andrea; Wunderlich, Bengt; Dingfelder, Fabian; Stüber, Jakob C; Plückthun, Andreas; Nettels, Daniel; Schuler, Benjamin

    2015-08-01

    Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function. PMID:26147918

  8. Visualizing odorant receptor trafficking in living cells down to the single-molecule level

    PubMed Central

    Jacquier, V.; Prummer, M.; Segura, J.-M.; Pick, H.; Vogel, H.

    2006-01-01

    Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of ≈190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception. PMID:16980412

  9. Successful comeback of the single-dose live oral cholera vaccine CVD 103-HgR.

    PubMed

    Herzog, Christian

    2016-01-01

    Effective and easy to administer cholera vaccines are in need more than ever, for at risk populations and travellers alike. In many parts of the world cholera is still endemic, causing outbreaks and constituting repeatedly serious public health problems. The oral live cholera vaccine CVD 103-HgR (Orochol, Mutachol), the first genetically modified organism (GMO) used as vaccine, was in its time (launched 1993, Switzerland) the ideal cholera vaccine: single-dose, protective efficacy of 80-100% against moderate to severe cholera, acting within 8 days and exhibiting excellent safety, indiscernible from placebo. However, there were strong headwinds: In the 1990s the indication for cholera vaccines was generally downplayed by experts and in 1997 the European Commission called for a moratorium of GMOs which blocked the registration in the European Union. Thus, demand for this vaccine remained low and in 2003 it was taken off the market for economic reasons. After a decade in obscurity it (Vaxchora) has resurfaced again, now produced in the U.S. and equipped with a U.S. FDA license (June 10, 2016). What had happened? This commentary gives a critical account of an almost unbelievable string of misadventures, emerging adverse circumstances and man-made failures which nearly killed this single-dose live oral cholera vaccine. The good news is that patience and persistence lead to success in the end, allowing good science to prevail for the benefit of those in need. PMID:27425792

  10. Nanowires: Quantitative Probing of Cu(2+) Ions Naturally Present in Single Living Cells (Adv. Mater. 21/2016).

    PubMed

    Lee, Junho; Lee, Hwa-Rim; Pyo, Jaeyeon; Jung, Youngseob; Seo, Ji-Young; Ryu, Hye Guk; Kim, Kyong-Tai; Je, Jung Ho

    2016-06-01

    Quantitative probing of the Cu(2+) ions naturally present in single living cells is accomplished by a probe made from a quantum-dot-embedded-nanowire waveguide. After inserting the active nanowire-based waveguide probe into single living cells, J. H. Je and co-workers directly observe photoluminescence (PL) quenching of the embedded quantum dots by the Cu(2+) ions diffused into the probe as described on page 4071. This results in quantitative measurement of intracellular Cu(2+) ions. PMID:27246918

  11. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging.

    PubMed

    Yan, Jing; Sharo, Andrew G; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

    2016-09-01

    Biofilms are surface-associated bacterial communities that are crucial in nature and during infection. Despite extensive work to identify biofilm components and to discover how they are regulated, little is known about biofilm structure at the level of individual cells. Here, we use state-of-the-art microscopy techniques to enable live single-cell resolution imaging of a Vibrio cholerae biofilm as it develops from one single founder cell to a mature biofilm of 10,000 cells, and to discover the forces underpinning the architectural evolution. Mutagenesis, matrix labeling, and simulations demonstrate that surface adhesion-mediated compression causes V. cholerae biofilms to transition from a 2D branched morphology to a dense, ordered 3D cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture in V. cholerae biofilms, and this growth pattern is controlled by a single gene, rbmA Competition analyses reveal that the dense growth mode has the advantage of providing the biofilm with superior mechanical properties. Our single-cell technology can broadly link genes to biofilm fine structure and provides a route to assessing cell-to-cell heterogeneity in response to external stimuli. PMID:27555592

  12. Ultrafast Tracking of a Single Live Virion During the Invagination of a Cell Membrane.

    PubMed

    Pan, Yangang; Wang, Shaowen; Shan, Yuping; Zhang, Dinglin; Gao, Jing; Zhang, Min; Liu, Shuheng; Cai, Mingjun; Xu, Haijiao; Li, Guohui; Qin, Qiwei; Wang, Hongda

    2015-06-01

    The first step in most viral infections is the penetration of the cell membrane via endocytosis. However, the underlying mechanism of this important process has not been quantitatively characterized; for example, the velocity and force of a single virion during invagination remain unknown. Here, the endocytosis of a single live virion (Singapore grouper iridovirus, SGIV) through the apical membranes of a host cell is monitored by developing and using a novel ultrafast (at the microsecond level) tracking technique: force tracing. For the first time, these results unambiguously reveal that the maximum velocity during the cell entry of a single SGIV by membrane invagination is approximately 200 nm s(-1), the endocytic force is approximately 60.8 ± 18.5 pN, and the binding energy density increases with the engulfment depth. This report utilizing high temporospatial resolution (subnanometer and microsecond levels) approaches provides new insight into the dynamic process of viral infection via endocytosis and the mechanism of membrane invagination at the single-particle level. PMID:25689837

  13. Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

    SciTech Connect

    Chan, J W; Winhold, H; Corzett, M H; Ulloa, J M; Cosman, M; Balhorn, R; Huser, T

    2007-01-09

    Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in

  14. Gastrointestinal parasites of free-living Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the Northern Red Sea, Egypt.

    PubMed

    Kleinertz, S; Hermosilla, C; Ziltener, A; Kreicker, S; Hirzmann, J; Abdel-Ghaffar, F; Taubert, A

    2014-04-01

    The present study represents the first report on the gastrointestinal parasite fauna infecting the free-living and alive Indo-Pacific bottlenose dolphins (Tursiops aduncus) inhabiting waters of the Red Sea at Hurghada, Egypt. A total of 94 individual faecal samples of the examined bottlenose dolphins were collected during several diving expeditions within their natural habitats. Using classical parasitological techniques, such as sodium acetate acetic acid formalin method, carbol fuchsin-stained faecal smears, coproantigen ELISA, PCR and macroscopical analyses, the study revealed infections with 21 different parasite species belonging to protozoans and metazoans with some of them bearing zoonotic and/or pathogenic potential. Four identified parasite species are potential zoonotic species (Giardia spp., Cryptosporidium spp., Diphyllobothrium spp., Ascaridida indet.); three of them are known to have high pathogenic potential for the examined dolphin species (Nasitrema attenuata, Zalophotrema spp. and Pholeter gastrophilus) and some appear to be directly associated with stranding events. In detail, the study indicates stages of ten protozoan species (Giardia spp., Sarcocystis spp., Isospora (like) spp., Cystoisospora (like) spp., Ciliata indet. I and II, Holotricha indet., Dinoflagellata indet., Hexamita (like) spp., Cryptosporidium spp.), seven trematode species (N. attenuata, Nasitrema spp. I and II, Zalophotrema curilensis, Zalophotrema spp., Pholeter gastrophilus, Trematoda indet.), one cestode species (Diphyllobothrium spp.), two nematode species (Ascaridida indet, Capillaria spp.) and one crustacean parasite (Cymothoidae indet.). Additionally, we molecularly identified adult worms of Anisakis typica in individual dolphin vomitus samples by molecular analyses. A. typica is a common parasite of various dolphin species of warmer temperate and tropical waters and has not been attributed as food-borne parasitic zoonoses so far. Overall, these parasitological findings

  15. Single Molecule Detection in Living Biological Cells using Carbon Nanotube Optical Probes

    NASA Astrophysics Data System (ADS)

    Strano, Michael

    2009-03-01

    Nanoscale sensing elements offer promise for single molecule analyte detection in physically or biologically constrained environments. Molecular adsorption can be amplified via modulation of sharp singularities in the electronic density of states that arise from 1D quantum confinement [1]. Single-walled carbon nanotubes (SWNT), as single molecule optical sensors [2-3], offer unique advantages such as photostable near-infrared (n-IR) emission for prolonged detection through biological media, single-molecule sensitivity and, nearly orthogonal optical modes for signal transduction that can be used to identify distinct classes of analytes. Selective binding to the SWNT surface is difficult to engineer [4]. In this lecture, we will briefly review the immerging field of fluorescent diagnostics using band gap emission from SWNT. In recent work, we demonstrate that even a single pair of SWNT provides at least four optical modes that can be modulated to uniquely fingerprint chemical agents by the degree to which they alter either the emission band intensity or wavelength. We validate this identification method in vitro by demonstrating detection and identification of six genotoxic analytes, including chemotherapeutic drugs and reactive oxygen species (ROS), which are spectroscopically differentiated into four distinct classes. We also demonstrate single-molecule sensitivity in detecting hydrogen peroxide, one of the most common genotoxins and an important cellular signal. Finally, we employ our sensing and fingerprinting method of these analytes in real time within live 3T3 cells, demonstrating the first multiplexed optical detection from a nanoscale biosensor and the first label-free tool to optically discriminate between genotoxins. We will also discuss our recent efforts to fabricate biomedical sensors for real time detection of glucose and other important physiologically relevant analytes in-vivo. The response of embedded SWNT in a swellable hydrogel construct to

  16. Pharmacokinetics of terbinafine after single oral dose administration in red-tailed hawks (Buteo jamaicensis).

    PubMed

    Bechert, Ursula; Christensen, J Mark; Poppenga, Robert; Fahmy, Sahar A; Redig, Patrick

    2010-06-01

    To determine pharmacokinetic parameters of orally administered terbinafine hydrochloride for potential treatment of aspergillosis in raptors, 10 adult red-tailed hawks (Buteo jamaicensis) were used in single dose trials by using 15, 30, and 60 mg/kg doses with a 2-week washout period between trials. After administration of 15 mg/kg terbinafine, mean (+/- SD) plasma concentration peaked in approximately 5 hours at 0.3 +/- 0.24 microg/mL, whereas a 30 mg/kg dose resulted in peak mean (+/- SD) plasma concentration of 1.2 +/- 0.40 microg/mL in 3 hours and a 60 mg/kg dose resulted in mean (+/- SD) concentration of 2.0 +/- 0.75 microg/mL in 5 hours. The volume of distribution decreased with increasing doses, averaging 76.8 +/- 38.06 mL/kg for the 15 mg/kg dose and falling to 55.2 +/- 17.4 mL/kg for the 30 mg/kg dose. This suggests that terbinafine accumulated in deep tissues, limiting further distribution at higher doses. The harmonic mean (+/- SD) half-life was biphasic, with initial values of 14.7 +/- 6.67 hours, 17.5 +/- 8.7 hours, and 13.3 +/- 5.03 hours for 15, 30, and 60 mg/kg doses, respectively. A rapid first-elimination phase was followed by a slower second phase, and final elimination was estimated to be 161 +/- 78.2 and 147 +/- 65.6 hours for 15 and 30 mg/kg doses, respectively. Linearity was demonstrated for the area under the curve but not for peak plasma concentrations for the 3 doses used. Calculations based on pharmacokinetic parameter values indicated that a dosage of 22 mg/kg terbinafine q24h would result in steady-state trough plasma concentrations above the minimum inhibitory concentration of terbinafine (0.8-1.6 microg/mL). This dosage is recommended as a potential treatment option for aspergillosis in raptors. However, additional research is required to determine both treatment efficacy and safety. PMID:20806657

  17. Diffusion dynamics of the Keap1–Cullin3 interaction in single live cells

    SciTech Connect

    Baird, Liam; Dinkova-Kostova, Albena T.

    2013-03-29

    Highlights: ► We developed a quantitative FRAP-based system to study the Keap1–Cul3 interaction. ► We show that Keap1–EGFP and mCherry–Cul3 interact in single live cells. ► We used inducers which target distinct cysteine sensors of Keap1 and differ 4000-fold in potency. ► Inducers cause Nrf2 stabilization, nuclear translocation, and target gene expression. ► Inducers of four different types do not dissociate the Keap1–EGFP:mCherry–Cul3 complex. -- Abstract: Transcription factor NF-E2 p45-related factor 2 (Nrf2) regulates the expression of a network of genes encoding drug-detoxification, anti-inflammatory, and metabolic enzymes, as well as proteins involved in the regulation of cellular redox homeostasis. Under basal conditions, Kelch-like ECH associated protein 1 (Keap1) targets Nrf2 for ubiquitination and proteasomal degradation via association with Cullin3 (Cul3)-based Rbx1 E3 ubiquitin ligase. Various small molecules (inducers) activate Nrf2 leading to upregulation of cytoprotective gene expression. Inducers chemically modify specific cysteine residues of Keap1 which ultimately loses its ability to target Nrf2 for degradation. Dissociation of the Keap1–Cul3 complex by inducers is one possible mechanism, but evidence in single live cells is lacking. To investigate the diffusion dynamics of the Keap1–Cul3 interaction and the effect of inducers, we developed a quantitative fluorescence recovery after photobleaching (FRAP)-based system using Keap1–EGFP and mCherry–Cul3 fusion proteins. We show that Keap1–EGFP and mCherry–Cul3 interact in single live cells. Exposure for 1 h to small-molecule inducers of 4 different types, the oleanane triterpenoid CDDO, the isothiocyanate sulforaphane, the sulfoxythiocarbamate STCA, and the oxidant hydrogen peroxide which target distinct cysteine sensors within Keap1 with potencies which differ by nearly 4000-fold, does not dissociate the Keap1–Cul3 complex. As inducers cause conformational changes

  18. Single-Cell, Time-Resolved Antimicrobial Effects of a Highly Cationic, Random Nylon-3 Copolymer on Live Escherichia coli.

    PubMed

    Choi, Heejun; Chakraborty, Saswata; Liu, Runhui; Gellman, Samuel H; Weisshaar, James C

    2016-01-15

    Synthetic random copolymers based on the nylon-3 (β-peptide) backbone show promise as inexpensive antimicrobial agents resistant to proteolysis. We present a time-resolved observational study of the attack of a particular copolymer MM63:CHx37 on single, live Escherichia coli cells. The composition and chain length of MM63:CHx37 (63% cationic subunits, 37% hydrophobic subunits, 35-subunit average length) were optimized to enhance antibacterial activity while minimizing lysis of human red blood cells. For E. coli cells that export GFP to the periplasm, we obtain alternating phase-contrast and green fluorescence images with a time resolution of 12 s over 60 min following initiation of copolymer flow. Within seconds, cells shrink and exhibit the same plasmolysis spaces that occur following abrupt external osmotic upshift. The osmoprotection machinery attempts to replenish cytoplasmic water, but recovery is interrupted by permeabilization of the cytoplasmic membrane (CM) to GFP. Evidently, the highly cationic copolymer and its counterions rapidly translocate across the outer membrane without permeabilizing it to GFP. The CM permeabilization event is spatially localized. Cells whose CM has been permeabilized never recover growth. The minimum inhibitory concentration (MIC) for cells lacking the osmolyte importer ProP is 4-fold smaller than for normal cells, suggesting that osmoprotection is an important survival strategy. In addition, at the time of CM permeabilization, we observe evidence of oxidative stress. The MIC under anaerobic conditions is at least 8-fold larger than under aerobic conditions, further implicating oxidative damage as an important bacteriostatic effect. Once the copolymer reaches the periplasm, multiple growth-halting mechanisms proceed in parallel. PMID:26493221

  19. Optoelectronic tweezers system for single cell manipulation and fluorescence imaging of live immune cells.

    PubMed

    Jeorrett, Abigail H; Neale, Steven L; Massoubre, David; Gu, Erdan; Henderson, Robert K; Millington, Owain; Mathieson, Keith; Dawson, Martin D

    2014-01-27

    A compact optoelectronic tweezers system for combined cell manipulation and analysis is presented. CMOS-controlled gallium nitride micro-LED arrays are used to provide simultaneous spatio-temporal control of dielectrophoresis traps within an optoelectronic tweezers device and fluorescence imaging of contrasting dye labelled cells. This capability provides direct identification, selection and controlled interaction of single T-lymphocytes and dendritic cells. The trap strength and profile for two emission wavelengths of micro-LED array have been measured and a maximum trapping force of 13.1 and 7.6 pN was achieved for projected micro-LED devices emitting at λmax 520 and 450 nm, respectively. A potential application in biological research is demonstrated through the controlled interaction of live immune cells where there is potential for this method of OET to be implemented as a compact device. PMID:24515144

  20. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

    PubMed Central

    Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments. PMID:19096510

  1. A general method to improve fluorophores for live-cell and single-molecule microscopy.

    PubMed

    Grimm, Jonathan B; English, Brian P; Chen, Jiji; Slaughter, Joel P; Zhang, Zhengjian; Revyakin, Andrey; Patel, Ronak; Macklin, John J; Normanno, Davide; Singer, Robert H; Lionnet, Timothée; Lavis, Luke D

    2015-03-01

    Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range. PMID:25599551

  2. Tracking surface glycans on live cancer cells with single molecule sensitivity**

    PubMed Central

    Jiang, Hao; English, Brian P.; Hazan, Rachel B.; Wu, Peng

    2015-01-01

    Using a combination of metabolically labeled glycans, bioorthogonal Cu(I)-catalyzed azide-alkyne cycloaddition and controlled bleaching of fluorescent probes conjugated to azide or alkyne tagged glycans, we achieve a sufficiently low spatial density of dye labeled glycans enabling dynamic single-molecule tracking and super-resolution imaging of N-linked sialic acids and O-linked GalNAc on the membrane of live cells. Analysis of the trajectories of these dye labeled glycans in mammary cancer cells reveal constrained diffusion of both N- and O-linked glycans which we interpret as reflecting the mobility of the glycan rather than caused by transient immobilization due to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging reveals the structure of dynamic membrane nanotubes. PMID:25515330

  3. Visualization of dynamics of single endogenous mRNA labeled in live mouse.

    PubMed

    Park, Hye Yoon; Lim, Hyungsik; Yoon, Young J; Follenzi, Antonia; Nwokafor, Chiso; Lopez-Jones, Melissa; Meng, Xiuhua; Singer, Robert H

    2014-01-24

    The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment. PMID:24458643

  4. Long-Lived, Coherent Acoustic Phonon Oscillations in GaN Single Crystals

    SciTech Connect

    Wu, S.; Geiser, P.; Jun, J.; Karpinski, J.; Park, J.-R.; Sobolewski, R.

    2006-01-31

    We report on coherent acoustic phonon (CAP) oscillations studied in high-quality bulk GaN single crystals with a two-color femtosecond optical pump-probe technique. Using a far-above-the-band gap ultraviolet excitation (~270 nm wavelength) and a near-infrared probe beam (~810 nm wavelength), the long-lived, CAP transients were observed within a 10 ns time-delay window between the pump and probe pulses, with a dispersionless (proportional to the probe-beam wave vector) frequency of ~45 GHz. The measured CAP attenuation corresponded directly to the absorption of the probe light in bulk GaN, indicating that the actual (intrinsic) phonon-wave attenuation in our crystals was significantly smaller than the measured 65.8 cm^-1 value. The velocity of the phonon propagation was equal to the velocity of sound in GaN.

  5. A general method to improve fluorophores for live-cell and single-molecule microscopy

    PubMed Central

    Grimm, Jonathan B.; English, Brian P.; Chen, Jiji; Slaughter, Joel P.; Zhang, Zhengjian; Revyakin, Andrey; Patel, Ronak; Macklin, John J.; Normanno, Davide; Singer, Robert H.; Lionnet, Timothée; Lavis, Luke D.

    2014-01-01

    Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here, we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range. PMID:25599551

  6. Quantitative imaging of single mRNA splice variants in living cells

    NASA Astrophysics Data System (ADS)

    Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

    2014-06-01

    Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

  7. Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity

    PubMed Central

    Martin, Robert M.; Rino, José; Carvalho, Célia; Kirchhausen, Tomas; Carmo-Fonseca, Maria

    2013-01-01

    SUMMARY Removal of introns from pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we have directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that beta-globin introns are transcribed and excised in 20-30 s. We further show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min-1, and that transcription can be rate limiting for splicing. These results have important implications for mechanistic understanding of co-transcriptional splicing regulation in the live-cell context. PMID:24035393

  8. Live cell imaging combined with high-energy single-ion microbeam.

    PubMed

    Guo, Na; Du, Guanghua; Liu, Wenjing; Guo, Jinlong; Wu, Ruqun; Chen, Hao; Wei, Junzhe

    2016-03-01

    DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10(-3) s(-1) and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10(-2) s(-1). PMID:27036791

  9. Live cell imaging combined with high-energy single-ion microbeam

    NASA Astrophysics Data System (ADS)

    Guo, Na; Du, Guanghua; Liu, Wenjing; Guo, Jinlong; Wu, Ruqun; Chen, Hao; Wei, Junzhe

    2016-03-01

    DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics to study early and fast cellular response to DNA damage after high linear energy transfer ion radiation. The HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online. The live cell imaging analysis shows that strand-break repair protein XRCC1 was recruited to the ion hit position within 20 s in the cells and formed bright foci in the cell nucleus. The fast recruitment of XRCC1 at the ion hits reached a maximum at about 200 s post-irradiation and then was followed by a slower release into the nucleoplasm. The measured dual-exponential kinetics of XRCC1 protein are consistent with the proposed consecutive reaction model, and the measurements obtained that the reaction rate constant of the XRCC1 recruitment to DNA strand break is 1.2 × 10-3 s-1 and the reaction rate constant of the XRCC1 release from the break-XRCC1 complex is 1.2 × 10-2 s-1.

  10. Face liveness detection from a single image via diffusion speed model.

    PubMed

    Wonjun Kim; Sungjoo Suh; Jae-Joon Han

    2015-08-01

    Spoofing using photographs or videos is one of the most common methods of attacking face recognition and verification systems. In this paper, we propose a real-time and nonintrusive method based on the diffusion speed of a single image to address this problem. In particular, inspired by the observation that the difference in surface properties between a live face and a fake one is efficiently revealed in the diffusion speed, we exploit antispoofing features by utilizing the total variation flow scheme. More specifically, we propose defining the local patterns of the diffusion speed, the so-called local speed patterns, as our features, which are input into the linear SVM classifier to determine whether the given face is fake or not. One important advantage of the proposed method is that, in contrast to previous approaches, it accurately identifies diverse malicious attacks regardless of the medium of the image, e.g., paper or screen. Moreover, the proposed method does not require any specific user action. Experimental results on various data sets show that the proposed method is effective for face liveness detection as compared with previous approaches proposed in studies in the literature. PMID:25879944

  11. Single-Chromophore-Based Photoswitchable Nanoparticles Enable Dual-Alternating-Color Fluorescence for Unambiguous Live Cell Imaging

    PubMed Central

    Tian, Zhiyuan; Wu, Wuwei; Wan, Wei; Li, Alexander D. Q.

    2009-01-01

    We have developed a class of spiropyran dyes and their fluorescence colors can be reversibly photoswitched from red color to green, blue, or nearly dark, thus alternating between two colors. Such individual dyes emit either one color or the other, but not both simultaneously. These photoswitchable dyes-enabled nanoparticles, however, emit either one pure color or a combination of both colors because the nanoparticle fluorescence originates from multiple dyes therein. As a result, the nanoparticle shines >30 times brighter than the state-of-the-art organic dyes such as fluorescein. Interestingly, these copolymer nanoparticles exhibit tunable non-specific interactions with live cells and nanoparticles containing properly balanced butyl acrylate and acrylamide monomers render essentially very little non-specific binding to live cells. Decorated with HMGA1 protein, these optically switchable dual-color nanoparticles undergo endocytosis and unambiguously identify themselves from fluorescence interference including autofluorescence, thus enabling a new tool for live cell imaging. PMID:19275146

  12. In situ observation of photo-bleaching in human single living cell excited by a NIR femtosecond laser

    NASA Astrophysics Data System (ADS)

    Cho, Sung-Hak; Chang, Won-Seok; Kim, Jae-Goo; Whang, Kyoung-Hyun; Choi, Kyeong-Sook; Sohn, Seong-Hyang

    2008-03-01

    The photo-bleaching of single living cells excited by femtosecond laser irradiation was observed in situ to study the nonlinear interaction between ultrafast laser pulses and living human breast MDA-MB-231 cells. We conducted a systematic study of the energy dependence of plasma-mediated photo-disruption of fluorescently labeled subcellular structures in the nucleus of living cells using near-infrared (NIR) femtosecond laser pulses through a numerical aperture objective lens (0.75 NA). The behavior of photo-bleached living cells with fluorescently labeled nuclei was observed for 18 h after femtosecond laser irradiation under a fluorescence microscope. The photo-bleaching of single living cells without cell disruption occurred at between 470 and 630 nJ. To study the photo-disruption of subcellular organelles in single living cells using the nonlinear absorption excited by a NIR femtosecond laser pulse, the process of photo-bleaching without photo-disruption provides key information for clarifying the nonlinear interaction between NIR ultrashort, high-intensity laser light and transparent fluorescently labeled living cells.

  13. Hemoglobin Aggregation in Single Red Blood Cells of Sickle Cell Anemia

    NASA Astrophysics Data System (ADS)

    Nishio, Izumi; Tanaka, Toyoichi; Sun, Shao-Tang; Imanishi, Yuri; Tsuyoshi Ohnishi, S.

    1983-06-01

    A laser light scattering technique was used to observe the extent of hemoglobin aggregation in solitary red blood cells of sickle cell anemia. Hemoglobin aggregation was confirmed in deoxygenated cells. The light scattering technique can also be applied to cytoplasmic studies of any biological cell.

  14. Spectroscopic study of red-light-emitting centers in K2Al2B2O7: Fe single crystals

    NASA Astrophysics Data System (ADS)

    Ogorodnikov, I. N.; Pustovarov, V. A.; Yakovlev, S. A.; Isaenko, L. I.

    2013-04-01

    We report on spectroscopic study of red-light-emitting centers in K2Al2B2O7 (KABO) single crystals containing ca. 2 ppm of Fe3+. Owing to the low Fe3+-concentration, KABO does not show noticeable absorption due to Fe3+d-d-transitions in the visible spectral region, but it exhibits the charge-transfer (CT) UV-absorption bands O-Fe at 4.7, 5.7 and 6.5 eV. The red photoluminescence at 1.675 eV (FWHM = 0.173 eV) is due to intracenter 4T1 (4G) → 6A1 (6S) transitions in Fe3+ ions. Because of partial overlapping of the fundamental absorption edge of the crystal, where mobile excitons are created, and a broad CT absorption band at 6.5 eV, the most intensive red emission occurs at 7 K upon excitation in the excitonic energy region. The presence of two nonequivalent Al2O7 clusters in KABO lattice provides two different types of red-light-emitting centers in the form of Fe3+ ion occupied the Al3+ tetrahedral site. Superposition of their luminescence bands determines both the spectrum and temperature dependence of red emission in KABO at T = 7-80 K: two bands with the ratio of intensities of ca. 2:1 are 20 meV-shifted relative to each other; two-stage thermal quenching obeys the Mott law with ET = 9 and 20 meV.

  15. In Silico Model-Driven Assessment of the Effects of Single Nucleotide Polymorphisms (SNPs) on Human Red Blood Cell Metabolism

    PubMed Central

    Jamshidi, Neema; Wiback, Sharon J.; Palsson, Bernhard Ø.

    2002-01-01

    The completion of the human genome project and the construction of single nucleotide polymorphism (SNP) maps have lead to significant efforts to find SNPs that can be linked to pathophysiology. In silico models of complete biochemical reaction networks relate a cell's individual reactions to the function of the entire network. Sequence variations can in turn be related to kinetic properties of individual enzymes, thus allowing an in silico model-driven assessment of the effects of defined SNPs on overall cellular functions. This process is applied to defined SNPs in two key enzymes of human red blood cell metabolism: glucose-6-phosphate dehydrogenase and pyruvate kinase. The results demonstrate the utility of in silico models in providing insight into differences between red cell function in patients with chronic and nonchronic anemia. In silico models of complex cellular processes are thus likely to aid in defining and understanding key SNPs in human pathophysiology. PMID:12421755

  16. Developing new optical imaging techniques for single particle and molecule tracking in live cells

    SciTech Connect

    Sun, Wei

    2010-01-01

    Differential interference contrast (DIC) microscopy is a far-field as well as wide-field optical imaging technique. Since it is non-invasive and requires no sample staining, DIC microscopy is suitable for tracking the motion of target molecules in live cells without interfering their functions. In addition, high numerical aperture objectives and condensers can be used in DIC microscopy. The depth of focus of DIC is shallow, which gives DIC much better optical sectioning ability than those of phase contrast and dark field microscopies. In this work, DIC was utilized to study dynamic biological processes including endocytosis and intracellular transport in live cells. The suitability of DIC microscopy for single particle tracking in live cells was first demonstrated by using DIC to monitor the entire endocytosis process of one mesoporous silica nanoparticle (MSN) into a live mammalian cell. By taking advantage of the optical sectioning ability of DIC, we recorded the depth profile of the MSN during the endocytosis process. The shape change around the nanoparticle due to the formation of a vesicle was also captured. DIC microscopy was further modified that the sample can be illuminated and imaged at two wavelengths simultaneously. By using the new technique, noble metal nanoparticles with different shapes and sizes were selectively imaged. Among all the examined metal nanoparticles, gold nanoparticles in rod shapes were found to be especially useful. Due to their anisotropic optical properties, gold nanorods showed as diffraction-limited spots with disproportionate bright and dark parts that are strongly dependent on their orientation in the 3D space. Gold nanorods were developed as orientation nanoprobes and were successfully used to report the self-rotation of gliding microtubules on kinesin coated substrates. Gold nanorods were further used to study the rotational motions of cargoes during the endocytosis and intracellular transport processes in live mammalian

  17. Opto-injection into single living cells by femtosecond near-infrared laser

    NASA Astrophysics Data System (ADS)

    Peng, Cheng

    This dissertation presents a novel technique to deliver membrane impermeable molecules into single living cells with the assistance of femtosecond (fs) near-infrared (NIR) laser pulses. This approach merges ultrafast laser technology with key biological, biomedical, and medical applications, such as gene transfection, gene therapy and drug delivery. This technique promises several major advantages, namely, very high transfection efficiency, high cell survival rate (≈100%) and fully preserved cell viabilities. It is also a promising method to deliver molecules into cells that are difficult or even completely resistant to established physical methods, such as microinjection by glass pipettes, electroporation, and biolistics. In this work, the system for fs NIR opto-injection was designed and built. Successful fs NIR opto-injection has been performed on several cell systems including single mammalian cells (bovine aortic endothelial cells), marine animal eggs (Spisula solidissima oocytes), and human cancer cells (fibrosarcoma HT1080) cultured in a tissue-like environment. The connections between laser parameters and cell responses were explored through further experiments and in-depth analyses, especially the relationship between dye uptake rate and incident laser intensity, and the relationship between pore size created on cell membranes and incident laser intensity. Dye uptake rate of the target cells was observed to depend on incident laser intensity. Pore size was found dependent on incident laser intensity. The conclusion was made that laser-induced breakdown and plasma-induced ablation in cell membrane are the physical principles that govern the process of fs NIR opto-injection.

  18. Revealing nonergodic dynamics in living cells from a single particle trajectory

    NASA Astrophysics Data System (ADS)

    Lanoiselée, Yann; Grebenkov, Denis S.

    2016-05-01

    We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

  19. Live Attenuated B. pertussis as a Single-Dose Nasal Vaccine against Whooping Cough

    PubMed Central

    Mielcarek, Nathalie; Debrie, Anne-Sophie; Raze, Dominique; Bertout, Julie; Rouanet, Carine; Younes, Amena Ben; Creusy, Colette; Engle, Jacquelyn; Goldman, William E; Locht, Camille

    2006-01-01

    Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV) in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth. PMID:16839199

  20. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Ben Arous, Juliette; Binding, Jonas; Léger, Jean-François; Casado, Mariano; Topilko, Piotr; Gigan, Sylvain; Claude Boccara, A.; Bourdieu, Laurent

    2011-11-01

    Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

  1. Effect of Viscoelasticity on the Analysis of Single-Molecule Force Spectroscopy on Live Cells

    PubMed Central

    Gupta, V.K.; Neeves, K.B.; Eggleton, C.D.

    2012-01-01

    Single-molecule force spectroscopy is used to probe the kinetics of receptor-ligand bonds by applying mechanical forces to an intermediate media on which the molecules reside. When this intermediate media is a live cell, the viscoelastic properties can affect the calculation of rate constants. We theoretically investigate the effect of media viscoelasticity on the common assumption that the bond force is equal to the instantaneous applied force. Dynamic force spectroscopy is simulated between two cells of varying micromechanical properties adhered by a single bond with a constant kinetic off-rate. We show that cell and microvilli deformation, and hydrodynamic drag contribute to bond forces that can be 28–90% lower than the applied force for loading rates of 103–107 pN/s, resulting in longer bond lifetimes. These longer bond lifetimes are not caused by changes in bond kinetics; rather, they are due to the mechanical response of the intermediate media on which the bonds reside. Under the assumption that the instantaneous bond force is equal to the applied force—thereby ignoring viscoelasticity—leads to 14–39% error in the determination of off-rates. We present an approach that incorporates viscoelastic properties in calculating the instantaneous bond force and kinetic dissociation parameter of the intermolecular bond. PMID:22828340

  2. Bright, Red Single-Molecule Emitters: Synthesis and Properties of Environmentally Sensitive Dicyanomethylenedihydrofuran (DCDHF) Fluorophores with Bisaromatic Conjugation

    PubMed Central

    Lu, Zhikuan; Liu, Na; Lord, Samuel J.; Bunge, Scott D.; Moerner, W. E.; Twieg, Robert. J.

    2009-01-01

    A group of new fluorescent dye materials for single-molecule imaging applications comprised of an amine donor, a π-system comprised of phenyl and thiophene rings and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran acceptor group have been synthesized. Relative to comparable single-ring compounds these doubly aromatic conjugated fluorophores have red-shifted absorption and emission usually accompanied by significantly increased quantum yields. Solvatochromism studies indicate that the photophysical properties of these dyes are sensitive to the solvent polarity and environmental rigidity. Photophysical studies demonstrate that these DCDHF dye materials are strong single-molecule emitters and the total number of detected photons per molecule is among the highest thus far for this family of fluorophores. PMID:20140061

  3. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

    PubMed Central

    2012-01-01

    Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry. Methods Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay. Results Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled

  4. Origin of the red sites and energy transfer rates in single MEH-PPV chains at low temperature.

    PubMed

    Feist, Florian A; Zickler, Martin F; Basché, Thomas

    2011-06-01

    Single poly[2-methoxy-5-(2'-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) molecules dispersed in thin poly(methylmethacrylate) films have been investigated by fluorescence emission, excitation and time-resolved spectroscopy at 1.2 K. For the molecular weight studied (∼200 kDa) a bimodal distribution of emission maxima is observed. Based on a comparison of the spectroscopic properties of blue and red sites and on polarisation-resolved measurements, we argue in agreement with recent quantum-chemical calculations that the red subpopulation most probably does not arise from interchromophoric excitation delocalisation but is to be attributed to longer chromophoric units originating from ordered regions of a polymer chain, where due to constraints on the chain conformation larger conjugation lengths can be realised. In excitation spectra within the red spectral region we can identify multiple chromophoric units, among them chromophores without correspondence in the emission spectrum-donors of the intramolecular energy transfer. Zero-phonon lines of donor chromophores proved to be significantly broadened, indicating fast excited-state population decay due to energy transfer. Thus, a distribution of energy transfer times within MEH-PPV chains could be determined from donor zero-phonon line widths, with an average value of 3.9 ps. Our study represents the first direct measurement of energy transfer times in conjugated polymers, parameters that are crucial for the performance of many technical applications based on this class of material. PMID:21472962

  5. Resonance Raman study of the oxygenation cycle of optically trapped single red blood cells in a microfluidic system

    NASA Astrophysics Data System (ADS)

    Ramser, Kerstin; Logg, Katarina; Enger, Jonas; Goksor, Mattias; Kall, Mikael; Hanstorp, Dag

    2004-10-01

    The average environmental response of red blood cells (RBCs) is routinely measured in ensemble studies, but in such investigations valuable information on the single cell level is obscured. In order to elucidate this hidden information is is important to enable the selection of single cells with certain properties while subsequent dynamics triggered by environmental stimulation are recorded in real time. It is also desirable to manipulate and control the cells under phsyiological conditions. As shown here, this can be achieved by combining optical tweezers with a confocal Raman set-up equipped with a microfluidic system. A micro-Raman set-up is combined with an optical trap with separate optical paths, lasers and objectives, which enables the acquisition of resonance Raman profils of single RBCs. The microfluidic system, giving full control over the media surrounding the cell, consists of a pattern of channels and reservoirs produced by electron beam lithography and moulded in PDMS. Fresh Hepes buffer or buffer containing sodium dithionite are transported through the channels using electro-osmotic flow, while the direct Raman response of the single optically trapped RBC is registered in another reservoir in the middle of the channel. Thus, it is possible to monitor the oxygenation cycle in a single cell and to study photo-induced chemistry. This experimental set-up has high potential for monitoring the drug response or conformational changes caused by other environmental stimuli for many types of single functional cells since "in vivo" conditions can be created.

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

    PubMed

    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

    2016-01-01

    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. PMID:26879144

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

    PubMed Central

    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

    2016-01-01

    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. PMID:26879144

  8. Imaging of single mRNA molecules moving within a living cell nucleus

    SciTech Connect

    Tadakuma, Hisashi; Ishihama, Yo; Shibuya, Toshiharu; Tani, Tokio; Funatsu, Takashi . E-mail: funatsu@mail.ecc.u-tokyo.ac.jp

    2006-06-09

    In eukaryotic cells, pre-mRNAs are transcribed in the nucleus, processed by 5' capping, 3'-polyadenylation, and splicing, and exported to the cytoplasm for translation. To examine the nuclear mRNA transport mechanism, intron-deficient mRNAs of truncated {beta}-globin and EGFP were synthesized, fluorescently labeled in vitro, and injected into the nucleus of living Xenopus A6 cells. The trajectories of single mRNA molecules in the nucleus were visualized using video-rate confocal microscopy. Approximately half the mRNAs moved by Brownian motion in the nucleoplasm, except the nucleoli, with an apparent diffusion coefficient of 0.2 {mu}m{sup 2}/s, about 1/150 of that in water. The slow diffusion could not be explained by simple diffusion obeying the Stokes-Einstein equation, suggesting interactions of the mRNAs with nuclear components. The remaining mRNAs were stationary with an average residence time of about 30 s, comparable to the time required for mRNA diffusion from the site of synthesis to nuclear pores.

  9. Single Adhesive Nanofibers from a Live Diatom Have the Signature Fingerprint of Modular Proteins

    PubMed Central

    Dugdale, T. M.; Dagastine, R.; Chiovitti, A.; Mulvaney, P.; Wetherbee, R.

    2005-01-01

    The adhesive and mechanical properties of a cell-substratum adhesive secreted by live diatom cells were examined in situ using atomic force microscopy. The resulting force curves have a regular saw-tooth pattern, the characteristic fingerprint of modular proteins, and when bridged between tip and surface can repeatedly be stretched and relaxed resulting in precisely overlaying saw-tooth curves (up to ∼600 successive cycles). The average rupture force of the peaks is 0.794 ± 0.007 (mean ± SE) nN at a loading rate of 0.8 μm/s and the average persistence length is 0.026 ± <0.001 (mean ± SE) nm (fit using the worm-like chain model). We propose that we are pulling on single adhesive nanofibers, each a cohesive unit composed of a set number of modular proteins aligned in register. Furthermore, we can observe and differentiate when up to three adhesive nanofibers are pulled based upon multimodal distributions of force and persistence length. The high force required for bond rupture, high extensibility (∼1.2 μm), and the accurate and rapid refolding upon relaxation, together provide strong and flexible properties ideally suited for the cell-substratum adhesion of this fouling diatom and allow us to understand the mechanism responsible for the strength of adhesion. PMID:16169972

  10. Compact and blinking-suppressed quantum dots for single-particle tracking in live cells.

    PubMed

    Lane, Lucas A; Smith, Andrew M; Lian, Tianquan; Nie, Shuming

    2014-12-11

    Quantum dots (QDs) offer distinct advantages over organic dyes and fluorescent proteins for biological imaging applications because of their brightness, photostability, and tunability. However, a major limitation is that single QDs emit fluorescent light in an intermittent on-and-off fashion called "blinking". Here we report the development of blinking-suppressed, relatively compact QDs that are able to maintain their favorable optical properties in aqueous solution. Specifically, we show that a linearly graded alloy shell can be grown on a small CdSe core via a precisely controlled layer-by-layer process, and that this graded shell leads to a dramatic suppression of QD blinking in both organic solvents and water. A substantial portion (>25%) of the resulting QDs does not blink (more than 99% of the time in the bright or "on" state). Theoretical modeling studies indicate that this type of linearly graded shell not only can minimize charge carrier access to surface traps but also can reduce lattice defects, both of which are believed to be responsible for carrier trapping and QD blinking. Further, we have evaluated the biological utility of blinking-suppressed QDs coated with polyethylene glycol (PEG)-based ligands and multidentate ligands. The results demonstrate that their optical properties are largely independent of surface coatings and solvating media, and that the blinking-suppressed QDs can provide continuous trajectories in live-cell receptor tracking studies. PMID:25157589

  11. Visualization of the movement of single histidine kinase molecules in live Caulobacter cells.

    PubMed

    Deich, J; Judd, E M; McAdams, H H; Moerner, W E

    2004-11-01

    The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 +/- 2 x 10(-3) microm(2)/s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported. PMID:15522969

  12. Compact and Blinking-Suppressed Quantum Dots for Single-Particle Tracking in Live Cells

    PubMed Central

    2015-01-01

    Quantum dots (QDs) offer distinct advantages over organic dyes and fluorescent proteins for biological imaging applications because of their brightness, photostability, and tunability. However, a major limitation is that single QDs emit fluorescent light in an intermittent on-and-off fashion called “blinking”. Here we report the development of blinking-suppressed, relatively compact QDs that are able to maintain their favorable optical properties in aqueous solution. Specifically, we show that a linearly graded alloy shell can be grown on a small CdSe core via a precisely controlled layer-by-layer process, and that this graded shell leads to a dramatic suppression of QD blinking in both organic solvents and water. A substantial portion (>25%) of the resulting QDs does not blink (more than 99% of the time in the bright or “on” state). Theoretical modeling studies indicate that this type of linearly graded shell not only can minimize charge carrier access to surface traps but also can reduce lattice defects, both of which are believed to be responsible for carrier trapping and QD blinking. Further, we have evaluated the biological utility of blinking-suppressed QDs coated with polyethylene glycol (PEG)-based ligands and multidentate ligands. The results demonstrate that their optical properties are largely independent of surface coatings and solvating media, and that the blinking-suppressed QDs can provide continuous trajectories in live-cell receptor tracking studies. PMID:25157589

  13. Placing Single-Molecule T4 Lysozyme Enzymes on a Bacterial Cell Surface: Toward Probing Single-Molecule Enzymatic Reaction in Living Cells

    SciTech Connect

    Hu, Dehong; Lu, H PETER.

    2004-07-01

    TheT4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics had been studied under physiological condition using purified E. Coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted cell wall by using a hydrodynamic micro-injection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than photobleaching time under physiological conditions.

  14. Progress of the Living with a Red Dwarf Program: Activity-Rotation-Age Relationships for M dwarfs and the Ages of Extrasolar Planets

    NASA Astrophysics Data System (ADS)

    Engle, Scott G.; Guinan, Edward Francis; Harper, Graham

    2015-08-01

    Red Dwarfs (M dwarfs or dM stars) make up over 75% of the local stellar population. This is among the reasons they are being targeted by an increasing number of planet-hunting programs. As such, developing a method to accurately estimate the age of a field M dwarf is of critical importance. However, due to their long lifetimes and very slow nuclear evolution, the best method for determining ages is likely through “magnetic tracers” such as X-UV activity levels and stellar rotation rates. The Living with a Red Dwarf program’s database of M dwarfs with photometrically determined rotation periods (via starspot modulations) is becoming substantial. Its expansion to include M dwarfs with well-detached WD companions - through which reliable ages can be determined - has had significant impacts on the reliability of the relations. When combined with M dwarfs possessing cluster/population memberships, or specific kinematics, a full range of “calibrators” is being realized. We report on our continuing efforts to build reliable Activity-Rotation-Age relationships for M dwarfs, utilizing X-UV measures obtained with HST, IUE Chandra and XMM (both proposed by us, and archival). Such relationships permit the assessment of the habitability of planets hosted by red dwarfs, by delineating the X-UV radiation environments these planets are exposed to, and have been exposed to in the past. After proper calibration, the relationships can also permit the age of a field red dwarf (and any hosted planets) to be determined through measures of either the stellar rotation period or X-UV activity level.We gratefully acknowledge the support from NSF/RUI Grant AST 1009903, Chandra Grant GO-13200633, HST Grants GO-12124X and GO-13020X.

  15. Wear Fast, Die Young: More Worn Teeth and Shorter Lives in Iberian Compared to Scottish Red Deer.

    PubMed

    Pérez-Barbería, F J; Carranza, J; Sánchez-Prieto, C

    2015-01-01

    Teeth in Cervidae are permanent structures that are not replaceable or repairable; consequently their rate of wear, due to the grinding effect of food and dental attrition, affects their duration and can determine an animal's lifespan. Tooth wear is also a useful indicator of accumulative life energy investment in intake and mastication and their interactions with diet. Little is known regarding how natural and sexual selection operate on dental structures within a species in contrasting environments and how these relate to life history traits to explain differences in population rates of tooth wear and longevity. We hypothesised that populations under harsh environmental conditions should be selected for more hypsodont teeth while sexual selection may maintain similar sex differences within different populations. We investigated the patterns of tooth wear in males and females of Iberian red deer (Cervus elaphus hispanicus) in Southern Spain and Scottish red deer (C. e. scoticus) across Scotland, that occur in very different environments, using 10343 samples from legal hunting activities. We found higher rates of both incisor and molar wear in the Spanish compared to Scottish populations. However, Scottish red deer had larger incisors at emergence than Iberian red deer, whilst molars emerged at a similar size in both populations and sexes. Iberian and Scottish males had earlier tooth depletion than females, in support of a similar sexual selection process in both populations. However, whilst average lifespan for Iberian males was 4 years shorter than that for Iberian females and Scottish males, Scottish males only showed a reduction of 1 year in average lifespan with respect to Scottish females. More worn molars were associated with larger mandibles in both populations, suggesting that higher intake and/or greater investment in food comminution may have favoured increased body growth, before later loss of tooth efficiency due to severe wear. These results

  16. Wear Fast, Die Young: More Worn Teeth and Shorter Lives in Iberian Compared to Scottish Red Deer

    PubMed Central

    Pérez-Barbería, F. J.; Carranza, J.; Sánchez-Prieto, C.

    2015-01-01

    Teeth in Cervidae are permanent structures that are not replaceable or repairable; consequently their rate of wear, due to the grinding effect of food and dental attrition, affects their duration and can determine an animal's lifespan. Tooth wear is also a useful indicator of accumulative life energy investment in intake and mastication and their interactions with diet. Little is known regarding how natural and sexual selection operate on dental structures within a species in contrasting environments and how these relate to life history traits to explain differences in population rates of tooth wear and longevity. We hypothesised that populations under harsh environmental conditions should be selected for more hypsodont teeth while sexual selection may maintain similar sex differences within different populations. We investigated the patterns of tooth wear in males and females of Iberian red deer (Cervus elaphus hispanicus) in Southern Spain and Scottish red deer (C. e. scoticus) across Scotland, that occur in very different environments, using 10343 samples from legal hunting activities. We found higher rates of both incisor and molar wear in the Spanish compared to Scottish populations. However, Scottish red deer had larger incisors at emergence than Iberian red deer, whilst molars emerged at a similar size in both populations and sexes. Iberian and Scottish males had earlier tooth depletion than females, in support of a similar sexual selection process in both populations. However, whilst average lifespan for Iberian males was 4 years shorter than that for Iberian females and Scottish males, Scottish males only showed a reduction of 1 year in average lifespan with respect to Scottish females. More worn molars were associated with larger mandibles in both populations, suggesting that higher intake and/or greater investment in food comminution may have favoured increased body growth, before later loss of tooth efficiency due to severe wear. These results

  17. A Single-center Experience in Splenic Diffuse Red Pulp Lymphoma Diagnosis.

    PubMed

    Julhakyan, Hunan L; Al-Radi, L S; Moiseeva, T N; Danishyan, K I; Kovrigina, A M; Glebova, S M; Lugovskaya, S A; Dvirnik, V N; Khvastunova, A N; Yakutik, I A; Savchenko, V G

    2016-08-01

    The World Health Organization 2008 classification highlighted a new nosology-splenic diffuse red pulp lymphoma (SDRPL) with clinical and laboratory features similar to both splenic marginal zone lymphoma and hairy cell leukemia (HCL) and variant form of HCL. Experience of hematologists on the diagnosis and differential diagnosis of SDRPL is extremely limited. The aim of our report was to characterize the clinical and immunomorphologic features of SDRPL on our own observations. During 2013-2014, in National Research Center for Hematology, 87 spleen specimens removed from various B-cell lymphomas were analyzed. In four (4.6%) cases, the diagnosis SDRPL was made based on morphologic, immunohistochemical, immunophenotypic, molecular examination of spleen biopsies, blood and bone marrow samples. In all cases of SDRPL were observed significant splenomegaly, lymphocytosis from 56% to 94% (in two cases with leukocytosis 55.000 and 75.000 109/l). The circulating "villous" lymphocytes phenotype was CD20+ (bright), CD11c+/±, CD103 (weakly)+/±, LAIR-1+, CD25-, CD5-, CD10-, and CD23-. Mutation BRAFV600E was not detected. Bone marrow with minor lymphoid CD20+, CD25-, Annexin1-, Cyclin D1- cell infiltration. The average weight of the spleen was 3900 g (1450-9500 g), and morphologically, there was revealed lymphoid infiltration of red pulp with phenotype CD20+, DBA.44+, CD25-, Annexin1-, Cyclin D1-, CD103-, CD123-, CD27-, focal SD11c± and TRAP±. Now patients are observed in remission: two patients after splenectomy, two after splenectomy and cladribine+rituximab chemotherapy. SRDPL-a rare lymphoma that is suspected in the cases with significant splenomegaly and lymphocytosis with villous lymphocytes forms that have only a part of the classic markers HCL, with minor bone marrow infiltration. The standard diagnosis and treatment is splenectomy. Differential diagnosis of SMZL and HCL has clear criteria, but criteria of differentiation with variant HCL are still unknown. PMID

  18. Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

    PubMed Central

    Hayot, Céline M.; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A.

    2012-01-01

    Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

  19. Aquaporin-4 Dynamics in Orthogonal Arrays in Live Cells Visualized by Quantum Dot Single Particle Tracking

    PubMed Central

    Crane, Jonathan M.; Van Hoek, Alfred N.; Skach, William R.

    2008-01-01

    Freeze-fracture electron microscopy (FFEM) indicates that aquaporin-4 (AQP4) water channels can assemble in cell plasma membranes in orthogonal arrays of particles (OAPs). We investigated the determinants and dynamics of AQP4 assembly in OAPs by tracking single AQP4 molecules labeled with quantum dots at an engineered external epitope. In several transfected cell types, including primary astrocyte cultures, the long N-terminal “M1” form of AQP4 diffused freely, with diffusion coefficient ∼5 × 10−10 cm2/s, covering ∼5 μm in 5 min. The short N-terminal “M23” form of AQP4, which by FFEM was found to form OAPs, was relatively immobile, moving only ∼0.4 μm in 5 min. Actin modulation by latrunculin or jasplakinolide did not affect AQP4-M23 diffusion, but deletion of its C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding domain increased its range by approximately twofold over minutes. Biophysical analysis of short-range AQP4-M23 diffusion within OAPs indicated a spring-like potential, with a restoring force of ∼6.5 pN/μm. These and additional experiments indicated that 1) AQP4-M1 and AQP4-M23 isoforms do not coassociate in OAPs; 2) OAPs can be imaged directly by total internal reflection fluorescence microscopy; and 3) OAPs are relatively fixed, noninterconvertible assemblies that do not require cytoskeletal or PDZ-mediated interactions for formation. Our measurements are the first to visualize OAPs in live cells. PMID:18495865

  20. Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Müller, Martin C.; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic

  1. Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation.

    PubMed

    Hayot, Céline M; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A

    2012-04-01

    Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

  2. Intracellular Calcium Gradients in Single Living Cells: Measurement and Analysis by Optical and Digital Techniques

    NASA Astrophysics Data System (ADS)

    Yelamarty, Rao Viswanadha

    Intracellular calcium (Ca^{2+ }) has been considered as a regulator of many cellular processes. In addition, Ca^{2+ } also plays a key role in mediating actions of many hormones, growth factors, and drugs. This thesis describes two general approaches, digital video and photomultiplier (PMT) based fluorescence microscopic systems, to measure such Ca^{2+} changes throughout the cell. They reveal the heterogeneous spatial and fast temporal changes of Ca^{2+} within a single isolated living cell. In order to measure spatial Ca^ {2+} in three dimensions (3-D), optical section microscopy (OSM) coupled to digital video imaging is introduced. With this approach, an increase in nuclear Ca^{2+} compared to cytosolic Ca^{2+} is detected in human erythroblasts and rat hepatocytes under the addition of growth factors: erythropoietin and epidermal growth factor respectively. In addition, the primary effect of non growth-promoting hormone vasopressin, raise in cytosolic Ca^{2+}, is also observed. These observations are the first to underscore the importance of nuclear Ca^{2+} increase in cell growth and differentiation. On the other hand, to track fast Ca^ {2+} transients (mesc) during excitation -contraction (EC) cycle and then examine alterations in Ca^{2+} transients in healthy and diseased (hypertensive) heart cells, a PMT based system is implemented. Significant alterations in Ca^{2+} transients in hypertensive heart cells were observed. This finding is compatible with the clinical finding that patients with hypertensive cardiomyopathy suffer a lack of adequate relaxation. Finally, to correlate the Ca^{2+} dynamics in an EC cycle with mechanical activity, a hybrid optical digital processor was developed. The performance of the hybrid processor is analyzed and applied simultaneously with the PMT based system. The mechanical contraction and relaxation of a single cardiac cell closely paralleled that of Ca^{2+} dynamics during an EC cycle. In summary, this thesis illustrates

  3. Living with a Red Dwarf: Rotation and X-Ray and Ultraviolet Properties of the Halo Population Kapteyn’s Star

    NASA Astrophysics Data System (ADS)

    Guinan, Edward F.; Engle, Scott G.; Durbin, Allyn

    2016-04-01

    As part of Villanova’s Living with a Red Dwarf program, we have obtained UV, X-ray, and optical data of the Population II red dwarf—Kapteyn’s Star. Kapteyn’s Star is noteworthy for its large proper motions and high radial velocity of ∼+245 km s‑1. As the nearest Pop II red dwarf, it serves as an old age anchor for calibrating activity/irradiance–rotation–age relations, and an important test bed for stellar dynamos and the resulting X-ray–UV emissions of slowly rotating, near-fully convective red dwarf stars. Adding to the notoriety, Kapteyn’s Star has recently been reported to host two super-Earth candidates, one of which (Kapteyn b) is orbiting within the habitable zone. However, Robertson et al. questioned the planet’s existence since its orbital period may be an artifact of activity, related to the star’s rotation period. Because of its large Doppler-shift, measures of the important, chromospheric H i Lyα 1215.67 Å emission line can be reliably made, because it is mostly displaced from ISM and geo-coronal sources. Lyα emission dominates the FUV region of cool stars. Our measures can help determine the X-ray–UV effects on planets hosted by Kapteyn’s Star, and planets hosted by other old red dwarfs. Stellar X-ray and Lyα emissions have strong influences on the heating and ionization of upper planetary atmospheres and can (with stellar winds and flares) erode or even eliminate planetary atmospheres. Using our program stars, we have reconstructed the past exposures of Kapteyn’s Star's planets to coronal—chromospheric XUV emissions over time. Based on observations made with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS 5-26555. These observations are associated with program #13020. This work is also based on observations obtained with the Chandra X-ray Observatory, a NASA science

  4. Light emission ranging from blue to red from a series of InGaN/GaN single quantum wells

    NASA Astrophysics Data System (ADS)

    Martin, R. W.; Edwards, P. R.; Pecharroman-Gallego, R.; Liu, C.; Deatcher, C. J.; Watson, I. M.; O'Donnell, K. P.

    2002-04-01

    In this paper, we describe the growth and characterization of InGaN single quantum wells with emission peaks in the blue, green, amber and red spectral regions, grown by metal-organic vapour phase epitaxy. Starting from the growth of a blue-emitting (peak ~430 nm) InGaN quantum well at 860°C the InGaN growth temperature was progressively reduced. The photoluminescence peak wavelength, measured at low temperature, shifts through the green and orange spectral regions and reaches 670 nm for an InGaN growth temperature of 760°C. This corresponds to an energy lower than the currently accepted band-gap of the binary compound, InN. Spectral characteristics of the luminescence peaks will be discussed, including an analysis of the phonon-assisted contribution. Low energy secondary ion mass spectrometry analysis provides information on the indium content and thickness of the `blue' and `red' quantum wells. The results are combined to discuss the origin of the `sub-band-gap' luminescence in terms of the combined influence of InN-GaN segregation and the effect of intense piezoelectric fields.

  5. Dynamics of a single red blood cell in simple shear flow.

    PubMed

    Sinha, Kushal; Graham, Michael D

    2015-10-01

    This work describes simulations of a red blood cell (RBC) in simple shear flow, focusing on the dependence of the cell dynamics on the spontaneous curvature of the membrane. The results show that an oblate spheroidal spontaneous curvature maintains the dimple of the RBC during tank-treading dynamics as well as exhibits off-shear-plane tumbling consistent with the experimental observations of Dupire et al. [J. Dupire, M. Socol, and A. Viallat, Proc. Natl. Acad. Sci. USA 109, 20808 (2012)] and their hypothesis of an inhomogeneous spontaneous shape. As the flow strength (capillary number Ca) is increased at a particular viscosity ratio between inner and outer fluid, the dynamics undergo transitions in the following sequence: tumbling, kayaking or rolling, tilted tank-treading, oscillating-swinging, swinging, and tank-treading. The tilted tank-treading (or spinning frisbee) regime has been previously observed in experiments but not in simulations. Two distinct classes of regime are identified: a membrane reorientation regime, where the part of membrane that is at the dimple at rest moves to the rim and vice versa, is observed in motions at high Ca such as tilted tank-treading, oscillating-swinging, swinging, and tank-treading, and a nonreorientation regime, where the part of the membrane starting from the dimple stays at the dimple, is observed in motions at low Ca such as rolling, tumbling, kayaking, and flip-flopping. PMID:26565275

  6. Dynamics of a single red blood cell in simple shear flow

    NASA Astrophysics Data System (ADS)

    Sinha, Kushal; Graham, Michael D.

    2015-10-01

    This work describes simulations of a red blood cell (RBC) in simple shear flow, focusing on the dependence of the cell dynamics on the spontaneous curvature of the membrane. The results show that an oblate spheroidal spontaneous curvature maintains the dimple of the RBC during tank-treading dynamics as well as exhibits off-shear-plane tumbling consistent with the experimental observations of Dupire et al. [J. Dupire, M. Socol, and A. Viallat, Proc. Natl. Acad. Sci. USA 109, 20808 (2012), 10.1073/pnas.1210236109] and their hypothesis of an inhomogeneous spontaneous shape. As the flow strength (capillary number Ca ) is increased at a particular viscosity ratio between inner and outer fluid, the dynamics undergo transitions in the following sequence: tumbling, kayaking or rolling, tilted tank-treading, oscillating-swinging, swinging, and tank-treading. The tilted tank-treading (or spinning frisbee) regime has been previously observed in experiments but not in simulations. Two distinct classes of regime are identified: a membrane reorientation regime, where the part of membrane that is at the dimple at rest moves to the rim and vice versa, is observed in motions at high Ca such as tilted tank-treading, oscillating-swinging, swinging, and tank-treading, and a nonreorientation regime, where the part of the membrane starting from the dimple stays at the dimple, is observed in motions at low Ca such as rolling, tumbling, kayaking, and flip-flopping.

  7. Voriconazole Disposition After Single and Multiple, Oral Doses in Healthy, Adult Red-tailed Hawks ( Buteo jamaicensis ).

    PubMed

    Gentry, Jordan; Montgerard, Christy; Crandall, Elizabeth; Cruz-Espindola, Crisanta; Boothe, Dawn; Bellah, Jamie

    2014-09-01

    Voriconazole is effective for treatment of aspergillosis, a common disease in captive red-tailed hawks ( Buteo jamaicensis ). To determine the disposition and safety of voriconazole after single and multiple, oral doses, 12 adult red-tailed hawks were studied in 2 phases. In phase 1, each bird received a single dose of voriconazole solution (10 mg/kg) by gavage. Blood samples were collected at 0, 0.5, 1, 3, 6, 9, 12, 16, 24, and 36 hours after treatment. In phase 2, each of 8 birds received voriconazole oral solution at 10 mg/kg PO q12h for 14 days. Plasma samples were collected on days 0, 5, and 10 and after the final dose and were processed as in phase 1. Plasma samples were submitted for analysis of voriconazole levels by high-performance liquid chromatography and ultraviolet spectrophotometry and for measurement of selected plasma biochemical parameters. After single dosing, voriconazole concentrations reached a (mean ± SD) peak (Cmax) of 4.7 ± 1.3 μg/mL at 2.0 ± 1.2 hours. The disappearance half-life (t1/2) was 2.8 ± 0.7 hours, and the mean residence time (MRT) was 4.6 ± 0.9 hours. After the last dose at 14 days, the mean Cmax of voriconazole was 4.5 ± 2.7 μg/mL at 2.4 ± 1.1 hours. The t1/2 was 2.1 ± 0.8 hours, and the MRT was 3.5 ± 1.1 hours. Although concentrations of several plasma biochemical parameters were significantly different at study end compared with prestudy concentrations, only plasma creatine kinase activity was outside the reference range. No adverse reactions were observed in any of the birds. After both single and multiple dosing at 10 mg/kg, voriconazole concentrations exceeded the minimum inhibitory concentration to inhibit 90% (MIC90) of Aspergillus species (1 μg/mL) by at least fourfold and remained above the MIC90 for 8.8 ± 1.1 hours after single dosing versus 6.5 ± 1.5 hours after multiple dosing (P = .003). This difference suggests that more frequent dosing (eg, up to q8h) may be necessary to maintain target

  8. Raman spectroscopic monitoring of the bioeffects of nitroglycerin on Hb-O II in single red blood cell

    NASA Astrophysics Data System (ADS)

    Chiang, Huihua Kenny; Ruan, Hung-Shiang; Cheng, Hung-You; Fang, Tung-Ting

    2007-02-01

    Raman spectroscopy has been shown to have the potential for providing oxygenated ability of erythrocytes. Raman line at 1638 cm-1 has also been reported as one significant oxygenic indicator for erythrocytes. In this research, we develop the Raman spectroscopic monitoring of the bioeffects of Nitroglycerin on hemoglobin oxygen saturation in a single red blood cell (RBC). Nitroglycerin has been frequently used in the management of angina pectoris. Nitroglycerin liberates nitric oxide (NO) to blood vessels. NO is an oxidizer that easily converts hemoglobin to methemoglobin. The conversion may cause the decrease of oxygenated ability of erythrocytes. In this study, we observed the oxidize state of erythrocytes caused by the over dosage of Nitroglycerin. When the dose of Nitroglycerin exceeds 2x10 -4 M, the oxygenic state of erythrocytes decreases significantly. The Raman spectroscopic results demonstrate the observation of the bioeffects of Nitroglycerin on hemoglobin.

  9. Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY.

    PubMed

    Oldemeyer, Sabine; Franz, Sophie; Wenzel, Sandra; Essen, Lars-Oliver; Mittag, Maria; Kottke, Tilman

    2016-07-01

    Cryptochromes constitute a group of flavin-binding blue light receptors in bacteria, fungi, plants, and insects. Recently, the response of cryptochromes to light was extended to nearly the entire visible spectral region on the basis of the activity of the animal-like cryptochrome aCRY in the green alga Chlamydomonas reinhardtii This finding was explained by the absorption of red light by the flavin neutral radical as the dark state of the receptor, which then forms the anionic fully reduced state. In this study, time-resolved UV-visible spectroscopy on the full-length aCRY revealed an unusually long-lived tyrosyl radical with a lifetime of 2.6 s, which is present already 1 μs after red light illumination of the flavin radical. Mutational studies disclosed the tyrosine 373 close to the surface to form the long-lived radical and to be essential for photoreduction. This residue is conserved exclusively in the sequences of other putative aCRY proteins distinguishing them from conventional (6-4) photolyases. Size exclusion chromatography showed the full-length aCRY to be a dimer in the dark at 0.5 mm injected concentration with the C-terminal extension as the dimerization site. Upon illumination, partial oligomerization was observed via disulfide bridge formation at cysteine 482 in close proximity to tyrosine 373. The lack of any light response in the C-terminal extension as evidenced by FTIR spectroscopy differentiates aCRY from plant and Drosophila cryptochromes. These findings imply that aCRY might have evolved a different signaling mechanism via a light-triggered redox cascade culminating in photooxidation of a yet unknown substrate or binding partner. PMID:27189948

  10. Single living cell detection of telomerase over-expression for cancer detection by an optical fiber nanobiosensor.

    PubMed

    Zheng, Xin Ting; Li, Chang Ming

    2010-02-15

    An optical fiber nanobiosensor was constructed to successfully detect a general cancer biomarker, telomerase at single cell level with its nanoscale tip. The nanotip immobilized with a specific antibody was inserted into a MCF-7 breast cancer cell nucleus to capture telomerases directly, after which an in vitro enzymatic sandwich immunoassay was performed to achieve sensitive single living cell detection. The nanotip inserted into MCF-7 cell nucleus provides significantly higher average (F-F(0))/F(0) ratio than that of human mesenchymal stem cell (hMSC) nucleus, demonstrating the successful detection of the telomerase over-expression in cancer cells as compared to normal cells. The detection in the cytoplasm shows much smaller average ratio than that in the nucleus of MCF-7 cells while clearly verifies the nuclear localization of telomerase. The successful detection of telomerase over-expression in a single living cell for the first time may provide a potential method for cancer detection, and also demonstrate a universal approach that can be used to detect other low expression proteins in a single living cell. PMID:19963365

  11. Measurement of ethanol formation in single living cells of Chlamydomonas reinhardtii using synchrotron Fourier Transform Infrared spectromicroscopy

    NASA Astrophysics Data System (ADS)

    Goff, Kira L.; Quaroni, Luca; Pedersen, Tor; Wilson, Kenneth E.

    2010-02-01

    We demonstrate the capability of Fourier-Transform Infra-Red (FITR) spectroscopy to detect metabolite formation by the unicellular algae Chlamydomonas reinhardtii in solution. We show that using a synchrotron source in the microscopy configuration provides a sufficient s/n ratio to detect small molecular species accumulating at a single cell, allowing an increased sensitivity relative to measurements of bulk cultures. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

  12. Measurement of ethanol formation in single living cells of Chlamydomonas reinhardtii using synchrotron Fourier Transform Infrared spectromicroscopy

    SciTech Connect

    Goff, Kira L.; Quaroni, Luca; Pedersen, Tor; Wilson, Kenneth E.

    2010-02-03

    We demonstrate the capability of Fourier-Transform Infra-Red (FITR) spectroscopy to detect metabolite formation by the unicellular algae Chlamydomonas reinhardtii in solution. We show that using a synchrotron source in the microscopy configuration provides a sufficient s/n ratio to detect small molecular species accumulating at a single cell, allowing an increased sensitivity relative to measurements of bulk cultures. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

  13. Understanding subcellular function on the nanometer scale in real time: Single-molecule imaging in living bacteria

    NASA Astrophysics Data System (ADS)

    Biteen, Julie

    It has long been recognized that microorganisms play a central role in our lives. By beating the diffraction limit that restricts traditional light microscopy, single-molecule fluorescence imaging is a precise, noninvasive way to sensitively probe position and dynamics, even in living cells. We are pioneering this super-resolution imaging method for unraveling important biological processes in live bacteria, and I will discuss how we infer function from subcellular dynamics (Tuson and Biteen, Analytical Chemistry 2015). In particular, we have understood the mechanism of membrane-bound transcription regulation in the pathogenic Vibrio cholerae, revealed an intimate and dynamic coupling between DNA mismatch recognition and DNA replication, and measured starch utilization in an important member of the human gut microbiome.

  14. Alterations in Red Blood Cells and Plasma Properties after Acute Single Bout of Exercise

    PubMed Central

    Gwozdzinski, Krzysztof; Pieniazek, Anna; Brzeszczynska, Joanna; Jegier, Anna

    2013-01-01

    The aim of this study was to investigate alterations in haemoglobin conformation and parameters related to oxidative stress in whole erythrocytes, membranes, and plasma after a single bout of exercise in a group of young untrained men. Venous blood samples from eleven healthy young untrained males (age = 22 ± 2 years, BMI = 23 ± 2.5 kg/m2) were taken from the antecubital vein before an incremental cycling exercise test, immediately after exercise, and 1 hour after exercise. Individual heart rate response to this exercise was 195 ± 12 beats/min and the maximum wattage was 292 ± 27 W. Immediately after exercise, significant increase in standard parameters (haemoglobin, haematocrit, lactate levels, and plasma volume) of blood was observed as well as plasma antioxidant capacity one hour after exercise. Reversible conformational changes in haemoglobin, measured using a maleimide spin label, were found immediately following exercise. The concentration of ascorbic acid inside erythrocytes significantly decreased after exercise. A significant decline in membrane thiols was observed one hour after exercise, but simultaneously an increase in plasma thiols immediately after and 1 h after exercise was also observed. This study shows that a single bout of exercise can lead to mobilization of defensive antioxidant systems in blood against oxidative stress in young untrained men. PMID:24453803

  15. The impact of reproduction on the stress axis of free-living male northern red backed voles (Myodes rutilus).

    PubMed

    Fletcher, Quinn E; Dantzer, Ben; Boonstra, Rudy

    2015-12-01

    Activation of the hypothalamic-pituitary-adrenal (HPA) axis culminates in the release of glucocorticoids (henceforth CORT), which have wide-reaching physiological effects. Three hypotheses potentially explain seasonal variation in CORT. The enabling hypothesis predicts that reproductive season CORT exceeds post-reproductive season CORT because CORT enables reproductive investment. The inhibitory hypothesis predicts the opposite because CORT can negatively affect reproductive function. The costs of reproduction hypothesis predicts that HPA axis condition declines over and following the reproductive season. We tested these hypotheses in wild male red-backed voles (Myodes rutilus) during the reproductive and post-reproductive seasons. We quantified CORT levels in response to restraint stress tests consisting of three blood samples (initial, stress-induced, and recovery). Mineralocorticoid (MR) and glucocorticoid (GR) receptor mRNA levels in the brain were also quantified over the reproductive season. Total CORT (tCORT) in the initial and stress-induced samples were greater in the post-reproductive than in the reproductive season, which supported the inhibitory hypothesis. Conversely, free CORT (fCORT) did not differ between the reproductive and post-reproductive seasons, which was counter to both the enabling and inhibitory hypotheses. Evidence for HPA axis condition decline in CORT as well as GR and MR mRNA over the reproductive season (i.e. costs of reproduction hypothesis) was mixed. Moreover, all of the parameters that showed signs of declining condition over the reproductive season did not also show signs of declining condition over the post-reproductive season suggesting that the costs resulting from reproductive investment had subsided. In conclusion, our results suggest that different aspects of the HPA axis respond differently to seasonal changes and reproductive investment. PMID:26188715

  16. Transverse mechanical properties of cell walls of single living plant cells probed by laser-generated acoustic waves.

    PubMed

    Gadalla, Atef; Dehoux, Thomas; Audoin, Bertrand

    2014-05-01

    Probing the mechanical properties of plant cell wall is crucial to understand tissue dynamics. However, the exact symmetry of the mechanical properties of this anisotropic fiber-reinforced composite remains uncertain. For this reason, biologically relevant measurements of the stiffness coefficients on individual living cells are a challenge. For this purpose, we have developed the single-cell optoacoustic nanoprobe (SCOPE) technique, which uses laser-generated acoustic waves to probe the stiffness, thickness and viscosity of live single-cell subcompartments. This all-optical technique offers a sub-micrometer lateral resolution, nanometer in-depth resolution, and allows the non-contact measurement of the mechanical properties of live turgid tissues without any assumption of mechanical symmetry. SCOPE experiments reveal that single-cell wall transverse stiffness in the direction perpendicular to the epidermis layer of onion cells is close to that of cellulose. This observation demonstrates that cellulose microfibrils are the main load-bearing structure in this direction, and suggests strong bonding of microfibrils by hemicelluloses. Altogether our measurement of the viscosity at high frequencies suggests that the rheology of the wall is dominated by glass-like dynamics. From a comparison with literature, we attribute this behavior to the influence of the pectin matrix. SCOPE's ability to unravel cell rheology and cell anisotropy defines a new class of experiments to enlighten cell nano-mechanics. PMID:24615232

  17. Phenotypic and Genotypic Comparison of Symbiotic and Free-Living Cyanobacteria from a Single Field Site

    PubMed Central

    West, N. J.; Adams, D. G.

    1997-01-01

    PCR amplification techniques were used to compare cyanobacterial symbionts from a cyanobacterium-bryophyte symbiosis and free-living cyanobacteria from the same field site. Thirty-one symbiotic cyanobacteria were isolated from the hornwort Phaeoceros sp. at several closely spaced locations, and 40 free-living cyanobacteria were isolated from the immediate vicinity of the same plants. One of the symbiotic isolates was a species of Calothrix, a genus not previously known to form bryophyte symbioses, and the remainder were Nostoc spp. Of the free-living strains, two were Calothrix spp., three were Chlorogloeopsis spp. and the rest were Nostoc spp. All of the symbiotic and all but one of the free-living strains were able to reconstitute the symbiosis with axenic cultures of both Phaeoceros and the liverwort Blasia sp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the regions flanking the 16S-23S rRNA internal transcribed spacer. With one exception, the two techniques produced complementary results and confirmed for the first time that a diversity of symbiotic cyanobacteria infect Phaeoceros in the field. Symbionts from adjacent colonies were different as often as they were the same, showing that the same thallus could be infected with many different cyanobacterial strains. Strains found to be identical by the techniques employed here were often found as symbionts in different thalli at the same locale but were never found free-living. Only one of the free-living strains, and none of the symbiotic strains, was found at more than one sample site, implying a highly localized distribution of strains. PMID:16535734

  18. Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

    2014-08-01

    Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

  19. Novel method of laparoendoscopic single-site and natural orifice specimen extraction for live donor nephrectomy: single-port laparoscopic donor nephrectomy and transvaginal graft extraction

    PubMed Central

    Jeong, Won Jun; Choi, Byung Jo; Hwang, Jeong Kye; Yuk, Seung Mo; Song, Min Jong

    2016-01-01

    Laparoscopic live donor nephrectomy (DN) has been established as a useful alternative to the traditional open methods of procuring kidneys. To maximize the advantages of the laparoendoscopic single-site (LESS) method, we applied natural orifice specimen extraction to LESS-DN. A 46-year-old woman with no previous abdominal surgery history volunteered to donate her left kidney to her husband and underwent single-port laparoscopic DN with transvaginal extraction. The procedure was completed without intraoperative complications. The kidney functioned well immediately after transplantation, and the donor and recipient were respectively discharged 2 days and 2 weeks postoperatively. Single-port laparoscopic DN and transvaginal graft extraction is feasible and safe. PMID:26878020

  20. The American Red Cross disaster mental health services: development of a cooperative, single function, multidisciplinary service model.

    PubMed

    Weaver, J D; Dingman, R L; Morgan, J; Hong, B A; North, C S

    2000-08-01

    Not until 1989 did the Red Cross officially recognize a need for a systematic and organized plan for the mental health needs of disaster survivors. Over the next decade, the Red Cross Disaster Mental Health Services program has developed and evolved to assist both disaster victims and the Red Cross workers who serve them to cope with the overwhelming stresses encountered by both groups in the aftermath of disasters. The Red Cross now coordinates a large and diverse group of mental health professionals from fields of psychology, psychiatry, nursing, social work, marriage and family therapy, and counseling who work together cooperatively. Cross-disciplinary conflicts are minimized by the Red Cross' generic approach to the various mental health professional specialties as functionally interchangeable in performing Red Cross duties. This article reviews the development of this process and describes one local Red Cross chapter's early experience as part of this effort. PMID:10932444

  1. Imaging single cells in a beam of live cyanobacteria with an X-ray laser

    DOE Data Explorer

    Schot, Gijs, vander

    2015-02-10

    Diffraction pattern of a micron-sized S. elongatus cell at 1,100 eV photon energy (1.13 nm wavelength) with ~10^11 photons per square micron on the sample in ~70 fs. The signal to noise ratio at 4 nm resolution is 3.7 with 0.24 photons per Nyquist pixel. The cell was alive at the time of the exposure. The central region of the pattern (dark red) is saturated and this prevented reliable image reconstruction.

  2. Biological aspects of the development and self-concept in adolescents living in single-parent families.

    PubMed

    Vecek, Andrea; Vidović, Vesna; Milicić, Jasna; Spoljar-Vrzina, Sanja; Vecek, Nenad; Arch-Vecek, Branka

    2009-09-01

    In this study we investigate whether there are differences between adolescents who grow up in single-parent families and those who grow up in nucleus families. We have decided that there are no differences in the physical development between the adolescents who are growing up in single parent families and those growing up in nucleus families. There is no difference in the self-concept between these two groups, except in the ethical and moral self-image of adolescents living with one parent. Adolescents living in single-parent families have a weaker moral self-image. It can thus be concluded that physical development and positive self-concept (a favorable image of oneself) in adolescents do not depend on whether an adolescent is growing up in a single-parent or a nucleus family, but on the different characteristics of parents and their relationship with children, whether they are married or not. For the children development the best is healthy marriage of their parents. PMID:19860118

  3. Single live cell topography and activity imaging with the shear-force-based constant-distance scanning electrochemical microscope.

    PubMed

    Schulte, Albert; Nebel, Michaela; Schuhmann, Wolfgang

    2012-01-01

    In recent years, scanning electrochemical microscopy (SECM) has become an important tool in topography and activity studies on single live cells. The used analytical probes ("SECM tips") are voltammetric micro- or nanoelectrodes. The tips may be tracked across a live cell in constant-height or constant-distance mode, while kept at potentials that enable tracing of the spatiotemporal dynamics of functional chemical species in the immediate environment. Depending on the type of single live cells studied, cellular processes addressable by SECM range from the membrane transport of metabolites to the stimulated release of hormones and neurotransmitters and processes such as cell respiration or cell death and differentiation. In this chapter, we provide the key practical details of the constant-distance mode of SECM, explaining the establishment, and operation of the tailored distance control unit that maintains a stable tip-to-cell separation during scanning. The continuously maintained tip positioning of the system takes advantage of the decreasing impact of very short-range hydrodynamic tip-to-surface shear-forces on the vibrational amplitude of an oscillating SECM tip, as the input for a computer-controlled feedback loop regulation. Suitable microelectrode probes that are nondestructive to soft cells are a prerequisite for the success of this methodology and their fabrication and successful application are the other topics covered. PMID:22264538

  4. Real-Time Imaging of Translation on Single mRNA Transcripts in Live Cells.

    PubMed

    Wang, Chong; Han, Boran; Zhou, Ruobo; Zhuang, Xiaowei

    2016-05-01

    Translation is under tight spatial and temporal controls to ensure protein production in the right time and place in cells. Methods that allow real-time, high-resolution visualization of translation in live cells are essential for understanding the spatiotemporal dynamics of translation regulation. Based on multivalent fluorescence amplification of the nascent polypeptide signal, we develop a method to image translation on individual mRNA molecules in real time in live cells, allowing direct visualization of translation events at the translation sites. Using this approach, we monitor transient changes of translation dynamics in responses to environmental stresses, capture distinct mobilities of individual polysomes in different subcellular compartments, and detect 3' UTR-dependent local translation and active transport of polysomes in dendrites of primary neurons. PMID:27153499

  5. Imaging single cells in a beam of live cyanobacteria with an X-ray laser

    DOE Data Explorer

    Schot, Gijs, vander

    2015-02-10

    This entry contains ten diffraction patterns, and reconstructions images, of individual living Cyanobium gracile cells, imaged using 517 eV X-rays from the LCLS XFEL. The Hawk software package was used for phasing. The Uppsala aerosol injector was used for sample injection, assuring very low noise levels. The cells come from various stages of the cell cycle, and were imaged in random orientations.

  6. Post-Operative Complications in Living Liver Donors: A Single-Center Experience in China

    PubMed Central

    Yu, Songfeng; Chen, Jihao; Wang, Jingqiao; Yang, Cheng; Jin, Mengmeng; Yan, Sheng; Zhang, Mangli; Zhang, Min; Zheng, Shusen

    2015-01-01

    The gap between the growing demand for available organs and the cadaveric organs facilitates the adoption of living donor liver transplantation. We retrospectively identified and evaluated the post-operative complications as per the modified Clavien classification system in 152 living liver donors at at the First Affiliated Hospital, College of Medicine, Zhejiang University between December, 2006 and June, 2014. Post-operative complications were observed in 61 patients (40.1%) in the present study, but no mortality was reported. Complications developed in 58 (40.0%) right, 1 (33.3%) left, and 2 (66.7%) lateral left hepatectomy donors. The prevalence of re-operation was 1.3%. Grade I and II complications were observed in 38 (25.0%) and 11 (7.2%) donors, respectively. Grade IIIa complications developed in 9 (5.9%) donors and only 3 (2.0%) patients reported grade IIIb complications. The most common complication was pleural effusion that occurred in 31 (20.4%) donors. No significant prognostic baseline factor was identified in this study. In conclusion, living donors experienced various complications, which were usually mild and had a good prognosis. PMID:26270475

  7. Imaging single cells in a beam of live cyanobacteria with an X-ray laser.

    PubMed

    van der Schot, Gijs; Svenda, Martin; Maia, Filipe R N C; Hantke, Max; DePonte, Daniel P; Seibert, M Marvin; Aquila, Andrew; Schulz, Joachim; Kirian, Richard; Liang, Mengning; Stellato, Francesco; Iwan, Bianca; Andreasson, Jakob; Timneanu, Nicusor; Westphal, Daniel; Almeida, F Nunes; Odic, Dusko; Hasse, Dirk; Carlsson, Gunilla H; Larsson, Daniel S D; Barty, Anton; Martin, Andrew V; Schorb, Sebastian; Bostedt, Christoph; Bozek, John D; Rolles, Daniel; Rudenko, Artem; Epp, Sascha; Foucar, Lutz; Rudek, Benedikt; Hartmann, Robert; Kimmel, Nils; Holl, Peter; Englert, Lars; Duane Loh, Ne-Te; Chapman, Henry N; Andersson, Inger; Hajdu, Janos; Ekeberg, Tomas

    2015-01-01

    There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential. PMID:25669616

  8. Ultrafast nanolaser device for detecting cancer in a single live cell.

    SciTech Connect

    Gourley, Paul Lee; McDonald, Anthony Eugene

    2007-11-01

    Emerging BioMicroNanotechnologies have the potential to provide accurate, realtime, high throughput screening of live tumor cells without invasive chemical reagents when coupled with ultrafast laser methods. These optically based methods are critical to advancing early detection, diagnosis, and treatment of disease. The first year goals of this project are to develop a laser-based imaging system integrated with an in- vitro, live-cell, micro-culture to study mammalian cells under controlled conditions. In the second year, the system will be used to elucidate the morphology and distribution of mitochondria in the normal cell respiration state and in the disease state for normal and disease states of the cell. In this work we designed and built an in-vitro, live-cell culture microsystem to study mammalian cells under controlled conditions of pH, temp, CO2, Ox, humidity, on engineered material surfaces. We demonstrated viability of cell culture in the microsystem by showing that cells retain healthy growth rates, exhibit normal morphology, and grow to confluence without blebbing or other adverse influences of the material surfaces. We also demonstrated the feasibility of integrating the culture microsystem with laser-imaging and performed nanolaser flow spectrocytometry to carry out analysis of the cells isolated mitochondria.

  9. TRICK: A Single-Molecule Method for Imaging the First Round of Translation in Living Cells and Animals.

    PubMed

    Halstead, J M; Wilbertz, J H; Wippich, F; Lionnet, T; Ephrussi, A; Chao, J A

    2016-01-01

    The life of an mRNA is dynamic within a cell. The development of quantitative fluorescent microscopy techniques to image single molecules of RNA has allowed many aspects of the mRNA lifecycle to be directly observed in living cells. Recent advances in live-cell multicolor RNA imaging, however, have now made it possible to investigate RNA metabolism in greater detail. In this chapter, we present an overview of the design and implementation of the translating RNA imaging by coat protein knockoff RNA biosensor, which allows untranslated mRNAs to be distinguished from ones that have undergone a round of translation. The methods required for establishing this system in mammalian cell lines and Drosophila melanogaster oocytes are described here, but the principles may be applied to any experimental system. PMID:27241753

  10. Noradrenergic modulation of glutamate-induced excitatory responses in single neurons of the red nucleus: an electrophysiological study.

    PubMed

    Bronzi, D; Licata, F; Li Volsi, G

    2015-08-01

    The effect induced by noradrenaline (NA) on the spiking activity evoked by glutamate (Glu) on single neurons of the mesencephalic red nucleus (RN) of the rat was studied extracellularly. Long-lasting microiontophoretic applications of the amine induced a significant and reversible depression of the responsiveness of RN neurons to Glu. This effect was mediated by noradrenergic alpha2 receptors since it was mimicked by application of clonidine, an alpha2 adrenoceptor agonist, and blocked or at least reduced by application of yohimbine, an antagonist of NA for the same receptors. The effect appears homogeneously throughout the nucleus and is independent of the effect of NA on baseline firing rate. Application of isoproterenol, a beta adrenoceptor agonist, either enhanced or depressed neuronal responses to Glu in a high percentage (86%) of the tested neurons. Moreover, application of timolol, a beta adrenoceptor antagonist, was able to strengthen the depressive effects induced by NA application on neuronal responsiveness to Glu. Although these data suggest some involvement of beta adrenergic receptors in the modulation of neuronal responsiveness to Glu, the overall results indicate a short-term depressive action of NA, mediated by alpha2 receptors, on the responsiveness of RN neurons and suggest that stress initially leads to an attenuation of the relay function of the RN. PMID:26012489

  11. A microfluidic system enabling Raman measurements of the oxygenation cycle in single optically trapped red blood cells.

    PubMed

    Ramser, Kerstin; Enger, Jonas; Goksör, Mattias; Hanstorp, Dag; Logg, Katarina; Käll, Mikael

    2005-04-01

    Using a lab-on-a-chip approach we demonstrate the possibility of selecting a single cell with certain properties and following its dynamics after an environmental stimulation in real time using Raman spectroscopy. This is accomplished by combining a micro Raman set-up with optical tweezers and a microfluidic system. The latter gives full control over the media surrounding the cell, and it consists of a pattern of channels and reservoirs defined by electron beam lithography that is moulded into rubber silicon (PDMS). Different buffers can be transported through the channels using electro-osmotic flow, while the resonance Raman response of an optically trapped red blood cell (RBC) is simultaneously registered. This makes it possible to monitor the oxygenation cycle of the cell in real time and to investigate effects like photo-induced chemistry caused by the illumination. The experimental set-up has high potential for in vivo monitoring of cellular drug response using a variety of spectroscopic probes. PMID:15791341

  12. Lateral migration and equilibrium shape and position of a single red blood cell in bounded Poiseuille flows

    NASA Astrophysics Data System (ADS)

    Shi, Lingling; Pan, Tsorng-Whay; Glowinski, Roland

    2012-11-01

    Lateral migration and equilibrium shape and position of a single red blood cell (RBC) in bounded two-dimensional Poiseuille flows are investigated by using an immersed boundary method. An elastic spring model is applied to simulate the skeleton structure of a RBC membrane. We focus on studying the properties of lateral migration of a single RBC in Poiseuille flows by varying the initial position, the initial angle, the swelling ratio (s*), the membrane bending stiffness of RBC (kb), the maximum velocity of fluid flow (umax), and the degree of confinement. The combined effect of the deformability, the degree of confinement, and the shear gradient of the Poiseuille flow make the RBCs migrate toward a certain cross-sectional equilibrium position, which lies either on the center line of the channel or off center line. For s*>0.8, the speed of the migration at the beginning decreases as one increases the swelling ratio s*. But for s*<0.8, the speed of the migration at the beginning is an increasing function of the swelling ratio s*. Two motions of oscillation and vacillating breathing (swing) of RBCs are observed. The distance Yd between the cell mass center of the equilibrium position and the center line of the channel increases with increasing the Reynolds number Re and reaches a peak, then decreases with increasing Re. The peak of Re is a decreasing function of the swelling ratio (s*<1.0). The cell membrane energy of the equilibrium position is an increasing function as Re increases. The slipper-shaped cell is more stable than the parachute-shaped one in the sense that the energy stored in the former is lower than that in the latter. For a given Re, the bigger the swelling ratio (s*<1.0), the lower the cell membrane energy.

  13. Monolithic integration of InGaN segments emitting in the blue, green, and red spectral range in single ordered nanocolumns

    SciTech Connect

    Albert, S.; Bengoechea-Encabo, A.; Sanchez-Garcia, M. A.; Calleja, E.

    2013-05-06

    This work reports on the selective area growth by plasma-assisted molecular beam epitaxy and characterization of InGaN/GaN nanocolumnar heterostructures. The optimization of the In/Ga and total III/V ratios, as well as the growth temperature, provides control on the emission wavelength, either in the blue, green, or red spectral range. An adequate structure tailoring and monolithic integration in a single nanocolumnar heterostructure of three InGaN portions emitting in the red-green-blue colors lead to white light emission.

  14. Long Term Prospective Assessment of Living Kidney Donors: Single Center Experience

    PubMed Central

    Nagib, Ayman Maher; Refaie, Ayman Fathi; Hendy, Yasser Abdelmoniem; Elfawal, Magdy Abass Mohmed; Shokeir, Ahmed Abdelrahman; Bakr, Mohamed Adel; Neamattala, Ahmed Hassan; Hamdy, Ahmed Farouk; Mahmoud, Khaled Mohamed; Ismail, Amani Mostafa; Ghoneim, Mohamed Ahmed

    2014-01-01

    Virtually, all studies reporting the outcomes of living kidney donation beyond the first year from donation were retrospective. In this prospective study, the outcome of 81 consecutive living kidney donors was thoroughly evaluated. Clinical, laboratory, and radiological assessments were carried out at predonation (basal), 3, 6, 12, and 24 months after donation. The mean age at time of donation was 37.8 ± 9.8 years and the majority was females (75.3%). The mean BMI increased significantly after donation (P < 0.04). The mean serum creatinine levels (mg/dl) were 0.75 ± 0.14, 1.01 ± 0.22, 0.99 ± 0.21, 0.98 ± 0.20, and 0.94 ± 0.20 (P < 0.0001). Likewise, the mean levels of measured creatinine clearance (mL/min) were 148.8 ± 35.7, 94.7 ± 26.6, 95.5 ± 24.6, 96.7 ± 20.2, and 101.6 ± 26.2 (P < 0.0001). The mean 24 hours urinary protein excretion (gm/dL) were 0.09 ± 0.03, 0.19 ± 0.18, 0.16 ± 0.09, 0.18 ± 0.25, and 0.17 ± 0.12 (P < 0.0001). There were significant increases in the means of the longitudinal and transverse diameters of the remaining kidney over time (P < 0.001). Out of 42 female donors, eleven female donors have got successful postdonation pregnancies. There were no reported surgical complications, either intra- or postoperative. Long-term follow-up is necessary for all living kidney donors through local institutional and world registries. This trial is registered with ClinicalTrials.gov NCT00813579. PMID:24967244

  15. Hematologic and Total Plasma Protein Values in Free-Living Red-tailed Amazon Parrot Nestlings (Amazona brasiliensis) in Paraná State, Brazil.

    PubMed

    Vaz, Frederico F; Locatelli-Dittrich, Rosangela; Sipinski, Elenise A B; Abbud, Maria C; Sezerban, Rafael M; Schmidt, Elizabeth M S; Dittrich, Jaqueline; Cavalheiro, Maria L

    2015-09-01

    The red-tailed Amazon parrot (Amazona brasiliensis) is an endangered psittacid species that is endemic in the south and southeast Brazilian Atlantic coastal region. Hematologic evaluation is important to monitor the health of these birds, and information about laboratory values for this species is scarce. Hematologic and total plasma protein profiles were determined for 33 free-living nestling parrots in Paraná state, Brazil. Parrots were temporarily removed from the nest and manually restrained to record body weight and collect blood samples. Mean body weight was <400 g in 13 birds (group 1) and >400 g in 20 birds (group 2). Significantly higher levels of mean corpuscular hemoglobin concentrations, white blood cell counts, monocytes, and basophils were observed in younger birds (group 1). A stress leukogram (high white blood cell and heterophil count) was found in all nestlings, suggesting stress induced by capture and restraint. Parameters obtained in this study will be essential to assess the physiologic and pathologic condition of wild parrots, to evaluate the effects of environmental changes on their health, and to contribute to conservation efforts of this endangered species. PMID:26378664

  16. A diode-end-pumped continuous-wave single-longitudinal-mode Nd:GdV O4-LBO red laser at 670 nm

    NASA Astrophysics Data System (ADS)

    Wang, Y. T.; Zhang, R. H.; Li, J. H.; Li, W. J.; Tan, C.; Zhang, B. L.

    2014-03-01

    A diode-end-pumped continuous-wave single-longitudinal-mode intracavity frequency-doubling Nd:GdV O4-LBO (lithium triborate) red laser at 670 nm is reported. A ring cavity was designed to enable single-longitudinal-mode operation of the laser. By optimizing the mode-to-pump ratio taking account of the influence of the laser beam radius inside the frequency-doubling crystal LBO on the frequency-doubling efficiency for the special cavity, a maximum output power of 1.3 W for a continuous-wave single-longitudinal-mode red laser at 670 nm was obtained, and the measured power stability was better than ±1.2% in 2 h. The experimental results are in good agreement with the theoretical calculation.

  17. Pharmacokinetics of meloxicam in red-eared slider turtles (Trachemys scripta elegans) after single intravenous and intramuscular injections.

    PubMed

    Uney, Kamil; Altan, Feray; Aboubakr, Mohammed; Cetin, Gul; Dik, Burak

    2016-05-01

    OBJECTIVE To determine the pharmacokinetics of meloxicam after single IV and IM injections in red-eared slider turtles (Trachemys scripta elegans). ANIMALS 8 healthy red-eared slider turtles. PROCEDURES Turtles received 1 dose of meloxicam (0.2 mg/kg) IV or IM (4 turtles/route), a 30-day washout period was provided, and then turtles received the same dose by the opposite route. Blood samples were collected at predetermined times for measurement of plasma meloxicam concentration. Pharmacokinetic values for each administration route were determined with a 2-compartment open model approach. RESULTS For IV administration, mean ± SD values of major pharmacokinetic variables were 1.02 ± 0.41 hours for distribution half-life, 9.78 ± 2.23 hours for elimination half-life, 215 ± 32 mL/kg for volume of distribution at steady state, 11.27 ± 1.44 μg•h/mL for area under the plasma concentration versus time curve, and 18.00 ± 2.32 mL/h/kg for total body clearance. For IM administration, mean values were 0.35 ± 0.06 hours for absorption half-life, 0.72 ± 0.06 μg/mL for peak plasma concentration, 1.5 ± 0.0 hours for time to peak concentration, 3.73 ± 2.41 hours for distribution half-life, 13.53 ± 1.95 hours for elimination half-life, 11.33 ± 0.92 μg•h/mL for area under the plasma concentration versus time curve, and 101 ± 6% for bioavailability. No adverse reactions were detected. CONCLUSIONS AND CLINICAL RELEVANCE Long half-life, high bioavailability, and lack of immediate adverse reactions of meloxicam administered IM at 0.2 mg/kg suggested the possibility of safe and effective clinical use in turtles. Additional studies are needed to establish appropriate administration frequency and clinical efficacy. PMID:27111010

  18. Electroporation and Microinjection Successfully Deliver Single-Stranded and Duplex DNA into Live Cells as Detected by FRET Measurements

    PubMed Central

    Bamford, Rosemary A.; Zhao, Zheng-yun; Hotchin, Neil A.; Styles, Iain B.; Nash, Gerard B.; Tucker, James H. R.; Bicknell, Roy

    2014-01-01

    Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells. PMID:24755680

  19. A single-cell scraper based on an atomic force microscope for detaching a living cell from a substrate

    NASA Astrophysics Data System (ADS)

    Iwata, Futoshi; Adachi, Makoto; Hashimoto, Shigetaka

    2015-10-01

    We describe an atomic force microscope (AFM) manipulator that can detach a single, living adhesion cell from its substrate without compromising the cell's viability. The micrometer-scale cell scraper designed for this purpose was fabricated from an AFM micro cantilever using focused ion beam milling. The homemade AFM equipped with the scraper was compact and standalone and could be mounted on a sample stage of an inverted optical microscope. It was possible to move the scraper using selectable modes of operation, either a manual mode with a haptic device or a computer-controlled mode. The viability of the scraped single cells was evaluated using a fluorescence dye of calcein-acetoxymethl ester. Single cells detached from the substrate were collected by aspiration into a micropipette capillary glass using an electro-osmotic pump. As a demonstration, single HeLa cells were selectively detached from the substrate and collected by the micropipette. It was possible to recultivate HeLa cells from the single cells collected using the system.

  20. A single-cell scraper based on an atomic force microscope for detaching a living cell from a substrate

    SciTech Connect

    Iwata, Futoshi; Adachi, Makoto; Hashimoto, Shigetaka

    2015-10-07

    We describe an atomic force microscope (AFM) manipulator that can detach a single, living adhesion cell from its substrate without compromising the cell's viability. The micrometer-scale cell scraper designed for this purpose was fabricated from an AFM micro cantilever using focused ion beam milling. The homemade AFM equipped with the scraper was compact and standalone and could be mounted on a sample stage of an inverted optical microscope. It was possible to move the scraper using selectable modes of operation, either a manual mode with a haptic device or a computer-controlled mode. The viability of the scraped single cells was evaluated using a fluorescence dye of calcein-acetoxymethl ester. Single cells detached from the substrate were collected by aspiration into a micropipette capillary glass using an electro-osmotic pump. As a demonstration, single HeLa cells were selectively detached from the substrate and collected by the micropipette. It was possible to recultivate HeLa cells from the single cells collected using the system.

  1. Factors Affecting Graft Survival among Patients Receiving Kidneys from Live Donors: A Single-Center Experience

    PubMed Central

    Ghoneim, Mohamed A.; Bakr, Mohamed A.; Refaie, Ayman F.; Akl, Ahmed I.; Shokeir, Ahmed A.; Shehab El-Dein, Ahmed B.; Ammar, Hesham M.; Ismail, Amani M.; Sheashaa, Hussein A.; El-Baz, Mahmoud A.

    2013-01-01

    Introduction. The aim of this report is to study the graft and patient survival in a large cohort of recipients with an analysis of factors that may affect the final outcomes. Methods. Between March 1976 and March 2008, 1967 consecutive live-donor renal transplants were carried out. Various variables that may have an impact on patients and/or graft survival were studied in two steps. Initially, a univariate analysis was carried out. Thereafter, significant variables were embedded in a stepwise regression analysis. Results. The overall graft survival was 86.7% and 65.5%, at 5 and 10 years, respectively. The projected half-life for grafts was 17.5 years and for patients was 22 years. Five factors had an independent negative impact on graft survival: donor's age, genetic considerations, the type of primary immunosuppression, number of acute rejection episodes, and total steroid dose during the first 3 months after transplantation. Conclusions. Despite refinements in tissue matching techniques and improvements in immunosuppression protocols, an important proportion of grafts is still lost following living donor kidney transplantation, presumably due to chronic allograft nephropathy. PMID:23878820

  2. Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry

    PubMed Central

    Roldán, M.; Thomas, F.; Castel, S.; Quesada, A.; Hernández-Mariné, M.

    2004-01-01

    A new imaging technique for the analysis of fluorescent pigments from a single cell is reported. It is based on confocal scanning laser microscopy coupled with spectrofluorometric methods. The setup allows simultaneous establishment of the relationships among pigment analysis in vivo, morphology, and three-dimensional localization inside thick intact microbial assemblages. PMID:15184183

  3. Spatiotemporal Mapping Of Fluorescence Paramaters In Single Living Cells The Cell's Detoxification Apparatus

    NASA Astrophysics Data System (ADS)

    Kohen, Elli; Prince, Jeffrey; Kohen, Cahide; Hirschberg, Joseph G.; Fried, Marek

    1988-06-01

    Our studies with quinacrine and benzo(a)pyrene suggest the formation of a multiorganelle detoxification complex (MODC) involving the endoplasmic reticulum, the Golgi apparatus, the lysosomes and the nuclear membrane. We have indications that not only is there a trapping of xenobiotics in the cytoplasmic components of the MODC, but there may also be a "nuclear pump" powered by postulated nuclear bioenergetic pathways involved in the ejection of these chemicals from the nucleus. We are using the microspectrofluorometric approach to study the extranuclear/nuclear energy metabolism related to these processes, and we have extended this method to investigate, in situ, within different components of the MODC, the blue/red spectral shifts associated with metabolites of fluorescent xenobiotics. Recording of fluorescence emission spectra, at different excitation wavelengths in L cells, allows the application of multivariate statistical methods to analyze complex (multicomponent spectra). Eluciadation of mechanisms, involved in the organization and activity of the MODC, can result in better targeting of gene modifiers and DNA-intercalating cancer chemotherapeutics towards their expected sites of action.

  4. Real-time visualization of prion transport in single live cells using quantum dots

    SciTech Connect

    Luo, Kan; Li, Shu; Xie, Min; Wu, Di; Wang, WenXi; Chen, Rui; Huang, Liqin; Huang, Tao; Pang, Daiwen; Xiao, Gengfu

    2010-04-09

    Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrP{sup C} to the infectious scrapie isoform PrP{sup Sc}. It is believed that such structural alteration may occur within the internalization pathway. However, there is no direct evidence to support this hypothesis. Employing quantum dots (QDs) as a probe, we have recorded a real-time movie demonstrating the process of prion internalization in a living cell for the first time. The entire internalization process can be divided into four discrete but connected stages. In addition, using methyl-beta-cyclodextrin to disrupt cell membrane cholesterol, we show that lipid rafts play an important role in locating cellular PrP{sup C} to the cell membrane and in initiating PrP{sup C} endocytosis.

  5. Multiparametric atomic force microscopy imaging of single bacteriophages extruding from living bacteria

    NASA Astrophysics Data System (ADS)

    Alsteens, David; Trabelsi, Heykel; Soumillion, Patrice; Dufrêne, Yves F.

    2013-12-01

    Force-distance (FD) curve-based atomic force microscopy is a valuable tool to simultaneously image the structure and map the biophysical properties of biological samples at the nanoscale. Traditionally, FD-based atomic force microscopy has been severely limited by its poor temporal and lateral resolutions. Here we report the use of advanced FD-based technology combined with biochemically sensitive tips to image filamentous bacteriophages extruding from living bacteria at unprecedented speed and resolution. Directly correlated multiparametric images of the structure, adhesion and elasticity of infected bacteria demonstrate that the sites of assembly and extrusion localize at the bacterial septum in the form of soft nanodomains surrounded by stiff cell wall material. The quantitative nano-bio-imaging method presented here offers a wealth of opportunities for mapping the physical properties and molecular interactions of complex biosystems, from viruses to tissues.

  6. Multiparametric atomic force microscopy imaging of single bacteriophages extruding from living bacteria.

    PubMed

    Alsteens, David; Trabelsi, Heykel; Soumillion, Patrice; Dufrêne, Yves F

    2013-01-01

    Force-distance (FD) curve-based atomic force microscopy is a valuable tool to simultaneously image the structure and map the biophysical properties of biological samples at the nanoscale. Traditionally, FD-based atomic force microscopy has been severely limited by its poor temporal and lateral resolutions. Here we report the use of advanced FD-based technology combined with biochemically sensitive tips to image filamentous bacteriophages extruding from living bacteria at unprecedented speed and resolution. Directly correlated multiparametric images of the structure, adhesion and elasticity of infected bacteria demonstrate that the sites of assembly and extrusion localize at the bacterial septum in the form of soft nanodomains surrounded by stiff cell wall material. The quantitative nano-bio-imaging method presented here offers a wealth of opportunities for mapping the physical properties and molecular interactions of complex biosystems, from viruses to tissues. PMID:24336094

  7. Adhesion and membrane tension of single vesicles and living cells using a micropipette-based technique.

    PubMed

    Colbert, M-J; Raegen, A N; Fradin, C; Dalnoki-Veress, K

    2009-10-01

    The fundamental study of the adhesion of cells to each other or to a substrate is a key research topic in cellular biophysics because cell adhesion is important to many biological processes. We report on the adhesion of a model cell, a liposome, and a living HeLa cell to a substrate measured with a novel experimental technique. The cells are held at the end of a micropipette mounted on a micromanipulator and brought into contact with a surface. The adhesion energy and membrane tension are measured directly using the deflection of the micropipette when binding or unbinding the cell from the substrate. Since the force applied on the cells is known throughout the experiment, the technique presented enables the measurement of dynamics such as changes in the adhesion, elasticity, and membrane tension with time. PMID:19777278

  8. Correlation of dual colour single particle trajectories for improved detection and analysis of interactions in living cells.

    PubMed

    Deschout, Hendrik; Martens, Thomas; Vercauteren, Dries; Remaut, Katrien; Demeester, Jo; De Smedt, Stefaan C; Neyts, Kristiaan; Braeckmans, Kevin

    2013-01-01

    Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a much stronger indication of interaction. However, frequently occurring phenomena in living cells, such as immobile phases or transient interactions, can limit the correlation to small parts of the trajectories. The method presented here, developed for the detection of interaction, is based on the correlation inside a window that is scanned along the trajectories, covering different subsets of the positions. This scanning window method was validated by simulations and, as an experimental proof of concept, it was applied to the investigation of the intracellular trafficking of polymeric gene complexes by endosomes in living retinal pigment epithelium cells, which is of interest to ocular gene therapy. PMID:23965965

  9. Comparison of the gravimetric, phenol red, and 14C-PEG-3350 methods to determine water absorption in the rat single-pass intestinal perfusion model.

    PubMed

    Sutton, S C; Rinaldi, M T; Vukovinsky, K E

    2001-01-01

    This study was undertaken to determine whether the gravimetric method provided an accurate measure of water flux correction and to compare the gravimetric method with methods that employ nonabsorbed markers (eg, phenol red and 14C-PEG-3350). Phenol red,14C-PEG-3350, and 4-[2-[[2-(6-amino-3-pyridinyl)-2-hydroxyethyl]amino]ethoxy]-, methyl ester, (R)-benzene acetic acid (Compound I) were co-perfused in situ through the jejunum of 9 anesthetized rats (single-pass intestinal perfusion [SPIP]). Water absorption was determined from the phenol red,14C-PEG-3350, and gravimetric methods. The absorption rate constant (ka) for Compound I was calculated. Both phenol red and 14C-PEG-3350 were appreciably absorbed, underestimating the extent of water flux in the SPIP model. The average +/- SD water flux microg/h/cm) for the 3 methods were 68.9 +/- 28.2 (gravimetric), 26.8 +/- 49.2 (phenol red), and 34.9 +/- 21.9 (14C-PEG-3350). The (average +/- SD) ka for Compound I (uncorrected for water flux) was 0.024 +/- 0.005 min(-1). For the corrected, gravimetric method, the average +/- SD was 0.031 +/- 0.001 min(-1). The gravimetric method for correcting water flux was as accurate as the 2 "nonabsorbed" marker methods. PMID:11741276

  10. Localization of Living-Bodies Using Single-Frequency Multistatic Doppler Radar System

    NASA Astrophysics Data System (ADS)

    Miwa, Takashi; Ogiwara, Shun; Yamakoshi, Yoshiki

    Recently, it has become important to rapidly detect human subjects buried under collapsed houses, rubble and soil due to earthquakes and avalanches to reduce the casualties in a disaster. Such detection systems have already been developed as one kind of microwave displacement sensors that are based on phase difference generated by the motion of the subject's breast. Because almost all the systems consist of single transmitter and receiver pair, it is difficult to rapidly scan a wide area. In this paper, we propose a single-frequency multistatic radar system to detect breathing human subjects which exist in the area surrounded by the transmitting and receiving array. The vibrating targets can be localized by the MUSIC algorithm with the complex amplitude in the Doppler frequency. This algorithm is validated by the simulated signals synthesized with a rigorous solution of a dielectric spherical target model. We show experimental 3D localization results using a developed multistatic Doppler radar system around 250MHz.

  11. Molecular extraction in single live cells by sneaking in and out magnetic nanomaterials

    PubMed Central

    Yang, Zhen; Deng, Liangzi; Lan, Yucheng; Zhang, Xiaoliu; Gao, Zhonghong; Chu, Ching-Wu; Cai, Dong; Ren, Zhifeng

    2014-01-01

    Extraction of intracellular molecules is crucial to the study of cellular signal pathways. Disruption of the cellular membrane remains the established method to release intracellular contents, which inevitably terminates the time course of biological processes. Also, conventional laboratory extractions mostly use bulky materials that ignore the heterogeneity of each cell. In this work, we developed magnetized carbon nanotubes that can be sneaked into and out of cell bodies under a magnetic force. Using a testing model with overexpression of GFP, the nanotubes successfully transported the intracellular GFP out at the single-cell level. The confined nanoscale invasiveness did not change cell viability or proliferation. This study presents the proof of concept of a previously unidentified real-time and single-cell approach to investigate cellular biology, signal messengers, and therapeutic effects with nanomaterials. PMID:25030447

  12. Real-time quantification of single RNA translation dynamics in living cells.

    PubMed

    Morisaki, Tatsuya; Lyon, Kenneth; DeLuca, Keith F; DeLuca, Jennifer G; English, Brian P; Zhang, Zhengjian; Lavis, Luke D; Grimm, Jonathan B; Viswanathan, Sarada; Looger, Loren L; Lionnet, Timothee; Stasevich, Timothy J

    2016-06-17

    Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds. Polysomes contain ~1 ribosome every 200 to 900 nucleotides and are globular rather than elongated in shape. By developing multicolor probes, we showed that most polysomes act independently; however, a small fraction (~5%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics. PMID:27313040

  13. Monofunctional stealth nanoparticle for unbiased single molecule tracking inside living cells.

    PubMed

    Lisse, Domenik; Richter, Christian P; Drees, Christoph; Birkholz, Oliver; You, Changjiang; Rampazzo, Enrico; Piehler, Jacob

    2014-01-01

    On the basis of a protein cage scaffold, we have systematically explored intracellular application of nanoparticles for single molecule studies and discovered that recognition by the autophagy machinery plays a key role for rapid metabolism in the cytosol. Intracellular stealth nanoparticles were achieved by heavy surface PEGylation. By combination with a generic approach for nanoparticle monofunctionalization, efficient labeling of intracellular proteins with high fidelity was accomplished, allowing unbiased long-term tracking of proteins in the outer mitochondrial membrane. PMID:24655019

  14. Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy

    NASA Astrophysics Data System (ADS)

    Renz, Marc; Rendler, Torsten; Börsch, Michael

    2012-03-01

    FoF1-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound enzymes which use an internal protondriven rotary double motor to catalyze the synthesis of adenosine triphosphate (ATP). According to the 'chemiosmotic hypothesis', a series of proton pumps generate the necessary pH difference plus an electric potential across the bacterial plasma membrane. These proton pumps are redox-coupled membrane enzymes which are possibly organized in supercomplexes, as shown for the related enzymes in the mitochondrial inner membrane. We report diffusion measurements of single fluorescent FoF1-ATP synthases in living E. coli by localization microscopy and single enzyme tracking to distinguish a monomeric enzyme from a supercomplex-associated form in the bacterial membrane. For quantitative mean square displacement (MSD) analysis, the limited size of the observation area in the membrane with a significant membrane curvature had to be considered. The E. coli cells had a diameter of about 500 nm and a length of about 2 to 3 μm. Because the surface coordinate system yielded different localization precision, we applied a sliding observation window approach to obtain the diffusion coefficient D = 0.072 μm2/s of FoF1-ATP synthase in living E. coli cells.

  15. The Impact of a Surgical Protocol for Enhanced Recovery on Living Donor Right Hepatectomy: A Single-Center Cohort Study.

    PubMed

    Kim, Seong Hoon; Kim, Young Kyu; Lee, Seung Duk; Lee, Eung Chang; Park, Sang Jae

    2016-04-01

    The concept of surgery for enhanced recovery (SFER) program has never been an issue in the context of living donor right hepatectomy (LDRH), much less its effects. The purpose of this study was to evaluate outcomes after the establishment of an SFER protocol for LDRH in a single center.A single-center cohort study was performed in 500 consecutive living donors who underwent right hepatectomy from January 2005 to June 2014 by analyzing the outcomes before and after an established SFER protocol that evolved with continuous refinements in surgical technique and management over 300 LDRHs, being in place on September 2011. Donor characteristics, operative outcomes, and postoperative complications divided into 2 groups (group 1, stepwise adjustment; group 2, complete adherence to the protocol) were compared.Donor characteristics were comparable in the 2 groups. Overall complication rate was 10.0% with no mortality. In group 2, operative time, hospital stay, and overall complication rate decreased significantly, and the morbidity was 1% and confined in grade I complication without reoperation, perioperative blood transfusion, or readmission. All donors in this series recovered fully and returned to the previous functional lifestyle.An SFER protocol on LDRH can be established by the gradual implementation of various refinements of surgical technique, and the recent outcomes achieved after the establishment of an SFER protocol could provide a current guidance on LDRH toward the ultimate goal of zero morbidity. PMID:27057855

  16. In vitro imaging of single living human umbilical vein endothelial cells with a clinical 3.0-T MRI scanner.

    PubMed

    Zhang, Z; van den Bos, E J; Wielopolski, P A; de Jong-Popijus, M; Bernsen, M R; Duncker, D J; Krestin, G P

    2005-09-01

    Iron oxide-labelled, single, living human umbilical vein endothelial cells (HUVECs) were imaged over time in vitro using a clinical 3.0-T magnetic resonance (MR) microscopy system. Labelling efficiency, toxicity, cell viability, proliferation and differentiation were assessed using flow cytometry, magnetic cell sorting and a phenanthroline assay. MR images were compared with normal light and fluorescence microscopy. Efficient uptake of iron oxide into HUVECs was shown, although with higher label uptake dose-dependent cytotoxic effects were observed, affecting cell viability. For MR imaging, a T2* weighted three-dimensional protocol was used with in-plane resolution of 39 x 48 microm2 and 100-microm slices with a scan time of 13 min. MRI could detect living cells in standard culture dishes at single-cell resolution, although label loss was observed that corresponded with the intracellular iron measurements. MR microscopy using iron oxide labels is a promising tool for studying HUVEC migration and cell biology in vitro and in vivo, but possible toxic effects of label uptake and loss of label over time should be taken into account. PMID:16096808

  17. Single Particle Tracking reveals two distinct environments for CD4 receptors at the surface of living T lymphocytes

    SciTech Connect

    Mascalchi, Patrice; Lamort, Anne Sophie; Salome, Laurence; Dumas, Fabrice

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer We studied the diffusion of single CD4 receptors on living lymphocytes. Black-Right-Pointing-Pointer This study reveals that CD4 receptors have either a random or confined diffusion. Black-Right-Pointing-Pointer The dynamics of unconfined CD4 receptors was accelerated by a temperature raise. Black-Right-Pointing-Pointer The dynamics of confined CD4 receptors was unchanged by a temperature raise. Black-Right-Pointing-Pointer Our results suggest the existence of two different environments for CD4 receptors. -- Abstract: We investigated the lateral diffusion of the HIV receptor CD4 at the surface of T lymphocytes at 20 Degree-Sign C and 37 Degree-Sign C by Single Particle Tracking using Quantum Dots. We found that the receptors presented two major distinct behaviors that were not equally affected by temperature changes. About half of the receptors showed a random diffusion with a diffusion coefficient increasing upon raising the temperature. The other half of the receptors was permanently or transiently confined with unchanged dynamics on raising the temperature. These observations suggest that two distinct subpopulations of CD4 receptors with different environments are present at the surface of living T lymphocytes.

  18. Tracking of mercury ions in living cells with a fluorescent chemodosimeter under single- or two-photon excitation.

    PubMed

    Lu, Zhou-Jun; Wang, Pei-Nan; Zhang, Yu; Chen, Ji-Yao; Zhen, Shen; Leng, Bing; Tian, He

    2007-08-01

    Tracking of Hg2+ in solutions as well as in living cells was conducted with a fluorescent chemodosimeter by measuring the spectral shift of its fluorescence under single- or two-photon excitation. The spectral hypsochromic shifts of this chemodosimeter when reacting with Hg2+ were found to be about 50 nm in acetonitrile/water solutions and 32 nm in Euglena gracilis 277 living cells. This chemodosimeter shows high sensitivity and selectivity, and is not influenced by the pH values. It can signal Hg2+ in solutions down to the ppb range under either single-photon excitation (SPE) at 405 nm or two-photon excitation (TPE) at 800 nm. However, with low cellular chemodosimeter concentrations, the SPE spectra were disturbed by the auto-fluorescence from the native fluorophore in the cell, while the TPE spectra were still of high quality since the two-photon absorption cross section of this chemodosimeter is much larger than that of the native fluorophores in the cell. PMID:17683744

  19. Biochemical measurements on single erythroid progenitor cells shed light on the combinatorial regulation of red blood cell production.

    PubMed

    Wang, Weijia; Akbarian, Vahe; Audet, Julie

    2013-02-01

    Adult bone marrow (BM) erythrocyte colony-forming units (CFU-Es) are important cellular targets for the treatment of anemia and also for the manufacture of red blood cells (RBCs) ex vivo. We obtained quantitative biochemical measurements from single and small numbers of CFU-Es by isolating and analyzing c-Kit(+)CD71(high)Ter119(-) cells from adult mouse BM and this allowed us to identify two mechanisms that can be manipulated to increase RBC production. As expected, maximum RBC output was obtained when CFU-Es were stimulated with a combination of Stem Cell Factor (SCF) and Erythropoietin (EPO) mainly because SCF supports a transient CFU-E expansion and EPO promotes the survival and terminal differentiation of erythroid progenitors. However, we found that one of the main factors limiting the output in RBCs was that EPO induces a downregulation of c-Kit expression which limits the transient expansion of CFU-Es. In the presence of SCF, the EPO-mediated downregulation of c-Kit on CFU-Es is delayed but still significant. Moreover, treatment of CFU-Es with 1-Naphthyl PP1 could partially inhibit the downregulation of c-Kit induced by EPO, suggesting that this process is dependent on a Src family kinase, v-Src and/or c-Fyn. We also found that CFU-E survival and proliferation was dependent on the level of time-integrated extracellular-regulated kinase (ERK) activation in these cells, all of which could be significantly increased when SCF and EPO were combined with mouse fetal liver-derived factors. Taken together, these results suggest two novel molecular strategies to increase RBC production and regeneration. PMID:23168618

  20. Living together: behavior and welfare in single and mixed species groups of capuchin (Cebus apella) and squirrel monkeys (Saimiri sciureus).

    PubMed

    Leonardi, Rebecca; Buchanan-Smith, Hannah M; Dufour, Valérie; MacDonald, Charlotte; Whiten, Andrew

    2010-01-01

    There are potential advantages of housing primates in mixed species exhibits for both the visiting public and the primates themselves. If the primates naturally associate in the wild, it may be more educational and enjoyable for the public to view. Increases in social complexity and stimulation may be enriching for the primates. However, mixed species exhibits might also create welfare problems such as stress from interspecific aggression. We present data on the behavior of single and mixed species groups of capuchin monkeys (Cebus apella) and squirrel monkeys (Saimiri sciureus) housed at the Living Links to Human Evolution Research Centre in the Royal Zoological Society of Scotland's Edinburgh Zoo. These species associate in the wild, gaining foraging benefits and decreased predation. But Cebus are also predators themselves with potential risks for the smaller Saimiri. To study their living together we took scan samples at > or =15 min intervals on single (n=109) and mixed species groups (n=152), and all occurrences of intraspecific aggression and interspecific interactions were recorded. We found no evidence of chronic stress and Saimiri actively chose to associate with Cebus. On 79% of scans, the two species simultaneously occupied the same part of their enclosure. No vertical displacement was observed. Interspecific interactions were common (>2.5/hr), and equally divided among mildly aggressive, neutral, and affiliative interactions such as play. Only one aggressive interaction involved physical contact and was non-injurious. Aggressive interactions were mostly (65%) displacements and vocal exchanges, initiated almost equally by Cebus and Saimiri. Modifications to the enclosure were successful in reducing these mildly aggressive interactions with affiliative interactions increasing in frequency and diversity. Our data suggest that in carefully designed, large enclosures, naturally associating monkeys are able to live harmoniously and are enriched by each other

  1. Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

    NASA Astrophysics Data System (ADS)

    Seyfert, K.; Oosaka, T.; Yaginuma, H.; Ernst, S.; Noji, H.; Iino, R.; Börsch, M.

    2011-03-01

    FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

  2. Non-invasive single-cell biomechanical analysis using live-imaging datasets.

    PubMed

    Pearson, Yanthe E; Lund, Amanda W; Lin, Alex W H; Ng, Chee P; Alsuwaidi, Aysha; Azzeh, Sara; Gater, Deborah L; Teo, Jeremy C M

    2016-09-01

    The physiological state of a cell is governed by a multitude of processes and can be described by a combination of mechanical, spatial and temporal properties. Quantifying cell dynamics at multiple scales is essential for comprehensive studies of cellular function, and remains a challenge for traditional end-point assays. We introduce an efficient, non-invasive computational tool that takes time-lapse images as input to automatically detect, segment and analyze unlabeled live cells; the program then outputs kinematic cellular shape and migration parameters, while simultaneously measuring cellular stiffness and viscosity. We demonstrate the capabilities of the program by testing it on human mesenchymal stem cells (huMSCs) induced to differentiate towards the osteoblastic (huOB) lineage, and T-lymphocyte cells (T cells) of naïve and stimulated phenotypes. The program detected relative cellular stiffness differences in huMSCs and huOBs that were comparable to those obtained with studies that utilize atomic force microscopy; it further distinguished naïve from stimulated T cells, based on characteristics necessary to invoke an immune response. In summary, we introduce an integrated tool to decipher spatiotemporal and intracellular dynamics of cells, providing a new and alternative approach for cell characterization. PMID:27422102

  3. Tracking Single Cells in Live Animals Using a Photoconvertible Near-Infrared Cell Membrane Label

    PubMed Central

    Wu, Juwell; Runnels, Judith M.; Turcotte, Raphaël; Celso, Cristina Lo; Scadden, David T.; Strom, Terry B.; Lin, Charles P.

    2013-01-01

    We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4+ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution. PMID:23990881

  4. Instrumentation for simultaneous kinetic imaging of multiple fluorophores in single living cells

    NASA Astrophysics Data System (ADS)

    Morris, Stephen J.; Beatty, Diane M.; Welling, Larry W.; Wiegmann, Thomas B.

    1991-05-01

    . Intracellular calcium increases rapidly when the bath Ca2+ is raised. The pH remains stable for several seconds, then suddenly collapses. The second example concerns fusion of human red blood cells (RBC) to fibroblasts expressing influenza hemagglutinin. Movement of soluble and membrane-bound dyes follow different kinetics, depending upon the molecular weight of the soluble dye. Furthermore, the swelling of the RBC occurs after the onset of fusion, and therefore cannot provide the driving force.

  5. Living at the border: A community and single-cell assessment of lake bacterioneuston activity

    PubMed Central

    Hörtnagl, Paul; Pérez, María Teresa; Sommaruga, Ruben

    2010-01-01

    We assessed the physicochemical properties of the surface microlayer (SML: first 900 μm) and its underlying water (ULW: 0.2–0.5-m depth) and compared the composition and activity of their bacterial communities in six lakes located across an altitude gradient. Activity was assessed at both the community level, by measuring leucine bulk incorporation, and at the single-cell level, by using microautoradiography. Catalyzed reporter deposition fluorescence in situ hybridization was used to quantitatively assess the structure of the bacterial assemblage. Dissolved organic matter at the SML was significantly enriched in small-size molecules as compared to the ULW. Bacterial abundance in the SML ranged from 3.2 × 105 cells mL−1 to 3.2 × 106 cells mL−1 and was enriched in four out of six lakes when compared to the ULW. The SML and ULW showed lake-specific differences in bacterial community composition, although in most cases, both layers were dominated by Betaproteobacteria. This group also contributed the most to total activity in both layers in all lakes, followed by Actinobacteria. Despite large differences in environmental conditions among lakes, the fraction of active neustonic bacteria was very similar in most of them. Both bulk and single-cell activities are not necessarily lower in the SML than in the ULW, and well-adapted bacteria exist in the extreme conditions found in this habitat. PMID:20401318

  6. Cooling and long-lived single-site localization of an ion in an optical lattice

    NASA Astrophysics Data System (ADS)

    Bylinskii, Alexei; Karpa, Leon; Gangloff, Dorian; Cetina, Marko; Vuletic, Vladan

    2013-05-01

    We report on localization of a continuously cooled single ion by a one-dimensional optical lattice. The ion is confined in a hybrid trap formed by an optical dipole potential produced by the standing-wave field of an optical cavity and a two-dimensional radio-frequency Paul trap transverse to the cavity axis. A lattice-assisted resolved Raman sideband process cools the ion to energies 20 times lower than the depth of the lattice potential, close to the vibrational ground state. We observe ion localization by measuring its displacement in the presence of a periodically driven electric field parallel to the lattice. We demonstrate full suppression of the driven ion motion due to optical localization to a single lattice site on a time-scale of 100 μs, which is 100 times longer than the vibrational period of the ion in the lattice site. At a longer time scale of 1 ms, driven motion is suppressed to 50%. The presented system paves the way to the realization of novel experiments studying classical and quantum friction models, and many-body physics with long-range interactions in periodic potentials. Army Research Office, National Science Foundation, National Science and Engineering Research Council of Canada, Alexander von Humboldt Foundation.

  7. Localization of bleomycin in a single living cell using three-photon excitation microscopy

    NASA Astrophysics Data System (ADS)

    Abraham, Anil T.; Brautigan, David L.; Hecht, Sidney M.; Periasamy, Ammasi

    2001-04-01

    Bleomycin has been used in the clinic as a chemotherapeutic agent for the treatment of several neoplasms, including non-Hodgkins lymphomas, squamous cell carcinomas, and testicular tumors. The effectiveness of bleomycin is believed to be derived from its ability to bind and oxidatively cleave DNA in the presence of a iron cofactor in vivo. A substantial amount of data on BLM has been collected, there is little information concerning the effects of bleomycin in living cells. In order to obtain data pertinent to the effects of BLM in intact cells, we have exploited the intrinsic fluorescence property of bleomycin to monitor the uptake of the drug in mammalian cells. We employed two light microscopy techniques, a wide-field and three-photon excitation (760 nm) fluorescence microscopy. Treatment of HeLa cells with bleomycin resulted in rapid to localization within the cells. In addition data collected from the wide field experiments, three-photon excitation of BLM which considerably reduced the phototoxic effect compared with UV light excitation in the wide-field microscopy indicated co-localization of the drug to regions of the cytoplasm occupied by the endoplasmic reticulum probe, DiOC5. The data clearly indicates that the cellular uptake of bleomycin after one minute includes the nucleus as well as in cytoplasm. Contrary to previous studies, which indicate chromosomal DNA as the target of bleomycin, the current findings suggest that the drug is distributed to many areas within the cell, including the endoplasmic reticulum, an organelle that is known to contain ribonucleic acids.

  8. Pancreas Transplantation From Living Donors: A Single Center Experience of 20 Cases.

    PubMed

    Choi, J Y; Jung, J H; Kwon, H; Shin, S; Kim, Y H; Han, D J

    2016-08-01

    Living donor pancreas transplantation (LDPT) has several advantages over deceased donor pancreas transplantation (DDPT), including better HLA matching, shorter ischemic time, and shorter waiting time. It remains an attractive option for diabetes mellitus (DM) patients with end stage renal disease. We reviewed 20 cases of LDPT performed in Asan Medical Center between October 1992 and March 2015. Six cases (30%) were pancreas transplantation alone (PTA), and the rest (70%) were simultaneous pancreas and kidney transplantation (SPK). Relations of donor and recipient were parents in 7 (35%), siblings in 6 (30%), spouse in 6 (30%), and cousin in 1 (5%). Graft survival in SPK at 1, 3, 5, and 10 years was 91.7%, 83.3%, 83.3%, and 83.3%, respectively, and that in PTA recipients was 50%, 33.3%, 16.7%, and 16.7%, respectively (p = 0.005). Causes of graft failure in SPK were thrombosis (one case), and rejection (one case), whereas those in PTA were noncompliance (two cases), thrombosis (one case), reflux pancreatitis (one case), and chronic rejection (one case). In terms of pancreas exocrine drainage, two grafts (25%) maintained their function in bladder drainage, while all grafts maintained in enteric drainage p < 0.05). Seven (35%) donors experienced minor pancreatic juice leakage and one underwent reoperation due to postoperative hematoma. Most donors maintained normoglycemia and normal renal function. However, two donors developed DM (at 1 and 90 months postdonation), and were treated with oral hypoglycemic agents. Graft survival in PTA recipients was poorer than in SPK due to poor compliance and bladder drainage-related problems. The surgical and metabolic complication rates of donors can be minimized by applying strict donor criteria. Therefore, LDPT with enteric drainage is an acceptable treatment for SPK. PMID:26833623

  9. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    PubMed Central

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2013-01-01

    Membrane proteins (MPs) mediate a variety of cellular responses to extracellular signals. While MPs are intensely studied for their values as disease biomarkers and therapeutic targets, in situ investigation of binding kinetics of MPs with their ligands has been a challenge. Traditional approaches isolate MPs and then study them ex situ, which does not accurately reflect their native structures and functions. We present here a label-free plasmonic microscopy method to map the local binding kinetics of MPs in their native environment. This new analytical method can perform simultaneous plasmonic and fluorescence imaging, thus making it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we have determined the distribution of MPs on the surface of single cells, and the local binding kinetic constants of different MPs. Furthermore, we have studied the polarization of the MPs on the cell surface during chemotaxis. PMID:23000999

  10. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  11. Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane*

    PubMed Central

    Anderluh, Andreas; Klotzsch, Enrico; Reismann, Alexander W. A. F.; Brameshuber, Mario; Kudlacek, Oliver; Newman, Amy Hauck; Sitte, Harald H.; Schütz, Gerhard J.

    2014-01-01

    The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane. PMID:24394416

  12. Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition.

    PubMed Central

    Di Lisa, F; Blank, P S; Colonna, R; Gambassi, G; Silverman, H S; Stern, M D; Hansford, R G

    1995-01-01

    1. The relation between mitochondrial membrane potential (delta psi m) and cell function was investigated in single adult rat cardiac myocytes during anoxia and reoxygenation. delta psi m was studied by loading myocytes with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetra-ethylbenzimidazolylcarbocyanine iodide), a fluorescent probe characterized by two emission peaks (539 and 597 nm with excitation at 490 nm) corresponding to monomer and aggregate forms of the dye. 2. De-energizing conditions applied to mitochondria, cell suspensions or single cells decreased the aggregate emission and increased the monomer emission. This latter result cannot be explained by changes of JC-1 concentration in the aqueous mitochondrial matrix phase indicating that hydrophobic interaction of the probe with membranes has to be taken into account to explain JC-1 fluorescence properties in isolated mitochondria or intact cells. 3. A different sensitivity of the two JC-1 forms to delta psi m changes was shown in isolated mitochondria by the effects of ADP and FCCP and the calibration with K+ diffusion potentials. The monomer emission was responsive to values of delta psi m below 140 mV, which hardly modified the aggregate emission. Thus JC-1 represents a unique double sensor which can provide semi-quantitative information in both low and high potential ranges. 4. At the onset of glucose-free anoxia the epifluorescence of individual myocytes studied in the single excitation (490 nm)-double emission (530 and 590 nm) mode showed a gradual decline of the aggregate emission, which reached a plateau while electrically stimulated (0.2 Hz) contraction was still retained. The subsequent failure of contraction was followed by the rise of the emission at 530 nm, corresponding to the monomer form of the dye, concomitantly with the development of rigor contracture. 5. The onset of the rigor was preceded by the increase in intracellular Mg2+ concentration ([Mg2+]i) monitored by mag-indo-1 epifluorescence

  13. Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-03-01

    Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

  14. Three-dimensional single-particle tracking in live cells: news from the third dimension

    NASA Astrophysics Data System (ADS)

    Dupont, A.; Gorelashvili, M.; Schüller, V.; Wehnekamp, F.; Arcizet, D.; Katayama, Y.; Lamb, D. C.; Heinrich, D.

    2013-07-01

    Single-particle tracking (SPT) is of growing importance in the biophysical community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its technical simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) analysis introduced previously. The 3D trajectories, and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells’ cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coefficient values derived from the 2D analysis were therefore biased.

  15. Tracking single Kv2.1 channels in live cells reveals anomalous subdiffusion and ergodicity breaking

    NASA Astrophysics Data System (ADS)

    Weigel, Aubrey; Simon, Blair; Tamkun, Michael; Krapf, Diego

    2011-03-01

    The dynamic organization of the plasma membrane is responsible for essential cellular processes, such as receptor trafficking and signaling. By studying the dynamics of transmembrane proteins a greater understanding of these processes as a whole can be achieved. It is broadly observed that the diffusion pattern of membrane protein displays anomalous subdiffusion. However, the mechanisms responsible for this behavior are not yet established. We explore the dynamics of the voltage gated potassium channel Kv2.1 by using single-particle tracking. We analyze Kv2.1 channel trajectories in terms of the time and ensemble distributions of square displacements. Our results reveal that all Kv2.1 channels experience anomalous subdiffusion and we observe that the Kv2.1 diffusion pattern is non-ergodic. We further investigated the role of the actin cytoskeleton in these channel dynamics by applying actin depolymerizing drugs. It is seen that with the breakdown of the actin cytoskeleton the Kv2.1 channel trajectories recover ergodicity.

  16. Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses.

    PubMed

    Ren, Linzhu; Peng, Zhiyuan; Chen, Xinrong; Ouyang, Hongsheng

    2016-04-01

    Cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. There are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand RNA virus infections. The first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. During infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. Using subgenomic replicon, which are genetically engineered viral RNA molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. The second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. The third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. This review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades. PMID:26728654

  17. QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

    EPA Science Inventory

    Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.

    ABSTRACT

    DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

  18. Effects of heat treatment on red gemstone spinel: single-crystal X-ray, Raman, and photoluminescence study

    NASA Astrophysics Data System (ADS)

    Widmer, Remo; Malsy, Anna-Kathrin; Armbruster, Thomas

    2015-04-01

    A red spinel, MgAl2O4, from Burma (Myanmar) containing as chromophores ca. 0.5 wt% of each Cr2O3 and V2O3, was sequentially heated for at least 72 h at temperatures ranging from 600 °C to 1,100 °C. The untreated and quenched samples were examined with single-crystal X-ray diffraction (XRD), Raman spectroscopy and photoluminescence spectroscopy. XRD results display a linear decrease of the cell parameter a and a continuous shift of the oxygen coordinate u, u, u at 3 m toward lower values with increasing temperature and associated Mg, Al disorder: T(Mg1- x Al x )M(Al2- x Mg x )O4. The natural spinel has x = 0.157(2) and reaches x = 0.286(4) after quenching from 1,100 °C. In its natural state, M-O and T-O distances are 1.9226(2) and 1.9361(4) Å. With increasing inversion of Mg from the tetrahedrally coordinated T to the octahedrally coordinated M site, M-O distances increase at 1,100 °C to 1.9333(4) Å and T-O distances decrease to 1.9130(8) Å. The crossover temperature, at which T-O and M-O distances become equal (i.e., 1.927 Å), is found to be at 650 °C and corresponds to an inversion parameter x = 0.208(3). With increasing heat treatment, Raman spectra of quenched samples become significantly broadened and a peak characteristic for Mg, Al disorder at 721 cm-1 firstly appears for a crystal quenched from 800 °C with x = 0.248(4). At room temperature, photoluminescence spectra are dominated by a strong R line at 684.5 nm accompanied by poorly resolved N lines: N1 (687 nm), N2 (688 nm), and N3 (689 nm). N lines are caused by different Mg, Al environments of Cr3+. With increasing inversion parameter ( x), the R line decreases in intensity and the N lines become prominent leading to strongly broadened peaks with a maximum shifted toward higher wave lengths (687.5 nm at 1,100 °C). Criteria for the detection of heat treatment on gemstone spinel applicable to gemological routine examination are provided. Extrapolation of u, a, and bond lengths from heat

  19. Real-Time Discrimination and Versatile Profiling of Spontaneous Reactive Oxygen Species in Living Organisms with a Single Fluorescent Probe.

    PubMed

    Zhang, Ruilong; Zhao, Jun; Han, Guangmei; Liu, Zhengjie; Liu, Cui; Zhang, Cheng; Liu, Bianhua; Jiang, Changlong; Liu, Renyong; Zhao, Tingting; Han, Ming-Yong; Zhang, Zhongping

    2016-03-23

    Fluorescent probes are powerful tools for the investigations of reactive oxygen species (ROS) in living organisms by visualization and imaging. However, the multiparallel assays of several ROS with multiple probes are often limited by the available number of spectrally nonoverlapping chromophores together with large invasive effects and discrepant biological locations. Meanwhile, the spontaneous ROS profilings in various living organs/tissues are also limited by the penetration capability of probes across different biological barriers and the stability in reactive in vivo environments. Here, we report a single fluorescent probe to achieve the effective discrimination and profiling of hydroxyl radicals (•OH) and hypochlorous acid (HClO) in living organisms. The probe is constructed by chemically grafting an additional five-membered heterocyclic ring and a lateral triethylene glycol chain to a fluorescein mother, which does not only turn off the fluorescence of fluorescein, but also create the dual reactive sites to ROS and the penetration capability in passing through various biological barriers. The reactions of probe with •OH and HClO simultaneously result in cyan and green emissions, respectively, providing the real-time discrimination and quantitative analysis of the two ROS in cellular mitochondria. Surprisingly, the accumulation of probes in the intestine and liver of a normal-state zebrafish and the transfer pathway from intestine-to-blood-to-organ/tissue-to-kidney-to-excretion clearly present the profiling of spontaneous •OH and HClO in these metabolic organs. In particular, the stress generation of •OH at the fresh wound of zebrafish is successfully visualized for the first time, in spite of its extremely short lifetime. PMID:26938117

  20. Viscoelastic Retraction of Single Living Stress Fibers and Its Impact on Cell Shape, Cytoskeletal Organization, and Extracellular Matrix Mechanics

    PubMed Central

    Kumar, Sanjay; Maxwell, Iva Z.; Heisterkamp, Alexander; Polte, Thomas R.; Lele, Tanmay P.; Salanga, Matthew; Mazur, Eric; Ingber, Donald E.

    2006-01-01

    Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale. PMID:16500961

  1. Mapping Cd²⁺-induced membrane permeability changes of single live cells by means of scanning electrochemical microscopy.

    PubMed

    Filice, Fraser P; Li, Michelle S M; Henderson, Jeffrey D; Ding, Zhifeng

    2016-02-18

    Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd(2+)-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd(2+) in varying concentrations. It is experimentally observed that 50 and 100 μM Cd(2+) caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd(2+) concentration. The Cd(2+) was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd(2+) stress is realized by the methodology presented. PMID:26826690

  2. Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single-Molecule Tracking Study.

    PubMed

    Möckl, Leonhard; Lindhorst, Thisbe K; Bräuchle, Christoph

    2016-03-16

    We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper-free click chemistry. Finally, these biotin-tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin-induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA-protein networks employing single-molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface. PMID:26698366

  3. Super-resolution imaging-based single particle tracking reveals dynamics of nanoparticle internalization by live cells.

    PubMed

    Li, Yiming; Shang, Li; Nienhaus, G Ulrich

    2016-04-14

    By combining super-resolution photoactivation localization microscopy with single particle tracking, we have visualized the endocytic process in the live-cell environment with nanoparticles (NPs) of different size and surface functionalization. This allowed us to analyze the dynamics of NPs interacting with cells with high spatial and temporal resolution. We identified two distinctly different types of pathways by which NPs are internalized via clathrin-coated pits (CCPs). Predominantly, NPs first bind to the membrane and, subsequently, CCPs form at this site. However, there are also instances where a NP diffuses on the membrane and utilizes a preformed CCP. Moreover, we have applied this new method to further explore the effects of size and surface functionalization on the NP dynamics on the plasma membrane and the ensuing endocytosis. PMID:27001905

  4. Hyperspectral multiplex single-particle tracking of different receptor subtypes labeled with quantum dots in live neurons

    NASA Astrophysics Data System (ADS)

    Labrecque, Simon; Sylvestre, Jean-Philippe; Marcet, Stephane; Mangiarini, Francesca; Bourgoin, Brice; Verhaegen, Marc; Blais-Ouellette, Sébastien; De Koninck, Paul

    2016-04-01

    The efficacy of existing therapies and the discovery of innovative treatments for central nervous system (CNS) diseases have been limited by the lack of appropriate methods to investigate complex molecular processes at the synaptic level. To improve our capability to investigate complex mechanisms of synaptic signaling and remodeling, we designed a fluorescence hyperspectral imaging platform to simultaneously track different subtypes of individual neurotransmitter receptors trafficking in and out of synapses. This imaging platform allows simultaneous image acquisition of at least five fluorescent markers in living neurons with a high-spatial resolution. We used quantum dots emitting at different wavelengths and functionalized to specifically bind to single receptors on the membrane of living neurons. The hyperspectral imaging platform enabled the simultaneous optical tracking of five different synaptic proteins, including subtypes of glutamate receptors (mGluR and AMPAR) and postsynaptic signaling proteins. It also permitted the quantification of their mobility after treatments with various pharmacological agents. This technique provides an efficient method to monitor several synaptic proteins at the same time, which could accelerate the screening of effective compounds for treatment of CNS disorders.

  5. Ratiometric Fluorescence Nanoprobes for Subcellular pH Imaging with a Single-Wavelength Excitation in Living Cells.

    PubMed

    Pan, Wei; Wang, Honghong; Yang, Limin; Yu, Zhengze; Li, Na; Tang, Bo

    2016-07-01

    Abnormal pH values in the organelles are closely associated with inappropriate cellular functions and many diseases. Monitoring subcellular pH values and their variations is significant in biological processes occurring in living cells and tissues. Herein, we develop a series of ratiometric fluorescence nanoprobes for quantification and imaging of pH values with a single-wavelength excitation in cytoplasm, lysosomes, and mitochondria. The nanoprobes consist of mesoporous silica nanoparticles assembled with aminofluorescein as the recognition unit for pH measurement and ethidium bromide as reference fluorophore. Further conjugation of subcellular targeting moiety enables the nanoprobes to specifically target lysosome and mitochondria. Confocal fluorescence imaging demonstrated that the nanoprobes could effectively monitor the pH fluctuations from 5.0 to 8.3 in living cells by ratio imaging with 488 nm excitation. Subcellular pH determination and imaging in lysosome and mitochondria could also be achieved in different conditions. The current method can offer a general strategy to determine subcellular analytes and investigate the interactions in biological samples. PMID:27295434

  6. Which Single Intervention Would Do the Most to Improve the Health of Those Living on Less Than $1 Per Day?

    PubMed Central

    Yamey, Gavin

    2007-01-01

    Background to the debate: PLoS Medicine is participating in the Council of Science Editors' global theme issue on poverty and human development on October 22, 2007 (http://www.councilscienceeditors.org/globalthemeissue.cfm). Over 200 scientific and medical journals are taking part. For our theme issue, we asked a wide variety of commentators worldwide—including clinicians, medical researchers, health reporters, policy makers, health activists, and development experts—to name the single intervention that they think would improve the health of those living in poverty. We also asked four individuals living in poor, rural agricultural communities in the Santillana district, province of Huanta, Ayacucho, Peru to give us their response to the question, “What do you think would do the most to improve your health and the health of your family?” (The four community members were Severino Rojas Poma, Mercedes Vargas Soto, Julián De La Cruz Chahua, and Martín Rojas Poma). Our October 2007 Editorial discusses this debate further. PMID:17958464

  7. Imaging Real-Time Gene Expression in Living Systems with Single-Transcript Resolution: Image Analysis of Single mRNA Transcripts

    PubMed Central

    Wells, Amber L.; Condeelis, John S.; Singer, Robert H.; Zenklusen, Daniel

    2016-01-01

    This protocol describes a method for establishing a green fluorescent protein (GFP) calibration curve using dilutions of recombinant GFP and blue fluorescent beads. The total fluorescence intensity (TFI) per mRNA molecule is first calculated by imaging serial dilutions of purified enhanced GFP (eGFP) to determine the TFI within a specific volume. A calibration curve of fluorescence intensity in a given voxel per molecule of GFP is then used to determine the number of GFP molecules in the sample of formaldehyde fixed cells to be imaged. This is followed by a method for detection of single molecules in formaldehyde-fixed and live cells. These cells have been cotransfected with mRNA reporter and MCP-xFP plasmids, where MCP-xFP refers to a fluorescent protein fused to the MS2 capsid protein. It is important to collect micrographs and establish the calibration curve on the same day that the cells are imaged, using the same equipment configuration, camera settings, and image acquisition parameters. Single-molecule measurements using GFP are performed as in Femino et al. (1998, 2003) and Fusco et al. (2003). PMID:21356979

  8. Super-resolution imaging-based single particle tracking reveals dynamics of nanoparticle internalization by live cells

    NASA Astrophysics Data System (ADS)

    Li, Yiming; Shang, Li; Nienhaus, G. Ulrich

    2016-03-01

    By combining super-resolution photoactivation localization microscopy with single particle tracking, we have visualized the endocytic process in the live-cell environment with nanoparticles (NPs) of different size and surface functionalization. This allowed us to analyze the dynamics of NPs interacting with cells with high spatial and temporal resolution. We identified two distinctly different types of pathways by which NPs are internalized via clathrin-coated pits (CCPs). Predominantly, NPs first bind to the membrane and, subsequently, CCPs form at this site. However, there are also instances where a NP diffuses on the membrane and utilizes a preformed CCP. Moreover, we have applied this new method to further explore the effects of size and surface functionalization on the NP dynamics on the plasma membrane and the ensuing endocytosis.By combining super-resolution photoactivation localization microscopy with single particle tracking, we have visualized the endocytic process in the live-cell environment with nanoparticles (NPs) of different size and surface functionalization. This allowed us to analyze the dynamics of NPs interacting with cells with high spatial and temporal resolution. We identified two distinctly different types of pathways by which NPs are internalized via clathrin-coated pits (CCPs). Predominantly, NPs first bind to the membrane and, subsequently, CCPs form at this site. However, there are also instances where a NP diffuses on the membrane and utilizes a preformed CCP. Moreover, we have applied this new method to further explore the effects of size and surface functionalization on the NP dynamics on the plasma membrane and the ensuing endocytosis. Electronic supplementary information (ESI) available: Experimental section, supporting figures and videos. See DOI: 10.1039/c6nr01495j

  9. Single-cell evaluation of red blood cell bio-mechanical and nano-structural alterations upon chemically induced oxidative stress.

    PubMed

    Sinha, Ameya; Chu, Trang T T; Dao, Ming; Chandramohanadas, Rajesh

    2015-01-01

    Erythroid cells, specifically red blood cells (RBCs), are constantly exposed to highly reactive radicals during cellular gaseous exchange. Such exposure often exceeds the cells' innate anti-oxidant defense systems, leading to progressive damage and eventual senescence. One of the contributing factors to this process are alterations to hemoglobin conformation and globin binding to red cell cytoskeleton. However, in addition to the aforementioned changes, it is possible that oxidative damage induces critical changes to the erythrocyte cytoskeleton and corresponding bio-mechanical and nano-structural properties of the red cell membrane. To quantitatively characterize how oxidative damage accounts for such changes, we employed single-cell manipulation techniques such as micropipette aspiration and atomic force microscopy (AFM) on RBCs. These investigations demonstrated visible morphological changes upon chemically induced oxidative damage (using hydrogen peroxide, diamide, primaquine bisphosphate and cumene hydroperoxide). Our results provide previously unavailable observations on remarkable changes in red cell cytoskeletal architecture and membrane stiffness due to oxidative damage. Furthermore, we also demonstrate that a pathogen that infects human blood cells, Plasmodium falciparum was unable to penetrate through the oxidant-exposed RBCs that have damaged cytoskeleton and stiffer membranes. This indicates the importance of bio-physical factors pertinent to aged RBCs and it's relevance to malaria infectivity. PMID:25950144

  10. Domain Structure of the Redβ Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo.

    PubMed

    Smith, Christopher E; Bell, Charles E

    2016-02-13

    Redβ is a component of the Red recombination system of bacteriophage λ that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Redβ interacts with λ exonuclease (λexo), the other component of the Red system, to form a "synaptosome" complex that somehow integrates the end resection and annealing steps of the reaction. Previous work using limited proteolysis and chemical modification revealed that Redβ consists of an N-terminal DNA binding domain, residues 1-177, and a flexible C-terminal "tail", residues 178-261. Here, we quantitatively compare the binding of the full-length protein (Redβ(FL)) and the N-terminal domain (Redβ(177)) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, Redβ(FL) binds more tightly to annealed duplex product than to ssDNA substrate, while Redβ(177) binds more tightly to ssDNA. In addition, the C-terminal region of Redβ corresponding to residues 182-261 was purified and found to fold into an α-helical domain that is required for the interaction with λexo to form the synaptosome complex. Deletion analysis of Redβ revealed that removal of just eleven residues from the C-terminus disrupts the interaction with λexo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Redβ appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of Redβ(FL) and Redβ(177) to ssDNA substrate and annealed duplex product may be important for Redβ to function as a SSA protein in vivo. PMID:26780547

  11. Living With A Red Dwarf: Rotation, Starspots, Activity Cycles, Coronal X-ray Activity And X-uv Irradiances Of Proxima Centauri

    NASA Astrophysics Data System (ADS)

    Jason, Merritt; Guinan, E.; Engle, S.; Pojmanski, G.

    2007-12-01

    As part of our Living with a Red Dwarf Program, we have carried out a detailed study of the radiative and plasma properties of the nearby dM5.5e star Proxima Centauri. Proxima Cen is noteworthy as the nearest star to the Sun. Because of its proximity ( 4.3 L.Y.) and membership in the α Cen system, Proxima Cen is an important star to use as a surrogate for solar-aged mid-dM stars. It is relatively bright (V = 11-mag) and has well determined observational and physical properties (MV, Teff, [Fe/H], angular diameter, mass and age). Importantly for our purposes, Proxima Cen has a reliable age of 5.5-6.0 Gyr from its association with the α Cen system in which α Cen A (G2 V) has a reliable isochronal age determination. We have analyzed 5 years of ASAS-3, V-band photometry to search for evidence of short- and long-term variations in brightness that could arise from magnetically related phenomenon (star spots, faculae, and possible UV flares). We also examine its coronal X-ray emission and variations as well as the stars chromospheric and transition regions in the UV from IUE and FUSE observations. The X-UV/optical data are combined and irradiances are calculated for use in extrasolar planet studies. From the photometry we find a rotational modulation of Prot = 83.5 days, in excellent agreement with the earlier HST/FGS study of Benedict et al. (1998). The character of its light variations indicates possible differential rotation as well as a probable long-term activity cycle of 6.9 +/- 0.5 yrs. Although Proxima Cen should be a fully convective star with a different magnetic dynamo (α2) than our Sun (αΩ), its overall magnetic behavior appears to be solar-like. This research is supported by grants from NSF/RUI AST-507536 and NASA Grants NNX06AD386 and NNG04G038G. We are grateful for this support.

  12. Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons

    PubMed Central

    Janning, Dennis; Igaev, Maxim; Sündermann, Frederik; Brühmann, Jörg; Beutel, Oliver; Heinisch, Jürgen J.; Bakota, Lidia; Piehler, Jacob; Junge, Wolfgang; Brandt, Roland

    2014-01-01

    The microtubule-associated phosphoprotein tau regulates microtubule dynamics and is involved in neurodegenerative diseases collectively called tauopathies. It is generally believed that the vast majority of tau molecules decorate axonal microtubules, thereby stabilizing them. However, it is an open question how tau can regulate microtubule dynamics without impeding microtubule-dependent transport and how tau is also available for interactions other than those with microtubules. Here we address this apparent paradox by fast single-molecule tracking of tau in living neurons and Monte Carlo simulations of tau dynamics. We find that tau dwells on a single microtubule for an unexpectedly short time of ∼40 ms before it hops to the next. This dwell time is 100-fold shorter than previously reported by ensemble measurements. Furthermore, we observed by quantitative imaging using fluorescence decay after photoactivation recordings of photoactivatable GFP–tagged tubulin that, despite this rapid dynamics, tau is capable of regulating the tubulin–microtubule balance. This indicates that tau's dwell time on microtubules is sufficiently long to influence the lifetime of a tubulin subunit in a GTP cap. Our data imply a novel kiss-and-hop mechanism by which tau promotes neuronal microtubule assembly. The rapid kiss-and-hop interaction explains why tau, although binding to microtubules, does not interfere with axonal transport. PMID:25165145

  13. RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression.

    PubMed

    Wang, Hailong; Li, Zhen; Jia, Ruonan; Hou, Yu; Yin, Jia; Bian, Xiaoying; Li, Aiying; Müller, Rolf; Stewart, A Francis; Fu, Jun; Zhang, Youming

    2016-07-01

    Full-length RecE and RecT from Rac prophage mediate highly efficient linear-linear homologous recombination that can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and screening. Homologous recombination mediated by Redαβ from lambda phage has been widely used for recombinant DNA engineering. Here we present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes from genomic DNA using one Escherichia coli host that harbors both RecET and Redαβ systems. The pipeline uses standardized cassettes for horizontal gene transfer options, as well as vectors with different replication origins configured to minimize recombineering background through the use of selectively replicating templates or CcdB counterselection. These optimized reagents and protocols facilitate fast acquisition of transgenes from genomic DNA preparations, which are ready for heterologous expression within 1 week. PMID:27254463

  14. Peptide-coated semiconductor quantum dots and their applications in biological imaging of single molecules in live cells and organisms

    NASA Astrophysics Data System (ADS)

    Pinaud, Fabien Florent

    2007-12-01

    A new surface chemistry has been developed for the solubilization and biofunctionalization of inorganic semiconductor nanocrystals fluorescent probes, also known as quantum dots. This chemistry is based on the surface coating of quantum dots with custom-designed polycysteine peptides and yields water-soluble, small, monodispersed and colloidally stable probes that remain bright and photostable in complex biological milieus. This peptide coating strategy was successfully tested on several types of core and core-shell quantum dots emitting from the visible (e.g. CdSe/ZnS) to the NIR spectrum range (e.g. CdTe/CdSe/ZnS). By taking advantage of the versatile physico-chemical properties of peptides, a peptide "toolkit" was designed and employed to impart several biological functions to individual quantum dots and control their biochemical activity at the nanometer scale. These biofunctionalized peptide-coated quantum dots were exploited in very diverse biological applications. Near-infrared emitting quantum dot probes were engineered with optimized blood circulation and biodistribution properties for in vivo animal imaging. Visible emitting quantum dots were used for single molecule tracking of raft-associated GPI-anchored proteins in live cells. This last application revealed the presence of discrete and non-caveolar lipid microdomains capable of impeding free lateral diffusions in the plasma membrane of Hela cells. Imaging and tracking of peptide-coated quantum dots provided the first direct evidence that microdomains having the composition and behavior expected for lipid rafts can induce molecular compartmentalization in the membrane of living cells.

  15. Transcription-Factor-Mediated DNA Looping Probed by High-Resolution, Single-Molecule Imaging in Live E. coli Cells

    PubMed Central

    Hensel, Zach; Xiao, Jie

    2013-01-01

    DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The “genetic switch” of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences OR and OL (separated by 2.3 kb), mediated by the λ repressor CI (accession number P03034), play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms. PMID:23853547

  16. Nature of red luminescence band in research-grade ZnO single crystals: A “self-activated” configurational transition

    SciTech Connect

    Chen, Y. N.; Xu, S. J. Zheng, C. C.; Ning, J. Q.; Ling, F. C. C.; Anwand, W.; Brauer, G.; Skorupa, W.

    2014-07-28

    By implanting Zn{sup +} ions into research-grade intentionally undoped ZnO single crystal for facilitating Zn interstitials (Zn{sub i}) and O vacancies (V{sub O}) which is revealed by precise X-Ray diffraction rocking curves, we observe an apparent broad red luminescence band with a nearly perfect Gaussian lineshape. This red luminescence band has the zero phonon line at ∼2.4 eV and shows distinctive lattice temperature dependence which is well interpreted with the configurational coordinate model. It also shows a low “kick out” thermal energy and small thermal quenching energy. A “self-activated” optical transition between a shallow donor and the defect center of Zn{sub i}-V{sub O} complex or V{sub Zn}V{sub O} di-vacancies is proposed to be responsible for the red luminescence band. Accompanied with the optical transition, large lattice relaxation simultaneously occurs around the center, as indicated by the generation of multiphonons.

  17. Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli.

    PubMed

    Stracy, Mathew; Jaciuk, Marcin; Uphoff, Stephan; Kapanidis, Achillefs N; Nowotny, Marcin; Sherratt, David J; Zawadzki, Pawel

    2016-01-01

    Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the 'proximal' and 'distal' UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA dissociates from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions. PMID:27562541

  18. Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli

    PubMed Central

    Stracy, Mathew; Jaciuk, Marcin; Uphoff, Stephan; Kapanidis, Achillefs N.; Nowotny, Marcin; Sherratt, David J.; Zawadzki, Pawel

    2016-01-01

    Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the ‘proximal' and ‘distal' UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA dissociates from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions. PMID:27562541

  19. A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

    PubMed Central

    Cox, Dianne; Hodgson, Louis

    2014-01-01

    Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized. PMID:24798463

  20. Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells

    PubMed Central

    Jose, Joyce; Tang, Jinghua; Taylor, Aaron B.; Baker, Timothy S.; Kuhn, Richard J.

    2015-01-01

    Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions. PMID:26633461

  1. Significance of Semiquantitative Assessment of Preformed Donor-Specific Antibody Using Luminex Single Bead Assay in Living Related Liver Transplantation

    PubMed Central

    Yoshizawa, Atsushi; Egawa, Hiroto; Yurugi, Kimiko; Hishida, Rie; Tsuji, Hiroaki; Ashihara, Eiji; Miyagawa-Hayashino, Aya; Teramukai, Satoshi; Maekawa, Taira; Haga, Hironori; Uemoto, Sinji

    2013-01-01

    Aim. To analyze the risks of preoperatively produced donor-specific antibody (DSA) in liver transplantation. Methods. DSA was assessed using direct complement-dependent cytotoxicity (CDC) and anti-human globulin- (AHG-) CDC tests, as well as the Luminex Single Antigen assay. Among 616 patients undergoing blood type identical or compatible living donor liver transplantation (LDLT), 21 patients were positive for CDC or AHG-CDC tests, and the preserved serum from 18 patients was examined to determine targeted Class I and II antigens. The relationships between the mean fluorescence intensity (MFI) of DSA and the clinical outcomes were analyzed. Results. Patients were divided into 3 groups according to the MFI of anti-Class I DSA: high (11 patients with MFI > 10,000), low (2 patients with MFI < 10,000), and negative (5 patients) MFI groups. Six of 11 patients with high Class-I DSA showed positive Class-II DSA. Hospital death occurred in 7 patients of the high MFI group. High MFI was a significant risk factor for mortality (P = 0.0155). Univariate analysis showed a significant correlation between MFI strength and C4d deposition (P = 0.0498). Conclusions. HLA Class I DSA with MFI > 10,000 had a significant negative effect on the clinical outcome of patients with preformed DSA in LDLT. PMID:23818917

  2. Quantum dot targeting with lipoic acid ligase and HaloTag for single-molecule imaging on living cells.

    PubMed

    Liu, Daniel S; Phipps, William S; Loh, Ken H; Howarth, Mark; Ting, Alice Y

    2012-12-21

    We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner. PMID:23181687

  3. Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.

    PubMed Central

    Iino, R; Koyama, I; Kusumi, A

    2001-01-01

    Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions). PMID:11371443

  4. Investigation of Cytotoxicity of Phosphoryl Choline Modified Single-Walled Carbon Nanotubes under a Live Cell Station

    PubMed Central

    Zhao, Yufeng; Mao, Qunlong; Liu, Yu; Zhang, Yan; Zhang, Tao; Jiang, Zhengsheng

    2014-01-01

    Single-walled carbon nanotubes (SWCNTs) and various modified SWCNTs have drawn a lot of attention due to their potential applications in biomedical field. Before further moving on to real clinical applications, hydrophobicity and toxicity of SWCNTs should be investigated thoroughly. In this paper, 2-methacryloyloxy ethyl phosphorylcholine (MPC) was adopted to modify SWCNTs and phosphoryl choline was grafted onto SWCNTs as small molecule moieties and polymeric chains, which made SWCNTs dispersed stably both in water and in cell culture medium for a long time. Cytotoxicity of pristine and modified SWCNTs were assayed upon successful preparation of the designed modified SWCNT. Furthermore, the internalization of SWCNTs by three cells was investigated using a live cell station under normal culture temperature (37°C) and low temperature (4°C). The results showed that the internalization of modified SWCNTs was related to both the active transport and the passive transport. Although the modification with phosphoryl choline remarkably reduced the cytotoxicity of SWCNTs, the results were probably due to other reasons such as the decrease in the ratio of cells which internalized modified SWCNTs since the cells without SWCNTs occupation still exhibited normal states. PMID:25057493

  5. Grafting Poly(ethylene glycol) Onto Single-Walled Carbon Nanotubes by Living Anionic Ring-Opening Polymerization.

    PubMed

    Li, Wei; Zhang, Guoxiang; Sheng, Wenbo; Liu, Zhiyong; Jia, Xin

    2016-01-01

    Recent years, many methods have been developed to widen the practical application of single-walled carbon nanotubes (SWCNTs). Among them, PEGylation is a general strategy to endow functionality, biocompatibility as well as its good solubility. In this paper, poly(ethylene glycol) (PEG) is successfully grafted onto SWCNTs through living anionic ring-opening polymerization of ethylene oxide (EO). By controlling the amount of monomer and initiator, a series of PEGylated SWCNTs with different PEG molecular weight and density are prepared. Then, the as-prepared SWCNTs have been verified by thermogravimetric analyses (TGA), Raman spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS). Finally, the protein resistance property of the PEGylated SWCNTs is investigated. It is found that these PEGylated SWCNTs have a good protein resistance property and the higher the content of PEG grafted on the SWCNTs, the less adsorption amount of BSA and the larger capacity to resist protein absorption. This work provides a novel method to prepare PEGylated SWCNTs. PMID:27398490

  6. 3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition

    NASA Astrophysics Data System (ADS)

    Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

    2015-11-01

    Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories.

  7. 3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition

    PubMed Central

    Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

    2015-01-01

    Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories. PMID:26526410

  8. 3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition.

    PubMed

    Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S

    2015-01-01

    Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories. PMID:26526410

  9. Incidence of single and mixed infections with Eimeria kofoidi, E. caucasica and E. legionensis on the health of experimentally infected red-legged partridges (Alectoris rufa).

    PubMed

    Naciri, M; Fort, G; Briant, J; Duperray, J; Benzoni, G

    2014-09-15

    Little is known about Eimeria-induced coccidiosis in partridges. After a coccidiosis outbreak in a farm rearing red-legged partridges (Alectoris rufa) in Brittany (France), three Eimeria species were identified as Eimeria kofoidi, Eimeria caucasica and Eimeria legionensis. This study aimed to reproduce the effects of the disease occurring in field conditions, in the absence of preventive treatments, to further build a coccidiosis model, helpful for coccidiostatic development. The pathogenic effects of a single infection with Eimeria kofoidi, E. caucasica and E. legionensis were evaluated, as well as the effects of multiple infections associating two or three of these species in red-legged partridges. Thirty-one-day-old birds were individually inoculated with Eimeria spp. and clinically followed up until 49 days of age. Mortality, lesion scores, daily oocyst production and growth were used as assessment criteria. Single infections with 250,000 E. kofoidi, 30,000 E. caucasica or 100,000 E. legionensis oocysts did not increase mortality rate compared to uninfected birds, whereas the combination of 3 species caused significant 28% mortality (P<0.05). Five days post inoculation, lesions scores in the proximal intestine (duodenum/jejunum) decreased in dual-infected birds and tended to decrease in triple-infected birds compared to lesions in mono-infected birds. The highest caecal lesion score was recorded in birds co-infected with E. kofoidi and E. legionensis. In multi-infected birds, the total number of oocysts excreted per gram of faeces was lower than the sum of oocysts excreted by mono-infected birds. Each single infection caused significant growth depression and even weight loss in E. legionensis infected birds (P<0.05). Dual or triple infections worsened this effect. Eighteen days post inoculation, only mono-infected birds had recovered. Their weight gains were not different from that of uninfected birds, whereas growth was significantly depressed in dual

  10. Associations of prepregnancy body mass index and gestational weight gain with pregnancy outcomes in nulliparous women delivering single live babies.

    PubMed

    Liu, Lu; Hong, Zhongxin; Zhang, Lihong

    2015-01-01

    The study was to assess the associations of prepregnancy body mass index (BMI) and gestational weight gain (GWG) with pregnancy outcomes. This was a retrospective analysis of 2973 nulliparous women who delivered single live babies. Prepregnancy BMI was categorized as underweight (<18.5 kg/m(2)), normal weight (18.5-24.9 kg/m(2)) or overweight/obese (≥25.0 kg/m(2)). GWG was categorized as inadequate, adequate or excessive. 567 (19.1%) women were overweight/obese, and 1600 (53.8%) exhibited excessive GWG. Compared with women of normal weight, overweight/obese women had a higher incidence of cesarean section (odds ratio, 95% confidence interval: 2.02, 1.59-2.56), postpartum hemorrhage (1.50, 1.05-2.14), preterm delivery (2.51, 1.83-3.45), preterm premature rupture of membranes (2.11, 1.32-3.38), gestational diabetes mellitus (2.04, 1.65-2.53), gestational hypertension (7.68, 4.21-14.00), preeclampsia (1.98, 1.18-3.33) and small for gestational age (2.81, 1.21-6.54). Compared with adequate GWG, excessive GWG increased the incidence of cesarean section (2.02, 1.59-2.56), preterm delivery (1.48, 1.05-2.71), preeclampsia (1.78, 1.34-4.27) and macrosomia (2.61, 1.61-4.25), and reduced the incidence of gestational diabetes mellitus (0.75, 0.62-0.92). High prepregnancy BMI and excessive GWG in nulliparous Chinese women are associated with adverse pregnancy outcomes. Weight control before and during pregnancy could reduce the complications of pregnancy. PMID:26242798

  11. Associations of prepregnancy body mass index and gestational weight gain with pregnancy outcomes in nulliparous women delivering single live babies

    PubMed Central

    Liu, Lu; Hong, Zhongxin; Zhang, Lihong

    2015-01-01

    The study was to assess the associations of prepregnancy body mass index (BMI) and gestational weight gain (GWG) with pregnancy outcomes. This was a retrospective analysis of 2973 nulliparous women who delivered single live babies. Prepregnancy BMI was categorized as underweight (<18.5 kg/m2), normal weight (18.5–24.9 kg/m2) or overweight/obese (≥25.0 kg/m2). GWG was categorized as inadequate, adequate or excessive. 567 (19.1%) women were overweight/obese, and 1600 (53.8%) exhibited excessive GWG. Compared with women of normal weight, overweight/obese women had a higher incidence of cesarean section (odds ratio, 95% confidence interval: 2.02, 1.59–2.56), postpartum hemorrhage (1.50, 1.05–2.14), preterm delivery (2.51, 1.83–3.45), preterm premature rupture of membranes (2.11, 1.32–3.38), gestational diabetes mellitus (2.04, 1.65–2.53), gestational hypertension (7.68, 4.21–14.00), preeclampsia (1.98, 1.18–3.33) and small for gestational age (2.81, 1.21–6.54). Compared with adequate GWG, excessive GWG increased the incidence of cesarean section (2.02, 1.59–2.56), preterm delivery (1.48, 1.05–2.71), preeclampsia (1.78, 1.34–4.27) and macrosomia (2.61, 1.61–4.25), and reduced the incidence of gestational diabetes mellitus (0.75, 0.62–0.92). High prepregnancy BMI and excessive GWG in nulliparous Chinese women are associated with adverse pregnancy outcomes. Weight control before and during pregnancy could reduce the complications of pregnancy. PMID:26242798

  12. Demonstration of specific binding of heparin to Plasmodium falciparum-infected vs. non-infected red blood cells by single-molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Valle-Delgado, Juan José; Urbán, Patricia; Fernàndez-Busquets, Xavier

    2013-04-01

    Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level.Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force

  13. Determination of single and repeated red cell volumes by the indicator dilution method using carbon monoxide as the indicator

    SciTech Connect

    Fukui, M.; Shigemi, K. )

    1989-11-01

    The use of radioactive isotopes limits clinical applications of blood volume measurement in the ICU. We measured red cell volumes with carbon monoxide-labeled RBC in six dogs and five human volunteers. The measured values obtained on the dogs were compared with the simultaneous measurements with the {sup 51}Cr method; the ratio of the carbon monoxide to {sup 51}Cr values ranged from 0.86 to 1.17, and the mean ratio was 1.0 +/- 0.1 (SD), r = .93. We infer from these results that the carbon monoxide method has several advantages over the {sup 51}Cr method: (a) the short labeling time (about 1 min), (b) rapidly decreasing background levels of carbon monoxide with FIO2 1.0, and (c) repeatability at intervals of several hours.

  14. Fibre-coupled red diode-pumped Alexandrite TEM00 laser with single and double-pass end-pumping

    NASA Astrophysics Data System (ADS)

    Arbabzadah, E. A.; Damzen, M. J.

    2016-06-01

    We report the investigation of an Alexandrite laser end-pumped by a fibre-coupled red diode laser module. Power, efficiency, spatial, spectral, and wavelength tuning performance are studied as a function of pump and laser cavity parameters. It is the first demonstration, to our knowledge, of greater than 1 W power and also highest laser slope efficiency (44.2%) in a diode-pumped Alexandrite laser with diffraction-limited TEM00 mode operation. Spatial quality was excellent with beam propagation parameter M 2 ~ 1.05. Wavelength tuning from 737–796 nm was demonstrated using an intracavity birefringent tuning filter. Using a novel double pass end-pumping scheme to get efficient absorption of both polarisation states of the scrambled fibre-delivered diode pump, a total output coupled power of 1.66 W is produced in TEM00 mode with 40% slope efficiency.

  15. A single phase, red emissive Mg2SiO4:Sm3+ nanophosphor prepared via rapid propellant combustion route.

    PubMed

    Naik, Ramachandra; Prashantha, S C; Nagabhushana, H; Sharma, S C; Nagaswarupa, H P; Anantharaju, K S; Nagabhushana, B M; Premkumar, H B; Girish, K M

    2015-04-01

    Mg2SiO4:Sm3+ (1-11 mol%) nanoparticles were prepared by a rapid low temperature solution combustion route. The powder X-ray diffraction (PXRD) patterns exhibit orthorhombic structure with α-phase. The average crystallite size estimated using Scherer's method, W-H plot and strain-size plots were found to be in the range 25-50 nm and the same was confirmed by Transmission Electron Microscopy (TEM). Scanning electron microscopy (SEM) pictures show porous structure and crystallites were agglomerated. The effect of Sm3+ cations on luminescence of Mg2SiO4 was well studied. Interestingly the samples could be effectively excited with 315 nm and emitted light in the red region, which was suitable for the demands of high efficiency WLEDs. The emission spectra consists of four main peaks which can be assigned to the intra 4-f orbital transitions of Sm3+ ions 4G5/2→6H5/2 (576 nm), 4G5/2→6H7/2 (611 nm), 4G5/2→6H9/2 (656 nm) and 4G5/2→6H11/2 (713 nm). The optimal luminescence intensity was obtained for 5 mol% Sm3+ ions. The CIE (Commission International de I'Eclairage) chromaticity co-ordinates were calculated from emission spectra, the values (0.588, 0.386) were close to the NTSC (National Television Standard Committee) standard value of red emission. Coordinated color temperature (CCT) was found to be 1756 K. Therefore optimized Mg2SiO4:Sm3+ (5 mol%) phosphor was quite useful for solid state lighting. PMID:25638435

  16. A single phase, red emissive Mg2SiO4:Sm3+ nanophosphor prepared via rapid propellant combustion route

    NASA Astrophysics Data System (ADS)

    Naik, Ramachandra; Prashantha, S. C.; Nagabhushana, H.; Sharma, S. C.; Nagaswarupa, H. P.; Anantharaju, K. S.; Nagabhushana, B. M.; Premkumar, H. B.; Girish, K. M.

    2015-04-01

    Mg2SiO4:Sm3+ (1-11 mol%) nanoparticles were prepared by a rapid low temperature solution combustion route. The powder X-ray diffraction (PXRD) patterns exhibit orthorhombic structure with α-phase. The average crystallite size estimated using Scherer's method, W-H plot and strain-size plots were found to be in the range 25-50 nm and the same was confirmed by Transmission Electron Microscopy (TEM). Scanning electron microscopy (SEM) pictures show porous structure and crystallites were agglomerated. The effect of Sm3+ cations on luminescence of Mg2SiO4 was well studied. Interestingly the samples could be effectively excited with 315 nm and emitted light in the red region, which was suitable for the demands of high efficiency WLEDs. The emission spectra consists of four main peaks which can be assigned to the intra 4-f orbital transitions of Sm3+ ions 4G5/2 → 6H5/2 (576 nm), 4G5/2 → 6H7/2 (611 nm), 4G5/2 → 6H9/2 (656 nm) and 4G5/2 → 6H11/2 (713 nm). The optimal luminescence intensity was obtained for 5 mol% Sm3+ ions. The CIE (Commission International de I'Eclairage) chromaticity co-ordinates were calculated from emission spectra, the values (0.588, 0.386) were close to the NTSC (National Television Standard Committee) standard value of red emission. Coordinated color temperature (CCT) was found to be 1756 K. Therefore optimized Mg2SiO4:Sm3+ (5 mol%) phosphor was quite useful for solid state lighting.

  17. Fracture mechanical behavior of red sandstone containing a single fissure and two parallel fissures after exposure to different high temperature treatments

    NASA Astrophysics Data System (ADS)

    Yang, Sheng-Qi; Jing, Hong-Wen; Huang, Yan-Hua; Ranjith, P. G.; Jiao, Yu-Yong

    2014-12-01

    A detailed understanding of the brittle deformation behavior of sandstone containing pre-existing flaws at elevated temperatures is a key concern in underground engineering. In this research, uniaxial compression tests were performed to evaluate the effect of high temperature treatments (300, 600 or 900 °C) on the strength, deformability and fracture coalescence behavior of a sandstone containing either a single fissure or two parallel fissures. All experiments focused on rectangular prismatic (80 × 160 × 30 mm) specimens of red sandstone. Constant strain rate experiments were performed on either: (1) specimens that contained a single 2 mm-wide fissure or (2) specimens that contained two 2 mm-wide parallel fissures. The specimens containing either one or two fissures were either left at room temperature (i.e., no heat treatment), or heat treated to 300, 600 or 900 °C prior to experimentation. The results demonstrated that, in all cases, the strength and stiffness of red sandstone was increased at 300 °C, before decreasing up to our maximum temperature of 900 °C. However, the peak strain at failure always showed an increase when the temperature was increased. The crack initiation, propagation and coalescence process were monitored during the deformation using both photographic monitoring and acoustic emission (AE) monitoring techniques. The monitoring results showed that the cracking process depended on both the fissure geometry and the heat treatment temperature. The potential mechanisms causing the differences in the mechanical behavior observed with increasing temperature are discussed, as is the influence of the single fissure and the two parallel fissures on the crack evolution process. These results are important and valuable to understand the fracture mechanism of rock engineering in deep underground mining excavations and nuclear waste depositories.

  18. Note: Multi-confocal fluorescence correlation spectroscopy in living cells using a complementary metal oxide semiconductor-single photon avalanche diode array

    NASA Astrophysics Data System (ADS)

    Kloster-Landsberg, M.; Tyndall, D.; Wang, I.; Walker, R.; Richardson, J.; Henderson, R.; Delon, A.

    2013-07-01

    Living cells are heterogeneous and rapidly changing biological samples. It is thus desirable to measure molecular concentration and dynamics in many locations at the same time. In this note, we present a multi-confocal setup capable of performing simultaneous fluorescence correlation spectroscopy measurements, by focusing the spots with a spatial light modulator and acquiring data with a monolithic 32 × 32 single-photon avalanche photodiode array. A post-processing method is proposed to correct cross-talk effects between neighboring spots. We demonstrate the applicability of our system by simultaneously measuring the diffusion of free enhanced Green Fluorescent Protein (eGFP) molecules at nine different points in living cells.

  19. Jupiter's Great Red spot

    NASA Technical Reports Server (NTRS)

    1979-01-01

    This color composite made from Voyager 2 narrow-angle camera frames shows the Great Red Spot during the late Jovian afternoon. North of the Red Spot lies a curious darker section of the South Equatorial Belt (SEB), the belt in which the Red Spot is located. A bright eruption of material passing from the SEB northward into the diffuse equatorial clouds has been observed on all occasions when this feature passes north of the Red Spot. The remnants of one such eruption are apparent in this photograph. To the lower left of the Red Spot lies one of the three long-lived White Ovals. This photograph was taken on June 29, 1979, when Voyager 2 was over 9 million kilometers (nearly 6 million miles) from Jupiter. The smallest features visible are over 170 kilometers (106 miles) across.

  20. Warm-white light-emitting diode with high color rendering index fabricated by combining trichromatic InGaN emitter with single red phosphor.

    PubMed

    Sheu, Jinn-Kong; Chen, Fu-Bang; Wang, Yen-Chin; Chang, Chih-Chiang; Huang, Shih-Hsien; Liu, Chun-Nan; Lee, Ming-Lun

    2015-04-01

    We present a trichromatic GaN-based light-emitting diode (LED) that emits near-ultraviolet (n-UV) blue and green peaks combined with red phosphor to generate white light with a low correlated color temperature (CCT) and high color rendering index (CRI). The LED structure, blue and green unipolar InGaN/GaN multiple quantum wells (MQWs) stacked with a top p-i-n structure containing an InGaN/GaN MQW emitting n-UV light, was grown epitaxially on a single substrate. The trichromatic LED chips feature a vertical conduction structure on a silicon substrate fabricated through wafer bonding and laser lift-off techniques. The blue and green InGaN/GaN MQWs were pumped with n-UV light to re-emit low-energy photons when the LEDs were electrically driven with a forward current. The emission spectrum included three peaks at approximately 405, 468, and 537 nm. Furthermore, the trichromatic LED chips were combined with red phosphor to generate white light with a CCT and CRI of approximately 2900 and 92, respectively. PMID:25968789

  1. [Raman spectra of single human living erythrocyte with the effect of pH and serum albumin].

    PubMed

    Wu, Zheng-Jie; Wang, Cheng; Lin, Zheng-Chun; Jiao, Qing-Ze

    2014-05-01

    In the present work, a cell environment which mimicked the real body environment according to the concentration radio between serum albumin and hemoglobin was built, and the cell morphology, the membrane deformation capacity, and the structure of intracellular hemoglobin of single human living erythrocyte under the effect of pH and serum albumin were studied. It was found that at different suspension pH, the magnitude of variations in cell shape and membrane deformation capacity changes with the structural changes of the intracellular hemoglobin. At pH 4. 14, 4. 76 and 10. 18, the loss of helical structure for hemoglobin, exposing of the hydrophobic amino acid in the globin chains, and changing of the combination of heme and globin, would completely destroy the stability of hemoglobin's structure, which seriously changes RBC's morphology and membrane deformation capacity. While at pH 6. 51 and 7. 80, the Raman spectra of erythrocytes are found to have no such changes, indicating that the structure of intracellular hemoglobin was not varied, thus the cell morphology and membrane deformation capacity are quite close to the normal values. At pH 5. 49 and 8. 76, RBC's morphology and membrane deformation capacity have different degrees of variation, but the structure of intracellular hemoglobin has not changed, suggesting that the cell morphology and membrane deformation capacity may be reversible. The results suggest that in the suspension solution containing serum albumin, erythrocytes have better ability to regulate and control the variation of the extracellular pH. In summary, upon building an environment which contains the same concentration radio of serum albumin to hemoglobin in the blood, this work performed systematic studies on the effect of pH on human erythrocytes. It can not only help to solve the problems about the mechanism of the structural and functional changes of erythrocytes induced by environmental pH, but also elucidates the possible variation of

  2. Efficacy of Korean Red Ginseng by Single Nucleotide Polymorphism in Obese Women: Randomized, Double-blind, Placebo-controlled Trial

    PubMed Central

    Kwon, Dong Hyun; Bose, Shambhunath; Song, Mi Young; Lee, Myeong Jong; Lim, Chi Yeon; Kwon, Bum Sun; Kim, Ho Jun

    2012-01-01

    This study examined the effects of Korean red ginseng (KRG) on obese women and aimed to confirm that the effects of KRG on obesity differ dependently on a gene. Fifty obese women were recruited and randomized to receive KRG (n=24) or placebo (n=26) for 8 wk. Measurements of blood pressure, height, weight, waist circumference, waist-hip ratio (WHR), total fat mass, percentage of body fat, resting metabolic rate, basal body temperature, and daily food intake (FI), blood test (serum lipid, liver and renal function), Korean version of obesity-related quality of life scale (KOQOL), and a gene examination were performed. Comparisons of subjects before and after the administration of KRG revealed significant improvements of obesity in terms of weight, body mass index (BMI), WHR, FI, and KOQOL. However, in the comparison between KRG group and placebo group, only KOQOL was significantly different. KRG displayed significant efficacy on BMI and KOQOL in the CT genotype of the G protein beta 3 gene, but not in the CC genotype, on blood sugar test in the Trp64/Arg genotype of the beta 3 adrenergic receptor gene, but not in Trp64/ Trp genotype, on KOQOL in the DD genotype of the angiotensin I converting enzyme gene, but not in the ID and DD genotypes. The effects of KRG on obesity were confirmed to some extent. However, a distinct effect compared to placebo was not confirmed. KRG is more effective for improving the secondary issues of the quality of life derived from obesity rather than having direct effects on the obesity-related anthropometric assessment and blood test indices. PMID:23717118

  3. Scan-Free Absorbance Spectral Imaging A(x, y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions.

    PubMed

    Isono, Takumi; Yamashita, Kyohei; Momose, Daisuke; Kobayashi, Hiroki; Kitamura, Masashi; Nishiyama, Yusuke; Hosoya, Takahiro; Kanda, Hiroaki; Kudo, Ayane; Okada, Norihide; Yagi, Takafumi; Nakata, Kazuaki; Mineki, Shigeru; Tokunaga, Eiji

    2015-01-01

    Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 μm × 1.2 μm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 μm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect. PMID:26061268

  4. Mining a database of single amplified genomes from Red Sea brine pool extremophiles—improving reliability of gene function prediction using a profile and pattern matching algorithm (PPMA)

    PubMed Central

    Grötzinger, Stefan W.; Alam, Intikhab; Ba Alawi, Wail; Bajic, Vladimir B.; Stingl, Ulrich; Eppinger, Jörg

    2014-01-01

    Reliable functional annotation of genomic data is the key-step in the discovery of novel enzymes. Intrinsic sequencing data quality problems of single amplified genomes (SAGs) and poor homology of novel extremophile's genomes pose significant challenges for the attribution of functions to the coding sequences identified. The anoxic deep-sea brine pools of the Red Sea are a promising source of novel enzymes with unique evolutionary adaptation. Sequencing data from Red Sea brine pool cultures and SAGs are annotated and stored in the Integrated Data Warehouse of Microbial Genomes (INDIGO) data warehouse. Low sequence homology of annotated genes (no similarity for 35% of these genes) may translate into false positives when searching for specific functions. The Profile and Pattern Matching (PPM) strategy described here was developed to eliminate false positive annotations of enzyme function before progressing to labor-intensive hyper-saline gene expression and characterization. It utilizes InterPro-derived Gene Ontology (GO)-terms (which represent enzyme function profiles) and annotated relevant PROSITE IDs (which are linked to an amino acid consensus pattern). The PPM algorithm was tested on 15 protein families, which were selected based on scientific and commercial potential. An initial list of 2577 enzyme commission (E.C.) numbers was translated into 171 GO-terms and 49 consensus patterns. A subset of INDIGO-sequences consisting of 58 SAGs from six different taxons of bacteria and archaea were selected from six different brine pool environments. Those SAGs code for 74,516 genes, which were independently scanned for the GO-terms (profile filter) and PROSITE IDs (pattern filter). Following stringent reliability filtering, the non-redundant hits (106 profile hits and 147 pattern hits) are classified as reliable, if at least two relevant descriptors (GO-terms and/or consensus patterns) are present. Scripts for annotation, as well as for the PPM algorithm, are available

  5. Pharmacokinetics of a single dose of intravenous and oral meloxicam in red-tailed hawks (Buteo jamaicensis) and great horned owls (Bubo virginianus).

    PubMed

    Lacasse, Claude; Gamble, Kathryn C; Boothe, Dawn M

    2013-09-01

    Pharmacokinetic data were determined after a single dose of meloxicam in red-tailed hawks (RTH; Buteo jamaicensis) and great horned owls (GHO; Bubo virginianus). In a nonrandomized crossover design, individual birds of each species received 1 dose of intravenous meloxicam (0.5 mg/kg i.v.; n = 7 for each species) followed by a 2-week washout period, and then each received 1 dose of oral meloxicam (0.5 mg/kg PO; n = 5 for each species). Blood samples were collected intermittently after administration, and meloxicam was detected in plasma by high-performance liquid chromatography. Time versus plasma concentration data were subjected to noncompartmental analysis. Red-tailed hawks were determined to have the shortest elimination half-life for meloxicam (0.49 +/- 0.5 hours) of any species documented. Great horned owls also eliminated meloxicam very rapidly (0.78 +/- 0.52 hours). Great horned owls achieved higher plasma concentrations (368 +/- 87 ng/mL) of meloxicam than RTH (182 +/- 167 ng/mL) after oral administration, although RTH had a markedly higher volume of distribution (832 +/- 711 mL/kg) than GHO (137.6 +/- 62.7 mL/kg). The differences in meloxicam pharmacokinetics between these 2 raptor species supports the need for species-dependent studies and underlines the challenges of extrapolating drug dosages between species. Results of this study suggest that the current recommended once-daily dosing interval of oral meloxicam is unlikely to maintain plasma concentrations anticipated to be therapeutic in either RTH or GHO, and practical dosing options are questionable for this nonsteriodal anti-inflammatory drug in these raptor species. PMID:24344511

  6. A low temperature X-ray single-crystal diffraction and polarised infra-red study of epidote

    NASA Astrophysics Data System (ADS)

    Gatta, G. Diego; Alvaro, Matteo; Bromiley, Geoffrey

    2012-01-01

    The effects of low-temperature on the crystal structure of a natural epidote [Ca1.925Fe0.745Al2.265Ti0.004Si3.037O12(OH), a = 8.8924(7), b = 5.6214(3), c = 10.1547(6)Å and β = 115.396(8)° at room conditions, Sp. Gr. P21 /m] have been investigated with a series of structure refinements down to 100 K on the basis of X-ray single-crystal diffraction data. The reflection conditions confirm that the space group is maintained within the T-range investigated. Structural refinements at all temperatures show the presence of Fe3+ at the octahedral M(3) site only [%Fe(M3) = 70.6(4)% at 295 K]. Only one independent proton site was located and two possible H-bonds occur, with O(10) as donor and O(4) and O(2) as acceptors. The H-bonding scheme is maintained down to 100 K and is supported by single crystal room- T polarised FTIR data. FTIR Spectra over the region 4,000-2,500 cm-1 are dominated by the presence of a strongly pleochroic absorption feature which can be assigned to protonation of O(10)-O(4). Previously unobserved splitting of this absorption features is consistent with a NNN influence due to the presence of Al and Fe3+ on the nearby M(3) site. An additional relatively minor absorption feature in FTIR spectra can be tentatively assigned to protonation of O(10)-O(2). Low- T does not affect significantly the tetrahedral and octahedral bond distances and angles, even when distances are corrected for "rigid body motions". A more significant effect is observed for the bond distances of the distorted Ca(1)- and Ca(2)-polyhedra, especially when corrected for "non-correlated motion". The main low- T effect is observed on the vibrational regime of the atomic sites, and in particular for the two Ca-sites. A significant reduction of the magnitude of the thermal displacement ellipsoids, with a variation of U eq (defined as one-third of the trace of the orthogonalised U ij tensor) by ~40% is observed for the Ca-sites between 295 and 100 K. Within the same T-range, the U eq of

  7. Public and Private Lives: Institutional Structures and Personal Supports in Low-Income Single Mothers' Educational Pursuits

    ERIC Educational Resources Information Center

    Cerven, Christine

    2013-01-01

    Drawing on a case study of 60 low-income single mothers in California, I present a grounded account of the barriers and supports single mothers encounter in their pursuit of postsecondary education (PSE) and detail what the women themselves attributed to their success. I highlight the role both significant others (peers, family, friends) and…

  8. Infra-red spectral microscopy of standing-wave resonances in single metal-dielectric-metal thin-film cavity

    NASA Astrophysics Data System (ADS)

    Nath, Janardan; Panjwani, Deep; Khalilzadeh-Rezaie, Farnood; Yesiltas, Mehmet; Smith, Evan M.; Ginn, James C.; Shelton, David J.; Hirschmugl, Carol; Cleary, Justin W.; Peale, Robert E.

    2015-09-01

    Resonantly absorbing thin films comprising periodically sub-wavelength structured metal surface, dielectric spacer, and metal ground plane are a topic of current interest with important applications. These structures are frequently described as "metamaterials", where effective permittivity and permeability with dispersion near electric and magnetic resonances allow impedance matching to free space for maximum absorption. In this paper, we compare synchrotron-based infrared spectral microscopy of a single isolated unit cell and a periodic array, and we show that the resonances have little to do with periodicity. Instead, the observed absorption spectra of usual periodically structured thin films are best described as due to standing-wave resonances within each independent unit cell, rather than as due to effective optical constants of a metamaterial. The effect of having arrays of unit cells is mainly to strengthen the absorption by increasing the fill factor, and such arrays need not be periodic. Initial work toward applying the subject absorbers to room-temperature bolometer arrays is presented.

  9. Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons.

    PubMed

    Widera, Darius; Klenke, Christin; Nair, Deepak; Heidbreder, Meike; Malkusch, Sebastian; Sibarita, Jean-Baptiste; Choquet, Daniel; Kaltschmidt, Barbara; Heilemann, Mike; Kaltschmidt, Christian

    2016-10-01

    Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient ([Formula: see text]) in neurites from [Formula: see text] to [Formula: see text] after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from [Formula: see text] to [Formula: see text], whereas a velocity increase from [Formula: see text] to [Formula: see text] was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons. PMID:27226975

  10. Microbead encapsulation of living plant protoplasts: A new tool for the handling of single plant cells1

    PubMed Central

    Grasso, Matthew S.; Lintilhac, Philip M.

    2016-01-01

    Premise of the study: Understanding plant cell biomechanics has been hampered by a lack of appropriate experimental tools. Here we introduce a protocol for the incorporation of individual plant protoplasts into precisely sized agarose microbeads. This technology may lead to new ways to manipulate the physical and chemical microenvironment of individual plant cells. Methods and Results: Living protoplasts obtained from BY-2 tobacco suspension cultures were continuously incorporated into a stream of agarose microdroplets, collected in cooled mineral oil as gelled microbeads, and then transferred into liquid MS medium for culture. In this first report, we show that spherical microbeads containing living protoplasts can be easily generated in quantity and that these encapsulated cells continue to grow and divide. Conclusions: Microbead encapsulation of protoplasts affords the opportunity to precisely control the physical microenvironment of individual plant cells. Ultimately, this method may help facilitate novel studies in plant biomechanics. PMID:27213126

  11. Eye redness

    MedlinePlus

    Bloodshot eyes; Red eyes; Scleral infection; Conjunctival infection ... There are many causes of a red eye or eyes. Some are medical emergencies and some are a cause for concern, but not an emergency. Others are nothing to worry about. ...

  12. Red Clover

    MedlinePlus

    ... 17):2057–2071. Red clover. Natural Medicines Comprehensive Database Web site. Accessed at www.naturaldatabase.com on July 22, 2009. Red clover ( Trifolium pratense ). Natural Standard Database Web site. Accessed at www.naturalstandard.com on July ...

  13. Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes.

    PubMed

    Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

    2016-01-01

    The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

  14. Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

    PubMed Central

    Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P.; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

    2016-01-01

    The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

  15. Determinants of graft survival in pediatric and adolescent live donor kidney transplant recipients: a single center experience.

    PubMed

    El-Husseini, Amr A; Foda, Mohamed A; Shokeir, Ahmed A; Shehab El-Din, Ahmed B; Sobh, Mohamed A; Ghoneim, Mohamed A

    2005-12-01

    To study the independent determinants of graft survival among pediatric and adolescent live donor kidney transplant recipients. Between March 1976 and March 2004, 1600 live donor kidney transplants were carried out in our center. Of them 284 were 20 yr old or younger (mean age 13.1 yr, ranging from 5 to 20 yr). Evaluation of the possible variables that may affect graft survival were carried out using univariate and multivariate analyses. Studied factors included age, gender, relation between donor and recipient, original kidney disease, ABO blood group, pretransplant blood transfusion, human leukocyte antigen (HLA) matching, pretransplant dialysis, height standard deviation score (SDS), pretransplant hypertension, cold ischemia time, number of renal arteries, ureteral anastomosis, time to diuresis, time of transplantation, occurrence of acute tubular necrosis (ATN), primary and secondary immunosuppression, total dose of steroids in the first 3 months, development of acute rejection and post-transplant hypertension. Using univariate analysis, the significant predictors for graft survival were HLA matching, type of primary urinary recontinuity, time to diuresis, ATN, acute rejection and post-transplant hypertension. The multivariate analysis restricted the significance to acute rejection and post-transplant hypertension. The independent determinants of graft survival in live-donor pediatric and adolescent renal transplant recipients are acute rejection and post-transplant hypertension. PMID:16269048

  16. Role of ICAM-1 polymorphisms (G241R, K469E) in mediating its single-molecule binding ability: Atomic force microscopy measurements on living cells

    SciTech Connect

    Bai, Rui; Yi, Shaoqiong; Zhang, Xuejie; Liu, Huiliang; Fang, Xiaohong

    2014-06-13

    Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP induced changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.

  17. Two-color RESOLFT nanoscopy with green and red fluorescent photochromic proteins.

    PubMed

    Lavoie-Cardinal, Flavie; Jensen, Nickels A; Westphal, Volker; Stiel, Andre C; Chmyrov, Andriy; Bierwagen, Jakob; Testa, Ilaria; Jakobs, Stefan; Hell, Stefan W

    2014-03-17

    Up to now, all demonstrations of reversible saturable optical fluorescence transitions (RESOLFT) superresolution microscopy of living cells have relied on the use of reversibly switchable fluorescent proteins (RSFP) emitting in the green spectral range. Here we show RESOLFT imaging with rsCherryRev1.4, a new red-emitting RSFP enabling a spatial resolution up to four times higher than the diffraction barrier. By co-expressing green and red RSFPs in living cells we demonstrate two-color RESOLFT imaging both for single ("donut") beam scanning and for parallelized versions of RESOLFT nanoscopy where an array of >23,000 "donut-like" minima are scanned simultaneously. PMID:24449030

  18. Red clover

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover (Trifolium pratense L.) is an important forage legume grown on approximately 4 million hectares worldwide. An estimated 2.8 million kg of red clover seed per year was produced worldwide in 2005-2007. This amount of seed would be enough to maintain approximately 4 million hectares of red...

  19. Red Sea

    Atmospheric Science Data Center

    2013-04-16

    article title:  The Red Sea     View Larger Image ... Imaging SpectroRadiometer (MISR) image of the Red Sea was acquired on August 13, 2000. Located between the East African coast and the Saudi Arabian peninsula, the Red Sea got its name because the blooms of a type of algae,  Trichodesmium ...

  20. iSBatch: a batch-processing platform for data analysis and exploration of live-cell single-molecule microscopy images and other hierarchical datasets.

    PubMed

    Caldas, Victor E A; Punter, Christiaan M; Ghodke, Harshad; Robinson, Andrew; van Oijen, Antoine M

    2015-10-01

    Recent technical advances have made it possible to visualize single molecules inside live cells. Microscopes with single-molecule sensitivity enable the imaging of low-abundance proteins, allowing for a quantitative characterization of molecular properties. Such data sets contain information on a wide spectrum of important molecular properties, with different aspects highlighted in different imaging strategies. The time-lapsed acquisition of images provides information on protein dynamics over long time scales, giving insight into expression dynamics and localization properties. Rapid burst imaging reveals properties of individual molecules in real-time, informing on their diffusion characteristics, binding dynamics and stoichiometries within complexes. This richness of information, however, adds significant complexity to analysis protocols. In general, large datasets of images must be collected and processed in order to produce statistically robust results and identify rare events. More importantly, as live-cell single-molecule measurements remain on the cutting edge of imaging, few protocols for analysis have been established and thus analysis strategies often need to be explored for each individual scenario. Existing analysis packages are geared towards either single-cell imaging data or in vitro single-molecule data and typically operate with highly specific algorithms developed for particular situations. Our tool, iSBatch, instead allows users to exploit the inherent flexibility of the popular open-source package ImageJ, providing a hierarchical framework in which existing plugins or custom macros may be executed over entire datasets or portions thereof. This strategy affords users freedom to explore new analysis protocols within large imaging datasets, while maintaining hierarchical relationships between experiments, samples, fields of view, cells, and individual molecules. PMID:26198886

  1. Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis.

    PubMed Central

    Hodgson, A L; Tachedjian, M; Corner, L A; Radford, A J

    1994-01-01

    An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector. Images PMID:7960105

  2. Mechanisms of Alizarin Red S and Methylene blue biosorption onto olive stone by-product: Isotherm study in single and binary systems.

    PubMed

    Albadarin, Ahmad B; Mangwandi, Chirangano

    2015-12-01

    The biosorption process of anionic dye Alizarin Red S (ARS) and cationic dye methylene blue (MB) as a function of contact time, initial concentration and solution pH onto olive stone (OS) biomass has been investigated. Equilibrium biosorption isotherms in single and binary systems and kinetics in batch mode were also examined. The kinetic data of the two dyes were better described by the pseudo second-order model. At low concentration, ARS dye appeared to follow a two-step diffusion process, while MB dye followed a three-step diffusion process. The biosorption experimental data for ARS and MB dyes were well suited to the Redlich-Peterson isotherm. The maximum biosorption of ARS dye, qmax = 16.10 mg/g, was obtained at pH 3.28 and the maximum biosorption of MB dye, qmax = 13.20 mg/g, was observed at basic pH values. In the binary system, it was indicated that the MB dye diffuses firstly inside the biosorbent particle and occupies the biosorption sites forming a monodentate complex and then the ARS dye enters and can only bind to untaken sites; forms a tridentate complex with OS active sites. PMID:26355260

  3. Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells.

    PubMed

    Grecco, H E; Lidke, K A; Heintzmann, R; Lidke, D S; Spagnuolo, C; Martinez, O E; Jares-Erijman, E A; Jovin, T M

    2004-11-01

    In this work, we characterized streptavidin-conjugated quantum dots (QDs) manufactured by Quantum Dot Corporation. We present data on: (1) two-photon excitation; (2) fluorescence lifetimes; (3) ensemble and single QD emission anisotropy; (4) QDs as donors for Forster resonance energy transfer (FRET); and (5) spectral conversion of QDs exposed to high-intensity illumination. We also demonstrate the utility of QDs for (1) imaging the binding and uptake of biotinylated transferrin on living cells, and (2) resolving by fluorescence lifetime imaging microscopy (FLIM) signals originating from QDs from those of spatially and spectrally overlapping visible fluorescent proteins (VFPs). PMID:15630694

  4. Confocal spectral imaging by microspectrofluorometry using two-photon excitation: application to the study of anticancer drugs within single living cancer cells

    NASA Astrophysics Data System (ADS)

    Chourpa, Igor; Pereira, Manuela; Millot, Jean-Marc; Morjani, Hamid; Manfait, Michel

    1999-06-01

    The use of the two-photon excitation (TPE) is believed to be prominent for fluorometric studies with cells. We evaluated the advantages and limitations of the two-photon technique compared to the single photon one when it used to detect potent anticancer drugs, camptothecins (CPTs), within single living cancer cells. The technique we used was confocal microspectrofluorometry amplified with possibility of the spectral imaging analysis. We have previously reported the use of the florescence emission of CPTs to study them qualitatively and quantitatively, namely, to follow the status of their hydrolyzable lactone moiety. However, the intracellular investigation of CPTs using microspectrofluorometry with single photon UV excitation (SPE) is hindered by significant interference of their fluorescence emission with cellular autofluorescence. We attempted to overcome these problems using the two-photon excitation. The intracellular single-photon- and two-photon-excited emission spectra from treated and control cells (HCT-116 line) were recorded using a spectral imaging approach. The obtained data demonstrate that, apart from intrinsically increased three- dimensional resolution, the two-photon approach was advantageous over the single-photon method with respect to selective fluorometric detection of intracellular CPTs. Nevertheless, much attention should be paid to avoid any excessive irradiation of the cells with UV and even NIR light.

  5. African American Single Mothers: Understanding Their Lives and Families. Sage Series on Race and Ethnic Relations, Volume 10.

    ERIC Educational Resources Information Center

    Dickerson, Bette J.

    Past research on African American single mothers and their families has been conducted using conventional paradigms based upon models of the dominant culture. This practice has resulted in the creation of stereotypes and misconceptions about "the Black family." In this collection of original work by an interdisciplinary group of scholars, the…

  6. Determination of the stoichiometry, structure, and distribution in living cells of protein complexes from analysis of single-molecular-complexes FRET

    NASA Astrophysics Data System (ADS)

    Stoneman, Michael R.; Patowary, S.; Roesch, M. T.; Singh, D. R.; Strogolov, V.; Oliver, J. A.; Raicu, V.

    2011-03-01

    Advances in two-photon microscopy with spectral resolution (TPM-SR) and the development of a simple theory of Förster Resonance Energy Transfer (FRET) for single molecular complexes recently lead to the development of a novel method for the determination of structure and localization in living cells of membrane protein complexes (Raicu et al., Nature Photon., 3, 2009). An appealing feature of this method is its ability to provide such important information while being unaffected by spurious signals originating from stochastic FRET (Singh and Raicu, Biophys. J., 98, 2010). We will present the results obtained from our recent studies of trimeric FRET calibration standards expressed in the cytoplasm of Chinese hamster ovary (CHO) cells, as well as a model G protein-coupled receptor expressed in the membrane of yeast. Emphasis will be placed on the measurement and analysis of single-molecular-complex FRET data for determination of the quaternary structure of some proteins (or the protein complex structure).

  7. A Microfluidic-Enabled Mechanical Microcompressor for the Immobilization of Live Single- and Multi-Cellular Specimens

    PubMed Central

    Yan, Yingjun; Jiang, Liwei; Aufderheide, Karl J.; Wright, Gus A.; Terekhov, Alexander; Costa, Lino; Qin, Kevin; McCleery, W. Tyler; Fellenstein, John J.; Ustione, Alessandro; Robertson, J. Brian; Johnson, Carl Hirschie; Piston, David W.; Hutson, M. Shane; Wikswo, John P.; Hofmeister, William; Janetopoulos, Chris

    2014-01-01

    A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila. PMID:24444078

  8. A microfluidic-enabled mechanical microcompressor for the immobilization of live single- and multi-cellular specimens.

    PubMed

    Yan, Yingjun; Jiang, Liwei; Aufderheide, Karl J; Wright, Gus A; Terekhov, Alexander; Costa, Lino; Qin, Kevin; McCleery, W Tyler; Fellenstein, John J; Ustione, Alessandro; Robertson, J Brian; Johnson, Carl Hirschie; Piston, David W; Hutson, M Shane; Wikswo, John P; Hofmeister, William; Janetopoulos, Chris

    2014-02-01

    A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila. PMID:24444078

  9. Evaluation of the insertion efficiencies of tapered silicon nanoneedles and invasiveness of diamond nanoneedles in manipulations of living single cells.

    PubMed

    Han, Sung-Woong; Ryu, Seunghwan; Kitagawa, Taro; Uetsuka, Hiroshi; Fujimori, Naoji; Aoki, Yukihiro; Ota, Ryo; Amemiya, Yosuke; Shimamoto, Nobuo; Nakamura, Chikashi; Miyake, Jun

    2009-01-01

    We have been developing a low invasive cell manipulation technology based on inserting an ultra-thin needle--"nanoneedle"--into a living cell by using an atomic force microscope (AFM). The nanoneedle, made from a silicon AFM tip by focused-ion-beam etching, has a diameter of several hundred nanometers and a length of about 10 microns. Successful insertion of the nanoneedle into the cell can be confirmed by the appearance of a steep relaxation of repulsive force in the force-distance curve as monitored by the AFM system. This technology, termed "cell surgery", can be applied for the detection of intracellular proteins in a living cell or for highly efficient gene transfer. The present study shows that the durability of a tapered nanoneedle is superior to that of a cylindrical nanoneedle, and that a proper aspect ratio for the tapered nanoneedle must be chosen to maintain sufficient insertion efficiency for a particular target cell: tapered nanoneedles of an aspect ratio over 20 showed high insertion efficiency for various kinds of mammalian cells. We then used diamond for the material of the nanoneedle because its specific properties, such as high stiffness, heat conductivity, and electrical conductivity capacitated by boron doping, were deemed useful for the analysis and manipulation of intracellular phenomena. We compared the capability of the diamond nanoneedle in cell manipulation with that of the silicon nanoneedle. Evaluation of the effect of the former on transcription efficiency and localization analysis of p53 expression revealed the low invasiveness for cell manipulation as was also the case for the silicon nanoneedle. We also succeeded in achieving highly efficient plasmid DNA delivery into a mouse fibroblast C3H10T1/2 using the diamond nanoneedle. The diamond nanoneedle is expected to contribute to the versatility of "cell surgery" technology. PMID:21471661

  10. Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level

    PubMed Central

    Monico, Carina; Capitanio, Marco; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S.

    2013-01-01

    The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates, explained in current models by a combination of 3D and 1D diffusion. Here, we present a review of the single-molecule approaches at the forefront of the study of protein-DNA interaction dynamics and target search in vitro and in vivo. Flow stretch, optical and magnetic manipulation, single fluorophore detection and localization as well as combinations of different methods are described and the results obtained with these techniques are discussed in the framework of the current facilitated diffusion model. PMID:23429188

  11. Seeing Red

    NASA Technical Reports Server (NTRS)

    2008-01-01

    This New Horizons image of Jupiter's volcanic moon Io was taken at 13:05 Universal Time during the spacecraft's Jupiter flyby on February 28, 2007. It shows the reddish color of the deposits from the giant volcanic eruption at the volcano Tvashtar, near the top of the sunlit crescent, as well as the bluish plume itself and the orange glow of the hot lava at its source. The relatively unprocessed image on the left provides the best view of the volcanic glow and the plume deposits, while the version on the right has been brightened to show the much fainter plume, and the Jupiter-lit night side of Io.

    New Horizons' color imaging of Io's sunlit side was generally overexposed because the spacecraft's color camera, the super-sensitive Multispectral Visible Imaging Camera (MVIC), was designed for the much dimmer illumination at Pluto. However, two of MVIC's four color filters, the blue and 'methane' filter (a special filter designed to map methane frost on the surface of Pluto at an infrared wavelength of 0.89 microns), are less sensitive than the others, and thus obtained some well-exposed views of the surface when illumination conditions were favorable. Because only two color filters are used, rather than the usual three, and because one filter uses infrared light, the color is only a rough approximation to what the human eye would see.

    The red color of the Tvashtar plume fallout is typical of Io's largest volcanic plumes, including the previous eruption of Tvashtar seen by the Galileo and Cassini spacecraft in 2000, and the long-lived Pele plume on the opposite side of Io. The color likely results from the creation of reddish three-atom and four-atom sulfur molecules (S3 and S4) from plume gases rich in two-atom sulfur molecules (S2 After a few months or years, the S3 and S4 molecules recombine into the more stable and familiar yellowish form of sulfur consisting of eight-atom molecules (S8), so these red deposits are only seen around recently-active Io

  12. Authenticity control of game meat products--a single method to detect and quantify adulteration of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon) by real-time PCR.

    PubMed

    Druml, Barbara; Grandits, Stephanie; Mayer, Walter; Hochegger, Rupert; Cichna-Markl, Margit

    2015-03-01

    This contribution presents a single real-time PCR assay allowing the determination of the deer content (the sum of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon)) in meat products to detect food adulteration. The PCR assay does not show cross-reactivity with 20 animal species and 43 botanical species potentially contained in game meat products. The limit of quantification is 0.5% for fallow deer and red deer and 0.1% for sika deer. The deer content in meat products is determined by relating the concentration obtained with the deer PCR assay to that obtained with a reference system which amplifies mammals and poultry DNA. The analysis of binary meat mixtures with pork, a meat mixture containing equal amounts of fallow deer, red deer and sika deer in pork and a model game sausage showed that the quantification approach is very accurate (systematic error generally <25%). PMID:25306377

  13. Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes

    PubMed Central

    Katz, Zachary B; English, Brian P; Lionnet, Timothée; Yoon, Young J; Monnier, Nilah; Ovryn, Ben; Bathe, Mark; Singer, Robert H

    2016-01-01

    Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality. DOI: http://dx.doi.org/10.7554/eLife.10415.001 PMID:26760529

  14. A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells

    NASA Astrophysics Data System (ADS)

    English, Brian P.; Singer, Robert H.

    2015-08-01

    Our aim is to develop quantitative single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a three-camera imaging microscope for fast tracking of multiple interacting molecules simultaneously, with high spatiotemporal resolution. The system was designed around an ASI RAMM frame using three separate tube lenses and custom multi-band dichroics to allow for enhanced detection efficiency. The frame times of the three Andor iXon Ultra EMCCD cameras are hardware synchronized to the laser excitation pulses of the three excitation lasers, such that the fluorophores are effectively immobilized during frame acquisitions and do not yield detections that are motion-blurred. Stroboscopic illumination allows robust detection from even rapidly moving molecules while minimizing bleaching, and since snapshots can be spaced out with varying time intervals, stroboscopic illumination enables a direct comparison to be made between fast and slow molecules under identical light dosage. We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. The three-color microscope combined with our co-movement algorithms have made it possible for instance to simultaneously image and track how the chromosome environment affects diffusion kinetics or determine how mRNAs diffuse during translation. Such multiplexed single-molecule measurements at a high spatiotemporal resolution inside living cells will provide a major tool for testing models relating molecular architecture and biological dynamics.

  15. The fluorescent bioprobe with aggregation-induced emission features for monitoring to carbon dioxide generation rate in single living cell and early identification of cancer cells.

    PubMed

    Chen, Didi; Wang, Huan; Dong, Lichao; Liu, Pai; Zhang, Yahui; Shi, Jianbing; Feng, Xiao; Zhi, Junge; Tong, Bin; Dong, Yuping

    2016-10-01

    A novel fluorescent probe, tris (2-(dimethylamino) ethyl)-4,4',4″-(1H-pyrrole-1,2,5-triyl) tribenzoate (TPP-TMAE), with aggregation-enhanced emission (AEE) feature showed a simple, highly selective, specific, and instant response to trace amount carbon dioxide (CO2). Because of this special characteristic, TPP-TMAE is ideal to be a biomarker for in-situ monitoring of the CO2 generation rate during the metabolism of single living cell. The rates in single living HeLa cell, MCF-7 cell, and MEF cell were 6.40 × 10(-6)±6.0 × 10(-8) μg/h, 5.78 × 10(-6)±6.0 × 10(-8) μg/h, and 4.27 × 10(-7)±4.0 × 10(-9) μg/h, respectively. The distinct responses of TPP-TMAE to CO2 generated from cancer cells and normal cells suggested TPP-TMAE as a useful tool for deeper understanding metabolism process and distinguishing cancer cells from normal cells during the early diagnosis of cancers. PMID:27372422

  16. A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells

    PubMed Central

    English, Brian P.; Singer, Robert H.

    2016-01-01

    Our aim is to develop quantitative single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a three-camera imaging microscope for fast tracking of multiple interacting molecules simultaneously, with high spatiotemporal resolution. The system was designed around an ASI RAMM frame using three separate tube lenses and custom multi-band dichroics to allow for enhanced detection efficiency. The frame times of the three Andor iXon Ultra EMCCD cameras are hardware synchronized to the laser excitation pulses of the three excitation lasers, such that the fluorophores are effectively immobilized during frame acquisitions and do not yield detections that are motion-blurred. Stroboscopic illumination allows robust detection from even rapidly moving molecules while minimizing bleaching, and since snapshots can be spaced out with varying time intervals, stroboscopic illumination enables a direct comparison to be made between fast and slow molecules under identical light dosage. We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. The three-color microscope combined with our co-movement algorithms have made it possible for instance to simultaneously image and track how the chromosome environment affects diffusion kinetics or determine how mRNAs diffuse during translation. Such multiplexed single-molecule measurements at a high spatiotemporal resolution inside living cells will provide a major tool for testing models relating molecular architecture and biological dynamics. PMID:26819489

  17. Free-Living Species of Carnivorous Mammals in Poland: Red Fox, Beech Marten, and Raccoon as a Potential Reservoir of Salmonella, Yersinia, Listeria spp. and Coagulase-Positive Staphylococcus.

    PubMed

    Nowakiewicz, Aneta; Zięba, Przemysław; Ziółkowska, Grażyna; Gnat, Sebastian; Muszyńska, Marta; Tomczuk, Krzysztof; Majer Dziedzic, Barbara; Ulbrych, Łukasz; Trościańczyk, Aleksandra

    2016-01-01

    The objective of the study was to examine a population of free-living carnivorous mammals most commonly found in Poland (red fox, beech marten, and raccoon) for the occurrence of bacteria that are potentially pathogenic for humans and other animal species and to determine their virulence potential (the presence of selected virulence genes). From the total pool of isolates obtained (n = 328), we selected 90 belonging to species that pose the greatest potential threat to human health: Salmonella spp. (n = 19; 4.51%), Yersinia enterocolitica (n = 10; 2.37%), Listeria monocytogenes and L. ivanovii (n = 21), and Staphylococcus aureus (n = 40; 9.5%). The Salmonella spp. isolates represented three different subspecies; S. enterica subsp. enterica accounted for a significant proportion (15/19), and most of the serotypes isolated (S. Typhimurium, S. Infantis, S. Newport and S. Enteritidis) were among the 10 non-typhoidal Salmonella serotypes that are most often responsible for infections in Europe, including Poland. Y. enterococlitica was detected in the smallest percentage of animals, but 60% of strains among the isolates tested possessed the ail gene, which is responsible for attachment and invasion. Potentially pathogenic Listeria species were isolated from approx. 5% of the animals. The presence of all tested virulence genes was shown in 35% of L. monocytogenes strains, while in the case of the other strains, the genes occurred in varying numbers and configurations. The presence of the inlA, inlC, hlyA, and iap genes was noted in all strains, whereas the genes encoding PI-PLC, actin, and internalin Imo2821 were present in varying percentages (from 80% to 55%). S. aureus was obtained from 40 individuals. Most isolates possessed the hla, hld (95% for each), and hlb (32.5%) genes encoding hemolysins as well as the gene encoding leukotoxin lukED (70%). In a similar percentage of strains (77.5%), the presence of at least one gene encoding enterotoxin was found, with 12

  18. Free-Living Species of Carnivorous Mammals in Poland: Red Fox, Beech Marten, and Raccoon as a Potential Reservoir of Salmonella, Yersinia, Listeria spp. and Coagulase-Positive Staphylococcus

    PubMed Central

    Nowakiewicz, Aneta; Zięba, Przemysław; Ziółkowska, Grażyna; Gnat, Sebastian; Muszyńska, Marta; Tomczuk, Krzysztof; Majer Dziedzic, Barbara; Ulbrych, Łukasz; Trościańczyk, Aleksandra

    2016-01-01

    The objective of the study was to examine a population of free-living carnivorous mammals most commonly found in Poland (red fox, beech marten, and raccoon) for the occurrence of bacteria that are potentially pathogenic for humans and other animal species and to determine their virulence potential (the presence of selected virulence genes). From the total pool of isolates obtained (n = 328), we selected 90 belonging to species that pose the greatest potential threat to human health: Salmonella spp. (n = 19; 4.51%), Yersinia enterocolitica (n = 10; 2.37%), Listeria monocytogenes and L. ivanovii (n = 21), and Staphylococcus aureus (n = 40; 9.5%). The Salmonella spp. isolates represented three different subspecies; S. enterica subsp. enterica accounted for a significant proportion (15/19), and most of the serotypes isolated (S. Typhimurium, S. Infantis, S. Newport and S. Enteritidis) were among the 10 non-typhoidal Salmonella serotypes that are most often responsible for infections in Europe, including Poland. Y. enterococlitica was detected in the smallest percentage of animals, but 60% of strains among the isolates tested possessed the ail gene, which is responsible for attachment and invasion. Potentially pathogenic Listeria species were isolated from approx. 5% of the animals. The presence of all tested virulence genes was shown in 35% of L. monocytogenes strains, while in the case of the other strains, the genes occurred in varying numbers and configurations. The presence of the inlA, inlC, hlyA, and iap genes was noted in all strains, whereas the genes encoding PI-PLC, actin, and internalin Imo2821 were present in varying percentages (from 80% to 55%). S. aureus was obtained from 40 individuals. Most isolates possessed the hla, hld (95% for each), and hlb (32.5%) genes encoding hemolysins as well as the gene encoding leukotoxin lukED (70%). In a similar percentage of strains (77.5%), the presence of at least one gene encoding enterotoxin was found, with 12

  19. A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells

    PubMed Central

    Huang, Peng; Chiu, Yi-Ting; Chen, Chongguang; Wang, Yujun; Liu-Chen, Lee-Yuan

    2013-01-01

    Introduction In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs available, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. Methods We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. Results KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4 min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. Discussion tdT is an alternative to, or even a better tool than, GFPs for fusing to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance. PMID:23856011

  20. Bias-corrected diagnostic performance of the naked-eye single-tube red-cell osmotic fragility test (NESTROFT): an effective screening tool for beta-thalassemia.

    PubMed

    Mamtani, Manju; Jawahirani, Anil; Das, Kishor; Rughwani, Vinky; Kulkarni, Hemant

    2006-08-01

    It is being increasingly recognized that a majority of the countries in the thalassemia-belt need a cost-effective screening program as the first step towards control of thalassemia. Although the naked eye single tube red cell osmotic fragility test (NESTROFT) has been considered to be a very effective screening tool for beta-thalassemia trait, assessment of its diagnostic performance has been affected with the reference test- and verification-bias. Here, we set out to provide estimates of sensitivity and specificity of NESTROFT corrected for these potential biases. We conducted a cross-sectional diagnostic test evaluation study using data from 1563 subjects from Central India with a high prevalence of beta-thalassemia. We used latent class modelling after ensuring its validity to account for the reference test bias and global sensitivity analysis to control the verification bias. We also compared the results of latent class modelling with those of five discriminant indexes. We observed that across a range of cut-offs for the mean corpuscular volume (MCV) and the hemoglobin A2 (HbA2) concentration the average sensitivity and specificity of NESTROFT obtained from latent class modelling was 99.8 and 83.7%, respectively. These estimates were comparable to those characterizing the diagnostic performance of HbA2, which is considered by many as the reference test to detect beta-thalassemia. After correction for the verification bias these estimates were 93.4 and 97.2%, respectively. Combined with the inexpensive and quick disposition of NESTROFT, these results strongly support its candidature as a screening tool-especially in the resource-poor and high-prevalence settings. PMID:17178668

  1. Donor Safety in Adult-Adult Living Donor Liver Transplantation: A Single-Center Experience of 356 Cases

    PubMed Central

    Meng, Haipeng; Yang, Jiayin; Yan, Lunan

    2016-01-01

    Background As an important means to tackle the worldwide shortage of liver grafts, adult-adult living donor liver transplantation (A-ALDLT) is the most massive operation a healthy person could undergo, so donor safety is of prime importance. However, most previous research focused on recipients, while complications in donors have not been fully described or investigated. Material/Methods To investigate donor safety in terms of postoperative complications, the clinical data of 356 A-ALDLT donors in our center from January 2002 to September 2015 were retrospectively analyzed. These patients were divided into a pre-2008 group (before January 2008) and a post-2008 group (after January 2008). Donor safety was evaluated with regard to the type, frequency, and severity of postoperative complications. Results There were no donor deaths in our center during this period. The overall complication rate was 23.0% (82/356). The proportion of Clavien I, II, III, and IV complications was 51.2% (42/82), 25.6% (21/82), 22.0% (18/82), and 1.2% (1/82), respectively. In all the donors, the incidence of Clavien I, II, III, and IV complications was 11.8% (42/356), 5.9% (21/356), 5.1% (18/356), and 0.3% (1/356), respectively. The overall complication rate in the post-2008 group was significantly lower than that in the pre-2008 group (18.1% (41/227) vs. 32.6% (42/129), P<0.01). Biliary complications were the most common, with an incidence of 8.4% (30/356). Conclusions The risk to A-ALDLT donors is controllable and acceptable with improvement in preoperative assessment and liver surgery. PMID:27178367

  2. Donor Safety in Adult-Adult Living Donor Liver Transplantation: A Single-Center Experience of 356 Cases.

    PubMed

    Meng, Haipeng; Yang, Jiayin; Yan, Lunan

    2016-01-01

    BACKGROUND As an important means to tackle the worldwide shortage of liver grafts, adult-adult living donor liver transplantation (A-ALDLT) is the most massive operation a healthy person could undergo, so donor safety is of prime importance. However, most previous research focused on recipients, while complications in donors have not been fully described or investigated. MATERIAL AND METHODS To investigate donor safety in terms of postoperative complications, the clinical data of 356 A-ALDLT donors in our center from January 2002 to September 2015 were retrospectively analyzed. These patients were divided into a pre-2008 group (before January 2008) and a post-2008 group (after January 2008). Donor safety was evaluated with regard to the type, frequency, and severity of postoperative complications. RESULTS There were no donor deaths in our center during this period. The overall complication rate was 23.0% (82/356). The proportion of Clavien I, II, III, and IV complications was 51.2% (42/82), 25.6% (21/82), 22.0% (18/82), and 1.2% (1/82), respectively. In all the donors, the incidence of Clavien I, II, III, and IV complications was 11.8% (42/356), 5.9% (21/356), 5.1% (18/356), and 0.3% (1/356), respectively. The overall complication rate in the post-2008 group was significantly lower than that in the pre-2008 group (18.1% (41/227) vs. 32.6% (42/129), P<0.01). Biliary complications were the most common, with an incidence of 8.4% (30/356). CONCLUSIONS The risk to A-ALDLT donors is controllable and acceptable with improvement in preoperative assessment and liver surgery. PMID:27178367

  3. Cytomegalovirus infection: its incidence and management in cytomegalovirus-seropositive living related liver transplant recipients: a single-center experience.

    PubMed

    Wadhawan, Manav; Gupta, Subash; Goyal, Neerav; Vasudevan, Karisangal R; Makki, Kausar; Dawar, Reetika; Sardana, Raman; Lal, Nand; Kumar, Ajay

    2012-12-01

    It is believed that antiviral prophylaxis decreases the incidence of cytomegalovirus (CMV) reactivation and disease. There are few data regarding weekly assays for CMV DNA after transplantation and the subsequent management of CMV. Here we report a cohort of living related liver transplantation (LRLT) patients who were treated for invasive CMV disease or for CMV infections if they were receiving steroids for rejection. Patients who underwent liver transplantation at our center between September 2006 and August 2010 and were recipient-positive/donor-positive (R(+) /D(+) ) were prospectively included. Patients were tested for CMV DNA 3 weeks after transplantation. CMV DNA-positive patients underwent weekly DNA monitoring until there were 2 consecutive negative reports. Those who developed CMV disease or had rising DNA titers while they were on treatment for rejection were treated. A Cox regression analysis was performed for factors predicting survival. Two hundred sixty-six of the 306 R(+) /D(+) patients were CMV DNA-negative 3 weeks after transplantation, and 40 had detectable DNA. One of the DNA-negative patients developed CMV disease after treatment for rejection with methylprednisolone. Thirty patients had <500 copies/mL, and 10 had ≥500 copies/mL. Two of the 30 patients with DNA levels < 500 copies/mL developed CMV disease. Six of the 10 patients with DNA levels ≥500 copies/mL developed disease. CMV disease occurred in 9 of the 306 patients (2.9%). One patient received treatment for a rise in DNA titers while he was receiving steroids. There was a significant correlation between steroid administration for acute cellular rejection (ACR) and CMV reactivation (P = 0.003) and disease (P = 0.002). A multivariate analysis showed that CMV reactivation/disease did not predict survival. There was no difference in survival between CMV DNA-positive patients and CMV DNA-negative patients (P = 0.68). In conclusion, CMV reactivation is common after LRLT (13%), but the

  4. Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

    PubMed Central

    2013-01-01

    The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses. PMID:24351207

  5. Single-molecule detection and tracking of RNA transcripts in living cells using phosphorothioate-optimized 2'-O-methyl RNA molecular beacons.

    PubMed

    Zhao, Dan; Yang, Yantao; Qu, Na; Chen, Mingming; Ma, Zhao; Krueger, Christopher J; Behlke, Mark A; Chen, Antony K

    2016-09-01

    Molecular Beacons (MBs) composed of 2'-O-methyl RNA (2Me) and phosphorothioate (PS) linkages throughout the backbone (2Me/PSFULL MBs) have enabled long-term imaging of RNA in living cells, but excess PS modification can induce nonspecific binding, causing false-positive signals. In this study, we evaluate the intracellular stability of MBs composed of 2Me with various PS modifications, and found that false-positive signals could be reduced to marginal levels when the MBs possess a fully PS-modified loop domain and a phosphodiester stem (2Me/PSLOOP MB). Additionally, 2Me/PSLOOP MBs exhibited uncompromised hybridization kinetics, prolonged functionality and >88% detection accuracy for single RNA transcripts, and could do so without interfering with gene expression or cell growth. Finally, 2Me/PSLOOP MBs could image the dynamics of single mRNA transcripts in the nucleus and the cytoplasm simultaneously, regardless of whether the MBs targeted the 5'- or the 3'-UTR. Together, these findings demonstrate the effectiveness of loop-domain PS modification in reducing nonspecific signals and the potential for sensitive and accurate imaging of individual RNAs at the single-molecule level. With the growing interest in the role of RNA localization and dynamics in health and disease, 2Me/PSLOOP MBs could enable new discoveries in RNA research. PMID:27261815

  6. Single-dose Live-attenuated Vesicular Stomatitis Virus-based Vaccine Protects African Green Monkeys from Nipah Virus Disease

    PubMed Central

    Prescott, Joseph; DeBuysscher, Blair L.; Feldmann, Friederike; Gardner, Donald J.; Haddock, Elaine; Martellaro, Cynthia; Scott, Dana; Feldmann, Heinz

    2015-01-01

    Nipah virus is a zoonotic paramyxovirus that causes severe respiratory and/or encephalitic disease in humans, often resulting in death. It is transmitted from pteropus fruit bats, which serve as the natural reservoir of the virus, and outbreaks occur on an almost annual basis in Bangladesh or India. Outbreaks are small and sporadic, and several cases of human-to-human transmission have been documented as an important feature of the epidemiology of Nipah virus disease. There are no approved countermeasures to combat infection and medical intervention is supportive. We recently generated a recombinant replication-competent vesicular stomatitis virus-based vaccine that encodes a Nipah virus glycoprotein as an antigen and is highly efficacious in the hamster model of Nipah virus disease. Herein, we show that this vaccine protects African green monkeys, a well-characterized model of Nipah virus disease, from disease one month after a single intramuscular administration of the vaccine. Vaccination resulted in a rapid and strong virus-specific immune response which inhibited virus shedding and replication. This vaccine platform provides a rapid means to afford protection from Nipah virus in an outbreak situation. PMID:25865472

  7. Single-dose live-attenuated vesicular stomatitis virus-based vaccine protects African green monkeys from Nipah virus disease.

    PubMed

    Prescott, Joseph; DeBuysscher, Blair L; Feldmann, Friederike; Gardner, Donald J; Haddock, Elaine; Martellaro, Cynthia; Scott, Dana; Feldmann, Heinz

    2015-06-01

    Nipah virus is a zoonotic paramyxovirus that causes severe respiratory and/or encephalitic disease in humans, often resulting in death. It is transmitted from pteropus fruit bats, which serve as the natural reservoir of the virus, and outbreaks occur on an almost annual basis in Bangladesh or India. Outbreaks are small and sporadic, and several cases of human-to-human transmission have been documented as an important feature of the epidemiology of Nipah virus disease. There are no approved countermeasures to combat infection and medical intervention is supportive. We recently generated a recombinant replication-competent vesicular stomatitis virus-based vaccine that encodes a Nipah virus glycoprotein as an antigen and is highly efficacious in the hamster model of Nipah virus disease. Herein, we show that this vaccine protects African green monkeys, a well-characterized model of Nipah virus disease, from disease one month after a single intramuscular administration of the vaccine. Vaccination resulted in a rapid and strong virus-specific immune response which inhibited virus shedding and replication. This vaccine platform provides a rapid means to afford protection from Nipah virus in an outbreak situation. PMID:25865472

  8. Single-molecule imaging of {beta}-actin mRNAs in the cytoplasm of a living cell

    SciTech Connect

    Yamagishi, Mai; Ishihama, Yo; Shirasaki, Yoshitaka; Kurama, Hideki; Funatsu, Takashi

    2009-04-15

    {beta}-Actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most {beta}-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D{sub MACRO}) at the leading edge was 0.3 {mu}m{sup 2}/s. On the other hand, D{sub MACRO} in the perinuclear region was 0.02 {mu}m{sup 2}/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize {beta}-actin mRNAs, led to an increase in D{sub MACRO} to 0.2 {mu}m{sup 2}/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of {beta}-actin mRNA.

  9. Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

    PubMed Central

    Marcus, Joshua M.; Burke, Russell T.; DeSisto, John A.; Landesman, Yosef; Orth, James D.

    2015-01-01

    Longitudinal tracking is a powerful approach to understand the biology of single cells. In cancer therapy, outcome is determined at the molecular and cellular scale, yet relationships between cellular response and cell fate are often unknown. The selective inhibitor of nuclear export, selinexor, is in development for the treatment of various cancers. Selinexor covalently binds exportin-1, causing nuclear sequestration of cargo proteins, including key regulators of the cell cycle and apoptosis. The cell cycle effects of selinexor and the relationships between cell cycle effects and cell fates, has not been described for individual cells. Using fluorescent cell cycle indicators we report the majority of cell death after selinexor treatment occurs from a protracted G1-phase and early S-phase. G1- or early S-phase treated cells show the strongest response and either die or arrest, while those treated in late S- or G2-phase progress to mitosis and divide. Importantly, the progeny of cell divisions also die or arrest, mostly in the next G1-phase. Cells that survive selinexor are negative for multiple proliferation biomarkers, indicating a penetrant, arrested state. Selinexor acts quickly, shows strong cell cycle selectivity, and is highly effective at arresting cell growth and inducing death in cancer-derived cells. PMID:26399741

  10. On birth single dose live attenuated OPV and BCG vaccination induces gut cathelicidin LL37 responses at 6 week of age: a natural experiment.

    PubMed

    Alam, Md Jahangir; Rashid, Md Mamunur; Kabir, Yearul; Raqib, Rubhana; Ahmad, Shaikh Meshbahuddin

    2015-01-01

    In a cross sectional study, we show that infants who received single dose of live attenuated OPV and BCG vaccines within 48h of birth, have higher excretion of human cathelicidin LL37 (p<0.05) in stool at 6wk of age. This response remained unchanged in multivariate analysis after adjusting for sex, mode of delivery, infant age, mother age birth weight and breast milk feeding pattern. This analysis also reveals that irrespective of vaccination, girl infants have higher human-beta-defencin2 (HBD2) and exclusively breastfed infants have higher total and anti-polio specific IgA to all three subtypes in stool (p<0.05). However, vaccination induces anti-polio IgA responses only to infants who are exclusively breastfed. Thus on-birth live attenuated vaccination may provide non-specific beneficial effect against infections while exclusive breastfeeding enhance protection by boosting vaccine induced IgA. The result also suggests that in polio endemic area, exclusive breastfeeding may be sufficient for mucosal anti-polio responses during early infancy. PMID:25444792

  11. Red Sky with Red Mesa

    SciTech Connect

    2011-04-14

    The Red Sky/Red Mesa supercomputing platform dramatically reduces the time required to simulate complex fuel models, from 4-6 months to just 4 weeks, allowing researchers to accelerate the pace at which they can address these complex problems. Its speed also reduces the need for laboratory and field testing, allowing for energy reduction far beyond data center walls.

  12. Red Sky with Red Mesa

    ScienceCinema

    None

    2014-06-23

    The Red Sky/Red Mesa supercomputing platform dramatically reduces the time required to simulate complex fuel models, from 4-6 months to just 4 weeks, allowing researchers to accelerate the pace at which they can address these complex problems. Its speed also reduces the need for laboratory and field testing, allowing for energy reduction far beyond data center walls.

  13. Red Capes, Red Herrings, and Red Flags.

    ERIC Educational Resources Information Center

    Fiske, Donald W.

    The argument that the personality structures obtained from retrospective ratings reflect semantic similarity structures has been as provocative as a red cape in the bull ring. High congruence between those two kinds of structures seems well established. What is less clear is how and why those structures differ from that for immediate judgments of…

  14. Long term follow-up study to evaluate immunogenicity and safety of a single dose of live attenuated hepatitis a vaccine in children

    PubMed Central

    Mitra, Monjori; Shah, Nitin; Faridi, MMA; Ghosh, Apurba; Sankaranarayanan, VS; Aggarwal, Anju; Chatterjee, Suparna; Bhattacharyya, Nisha; Kadhe, Ganesh; Vishnoi, Gaurav; Mane, Amey

    2015-01-01

    Worldwide, viral hepatitis continues to be a cause of considerable morbidity and mortality. Mass immunization with a single dose of live attenuated HAV has been shown to significantly reduce disease burden in the community. This was a phase IV, 5-year follow up study carried out at 4 centers (Kolkata, Delhi, Mumbai and Chennai) across India. The subjects with antibody titer <20 mIU/mL at baseline were evaluated for long term immunogenicity. Of the 503 subjects enrolled, 349 subjects were baseline seronegative with an anti-HAV antibody titer <20 mIU/mL. Overall, 343 subjects could be followed up at some point of time during this 5 y post vaccination period. In the last year (60 months) of follow-up, 108 subjects (97.3%) of 111 subjects (who came for follow-up at the end of 5 y) had a protective antibody titer (anti-HAV antibody titer >20 mIU/mL). The seroconversion rates considering seroprotection levels of anti-HAV antibody titer >20 mIU/mL, following vaccination starting from 6 weeks, 6 months, 12 months, 24 months, 36 months, 48 months and 60 months were 95.1%, 97.9%, 98.3%, 96.2%, 97.8%, 92.6% and 97.3%, respectively. The geometric mean concentration (GMC) over the years increased from 64.9 mIU/mL at 6 weeks to 38.1 mIU/mL and 135.2 mIU/mL at 6 months and 12 months, respectively and was maintained at 127.1 mIU/mL at 60 months. In conclusion, the result of this 5-year follow up study showed that the single dose of live attenuated vaccine is well tolerated and provides long-term immunogenicity in healthy Indian children. PMID:26018443

  15. Long term follow-up study to evaluate immunogenicity and safety of a single dose of live attenuated hepatitis a vaccine in children.

    PubMed

    Mitra, Monjori; Shah, Nitin; Faridi, Mma; Ghosh, Apurba; Sankaranarayanan, V S; Aggarwal, Anju; Chatterjee, Suparna; Bhattacharyya, Nisha; Kadhe, Ganesh; Vishnoi, Gaurav; Mane, Amey

    2015-01-01

    Worldwide, viral hepatitis continues to be a cause of considerable morbidity and mortality. Mass immunization with a single dose of live attenuated HAV has been shown to significantly reduce disease burden in the community. This was a phase IV, 5-year follow up study carried out at 4 centers (Kolkata, Delhi, Mumbai and Chennai) across India. The subjects with antibody titer <20 mIU/mL at baseline were evaluated for long term immunogenicity. Of the 503 subjects enrolled, 349 subjects were baseline seronegative with an anti-HAV antibody titer <20 mIU/mL. Overall, 343 subjects could be followed up at some point of time during this 5 y post vaccination period. In the last year (60 months) of follow-up, 108 subjects (97.3%) of 111 subjects (who came for follow-up at the end of 5 y) had a protective antibody titer (anti-HAV antibody titer >20 mIU/mL). The seroconversion rates considering seroprotection levels of anti-HAV antibody titer >20 mIU/mL, following vaccination starting from 6 weeks, 6 months, 12 months, 24 months, 36 months, 48 months and 60 months were 95.1%, 97.9%, 98.3%, 96.2%, 97.8%, 92.6% and 97.3%, respectively. The geometric mean concentration (GMC) over the years increased from 64.9 mIU/mL at 6 weeks to 38.1 mIU/mL and 135.2 mIU/mL at 6 months and 12 months, respectively and was maintained at 127.1 mIU/mL at 60 months. In conclusion, the result of this 5-year follow up study showed that the single dose of live attenuated vaccine is well tolerated and provides long-term immunogenicity in healthy Indian children. PMID:26018443

  16. Long and short-term spatial processes analysis of an acacia tree population using a single aerial photograph with near infra-red band

    NASA Astrophysics Data System (ADS)

    Isaacson, Sivan; Blumberg, Dan G.; Rachmilevitch, Shimon; Ephrath, Jhonathan E.

    2014-05-01

    Hyper-arid zones are characterized by highly sparse vegetation cover. Monitoring vegetation dynamics in hyper-arid zones is important because any reduction in the vegetation cover in these areas can lead to a considerable reduction in the carrying capacity of the ecological system. Remote sensing expands the spatial and temporal database and is thus a powerful tool for long-term monitoring in arid zones, where access is limited and long-term ground data are rarely available. The main goal of this research was to study both the long-term and short-term spatial processes affecting the acacia population, by using information from a single, three bands color infrared (CIR) aerial photograph (green, red and near infrared). CIR images enable us to obtain information about photosynthetically active biomass by using vegetation indices such as NDVI. A map of individual acacia trees that was extracted from a CIR aerial photograph of Wadi Ktora allowed us to examine the distribution pattern of the trees size and foliage health status (NDVI). Tree size distribution was used as an indicator of long-term (decades) geo-hydrologic spatial processes effecting the acacia population. The tree health status distribution was used as an indicator for short-term (months to a few years) geo-hydrologic spatial processes, such as the paths of recent flashfloods events. Comparison of the tree size distribution and NDVI values distribution enabled us to differentiate between long-term and short-term processes that brought the population to its present state. The spatial analysis revealed that both the tree size and NDVI distribution patterns were significantly clustered, suggesting that the processes responsible for tree size and tree health status do have a spatial expression. Furthermore, each of the attributes has a different distribution and unique clustering location. We suggest that the lack of spatial correlation between tree size and health status is a result of spatial

  17. A living cell quartz crystal microbalance biosensor for continuous monitoring of cytotoxic responses of macrophages to single-walled carbon nanotubes

    PubMed Central

    2011-01-01

    Background Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Δf). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses of single-walled carbon nanotubes (SWCNTs). Results We analyzed macrophage responses to additions of Zymosan A, polystyrene beads (PBs) (benign substances) or SWCNT (3-150 μg/ml) in the QCMB over 18 hrs. In parallel, toxicity was monitored over 24/48 hrs using conventional viability assays and histological stains to detect apoptosis. In the QCMB, a stable unchanging oscillation frequency occurred when cells alone, Zymosan A alone, PBs alone or SWCNTs without cells at the highest dose alone were used. With living cells in the QCMB, when Zymosan A, PBs or SWCNTs were added, a significant decrease in frequency occurred from 1-6 hrs. For SWCNTs, this Δf was dose-dependent. From 6-18 hrs, benign substances or low dose SWCNT (3-30 μg/ml) treatments showed a reversal of the decrease of oscillation frequency, returning to or exceeding pre-treatment levels. Cell recovery was confirmed in conventional assays. The lag time to see the Δf reversal in QCMB plots was linearly SWCNT

  18. Living with a Single Parent

    MedlinePlus

    ... too. Other choices include going to an after-school program or staying with a neighbor or relative. During the summer, many schools and towns offer summer programs and camps. continue Time Troubles It can be ...

  19. Single and repeated moderate consumption of native or dealcoholized red wine show different effects on antioxidant parameters in blood and DNA strand breaks in peripheral leukocytes in healthy volunteers: a randomized controlled trial [ISRCTN68505294

    PubMed Central

    Arendt, Bianca M; Ellinger, Sabine; Kekic, Klaudia; Geus, Leonie; Fimmers, Rolf; Spengler, Ulrich; Müller, Wolfgang-Ulrich; Goerlich, Roland

    2005-01-01

    Background Red wine (RW) is rich in antioxidant polyphenols that might protect from oxidative stress related diseases, such as cardiovascular disease and cancer. Antioxidant effects after single ingestion of RW or dealcoholized RW (DRW) have been observed in several studies, but results after regular consumption are contradictory. Thus, we examined if single or repeated consumption of moderate amounts of RW or DRW exert antioxidant activity in vivo. Methods Total phenolic content and concentration of other antioxidants in plasma/serum, total antioxidant capacity (TEAC) in plasma as well as DNA strand breaks in peripheral leukocytes were measured in healthy non-smokers A) before, 90 and 360 min after ingestion of one glass of RW, DRW or water; B) before and after consumption of one glass of RW or DRW daily for 6 weeks. DNA strand breaks (SB) were determined by single cell gel electrophoresis (Comet Assay) in untreated cells and after induction of oxidative stress ex vivo with H2O2 (300 μM, 20 min). Results Both RW and DRW transiently increased total phenolic content in plasma after single consumption, but only RW lead to a sustained increase if consumed regularly. Plasma antioxidant capacity was not affected by single or regular consumption of RW or DRW. Effects of RW and DRW on DNA SB were conflicting. DNA strand breaks in untreated cells increased after a single dose of RW and DRW, whereas H2O2 induced SB were reduced after DRW. In contrast, regular RW consumption reduced SB in untreated cells but did not affect H2O2 induced SB. Conclusion The results suggest that consumption of both RW and DRW leads to an accumulation of phenolic compounds in plasma without increasing plasma antioxidant capacity. Red wine and DRW seem to affect the occurrence of DNA strand breaks, but this cannot be referred to antioxidant effects. PMID:16287499

  20. Imaging Fos-Jun transcription factor mobility and interaction in live cells by single plane illumination-fluorescence cross correlation spectroscopy.

    PubMed

    Pernuš, Agata; Langowski, Jörg

    2015-01-01

    We collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. To monitor this process, we used fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS), which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just one point on conventional confocal FCS. We find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing either an eGFP-mRFP dimer, separately expressing eGFP and mRPF, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation. These results extend our earlier findings from confocal FCCS to include spatial information. PMID:25875593

  1. Improving the drug delivery characteristics of graphene oxide based polymer nanocomposites through the "one-pot" synthetic approach of single-electron-transfer living radical polymerization

    NASA Astrophysics Data System (ADS)

    Gao, Peng; Liu, Meiying; Tian, Jianwen; Deng, Fengjie; Wang, Ke; Xu, Dazhuang; Liu, Liangji; Zhang, Xiaoyong; Wei, Yen

    2016-08-01

    Graphene oxide (GO) based polymer nanocomposites have attracted extensive research interest recently for their outstanding physicochemical properties and potential applications. However, surface modification of GO with synthetic polymers has demonstrated to be trouble for most polymerization procedures are occurred under non-aqueous solution, which will in turn lead to the restacking of GO. In this work, a facile and efficient "one-pot" strategy has been developed for surface modification of GO with synthetic polymers through single-electron-transfer living radical polymerization (SET-LRP). The GO based polymer nanocomposites were obtained via SET-LRP in aqueous solution using poly(ethylene glycol) methyl ether methacrylate (PEGMA) as the monomer and 11-bromoundecanoic acid as the initiator, which could be effectively adsorbed on GO through hydrophobic interaction. The successful preparation of GO based polymer nanocomposites was confirmed by a series of characterization techniques such as 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy, thermogravimetric analysis, transmission electron microscopy and X-ray photoelectron spectroscopy. The resultant products exhibit high water disperisibility, excellent biocompatibility and high efficient drug loading capability, making these PEGylated GO nanocomposites promising candidates for biomedical applications.

  2. Striatal and extrastriatal imaging of dopamine D2 receptors in the living human brain with [123I]epidepride single-photon emission tomography.

    PubMed

    Kuikka, J T; Akerman, K K; Hiltunen, J; Bergström, K A; Räsänen, P; Vanninen, E; Halldin, C; Tiihonen, J

    1997-05-01

    The iodine-123 labelled ligand benzamide epidepride was evaluated as a probe for in vivo imaging of striatal and extrastriatal dopamine D2 receptor sites in the human brain. Four healthy males were imaged with a high-resolution single-photon emission tomography scanner. Striatal radioactivity peaked at 3 h after injection. The specific binding in the striatum was 0.91+/-0.03 at 3 h and this ratio steadily increased with time. Extrastriatal radioactivity was highest in the thalamus, in the midbrain and in the temporal cortex, and peaked at 45-60 min after injection of tracer. A smaller amount of radioactivity was found in the parietal, frontal and occipital cortices. Two radioactive metabolites were observed, of which one was more lipophilic than the parent compound. The radiation burden to the patient was 0.035 mSv/MBq (effective dose equivalent). The preliminary results showed that [123I]epidepride can be used for imaging striatal and extrastriatal dopamine D2 receptor sites in the living human brain. PMID:9142727

  3. Development of single shot soft x-ray contact microscopy system for nano-scale dynamics measurement of living biological specimen

    NASA Astrophysics Data System (ADS)

    Kishimoto, Maki; Kado, Masataka; Ishino, Masahiko; Tamotsu, Satoshi; Yasuda, Keiko; Shinohara, Kunio

    2012-07-01

    We have been developing a picosecond single shot soft x-ray contact microscopy system for observing the nanometer-scale inner structure of the living biological specimen in a hydrated condition. The microscopy system consists of an intense IR pump laser system for generating laser-induced plasma as a soft x-ray source and x-ray microscope chamber. The pump laser system employs OPCPA (Optical Parametric Chirped Pulse Amplification) technique to obtain a high contrast pump laser pulse, and we can generate water-window x-rays effectively by combining it to an ultra-thin metal target. The x-ray microscope chamber is composed of a vacuum chamber, a focusing lens, a metal film target, an in-vacuum type sample holder. The pump laser pulse is focused on the metal film target with a focusing lens. The soft x-rays from the laser-induced plasma illuminates bio-specimens on the PMMA photo resist set in the in-vacuum sample holder. The photo resist is developed and the x-ray transmission image recorded on the photo resist is read out by AFM. We took x-ray images of hydrated Leydig cells from mouse testicle and demonstrated that the developed x-ray microscopy system has a spatial resolution of about 100 nm.

  4. Benefits of combined preventive therapy with co-trimoxazole and isoniazid in adults living with HIV: time to consider a fixed-dose, single tablet coformulation.

    PubMed

    Harries, Anthony D; Lawn, Stephen D; Suthar, Amitabh B; Granich, Reuben

    2015-12-01

    Antiretroviral therapy (ART) is the main intervention needed to reduce morbidity and mortality and to prevent tuberculosis in adults living with HIV. However, in most resource-limited countries, especially in sub-Saharan Africa, ART is started too late to have an effect with substantial early morbidity and mortality, and in high tuberculosis burden settings ART does not reduce the tuberculosis risk to that reported in individuals not infected with HIV. Co-trimoxazole preventive therapy started before or with ART, irrespective of CD4 cell count, reduces morbidity and mortality with benefits that continue indefinitely. Isoniazid preventive therapy as an adjunct to ART prevents tuberculosis in high-exposure settings, with long-term treatment likely to be needed to sustain this benefit. Unfortunately, both preventive therapies are underused in low-income and high-burden settings. ART development has benefited from patient-centred simplification with several effective regimens now available as a one per day pill. We argue that co-trimoxazole and isoniazid should also be combined into a single fixed-dose pill, along with pyridoxine (vitamin B6), that would be taken once per day to help with individual uptake and national scale-up of therapies. PMID:26515525

  5. A Fluorogenic Red Fluorescent Protein Heterodimer

    PubMed Central

    Alford, Spencer C.; Abdelfattah, Ahmed S.; Ding, Yidan; Campbell, Robert E.

    2012-01-01

    SUMMARY The expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green fluorescent protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red fluorescent protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed protein evolution led to the discovery of a ddRFP with a Kd of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a protein-protein interaction in vitro, imaging of the reversible Ca2+-dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis. PMID:22444590

  6. Role of basiliximab in the prevention of acute cellular rejection in adult to adult living-related liver transplantation: a single center experience

    PubMed Central

    Gruttadauria, S; Mandalà, L; Biondo, D; Spampinato, M; Lamonaca, V; Volpes, R; Vizzini, G; Marsh, JW; Marcos, A; Gridelli, B

    2007-01-01

    We report our single center experience with the use of basiliximab, a chimeric monoclonal antibody directed against the alpha chain of the interleukin-2 (IL-2) receptor (CD25), in combination with a steroid- and tacrolimus-based regimen in adult to adult living-related liver transplantation (ALRLT). Sixty consecutive ALRLTs were analyzed. All patients received two 20-mg doses of basiliximab (days 0 and 4 after transplantation) followed by tacrolimus (0.15 mg/kg/day; 10–15 ng/mL target trough levels) and a dose regimen of steroids (starting with 20 mg iv, switched to po as soon as the patient was able to eat, and weaned off within 1–2 months). Follow-up ranged from 6 to 1699.4 days after transplantation (mean 517.5 days, SD ± 413.4; median 424 days). Of the recipients, 95% remained rejection-free during follow-up, with an actuarial rejection-free probability of 96.61% within 3 months. Three patients had episodes of biopsy-proven acute cellular rejection (ACR). Actuarial patient and graft survival rates at 3 years were 82.09% and 75.61%. Six patients (10%) experienced sepsis. There was no evidence of cytomegalovirus infections or side-effects related to the basiliximab. We found zero de novo malignancy, although we observed 5 patients with metastatic spread of their primary malignancy during the follow-up. Basiliximab in association with tacrolimus and steroids is effective in reducing episodes of ACR and increasing ACR-free survival after ALRLT. PMID:19707350

  7. Microwave-promoted pure host phase for red emission CaS:Eu2+ phosphor from single CaSO4 precursor and the photoluminescence property

    NASA Astrophysics Data System (ADS)

    Ma, Jian; Lu, Qi-Fei; Wang, Yan-Ze; Lu, Zhi-Juan; Sun, Liang; Dong, Xiao-Fei; Wang, Da-Jian

    2014-08-01

    We report a novel approach to obtaining a classical blue-green excitable CaS:Eu2+ phosphor with desired red emission by microwave (MW) firing procedure in the absence of adding elemental sulphur. The disturbing effect of MW electromagnetic field on decomposition of CaSO4 into CaS activated by europium is distinctly observed to give pure host phase without adding any elemental sulphur and carbon. The host phase evolution is observed to be highly dependent on the variation of applied MW power from X-ray diffraction (XRD) patterns and the corresponding photoluminescence (PL), and a maximum PL intensity at 1100 W of MW power is acquired for the obtained purer host phase. The non-thermal and non-equilibrium effects by MW are revealed to correlate with the interaction between polar structure of the host and applied electromagnetic field. The results demonstrate an optional procedure to prepare this red-emitting phosphor in an effective, environment-friendly and scalable approach for phosphor production in the application of bio-illumination for plant cultivation and artificial photosynthesis.

  8. Effect of a Single Water Molecule on the Electronic Absorption by o- and p-Nitrophenolate: A Shift to the Red or to the Blue?

    PubMed

    Houmøller, Jørgen; Wanko, Marius; Rubio, Angel; Nielsen, Steen Brøndsted

    2015-11-25

    Many photoactive biomolecules are anions and exhibit ππ* optical transitions but with a degree of charge transfer (CT) character determined by the local environment. The phenolate moiety is a common structural motif among biochromophores and luminophores, and nitrophenolates are good model systems because the nitro substituent allows for CT-like transitions. Here we report gas-phase absorption spectra of o- and p-nitrophenolate·H2O complexes to decipher the effect of just one H2O and compare them with ab initio calculations of vertical excitation energies. The experimental band maximum is at 3.01 and 3.00 eV for ortho and para isomers, respectively, and is red-shifted by 0.10 and 0.13 eV relative to the bare ions, respectively. These shifts indicate that the transition has become more CT-like because of localization of negative charge on the phenolate oxygen, i.e., diminished delocalization of the negative excess charge. However, the transition bears less CT than that of m-nitrophenolate·H2O because this complex absorbs further to the red (2.56 eV). Our work emphasizes the importance of local perturbations: one water causes a larger shift than experienced in bulk for para isomer and almost the full shift for ortho isomer. Predicting microenvironmental effects in the boundary between CT and non-CT with high accuracy is nontrivial. However, in agreement with experiment, our calculations show a competition between the effects of electronic delocalization and electrostatic interaction with the solvent molecule. As a result, the excitation energy of ortho and para isomers is less sensitive to hydration than that of the meta isomer because donor and acceptor orbitals are only weakly coupled in the meta isomer. PMID:26549521

  9. Randomized, Controlled Human Challenge Study of the Safety, Immunogenicity, and Protective Efficacy of a Single Dose of Peru-15, a Live Attenuated Oral Cholera Vaccine

    PubMed Central

    Cohen, Mitchell B.; Giannella, Ralph A.; Bean, Judy; Taylor, David N.; Parker, Susan; Hoeper, Amy; Wowk, Stephen; Hawkins, Jennifer; Kochi, Sims K.; Schiff, Gilbert; Killeen, Kevin P.

    2002-01-01

    Peru-15 is a live attenuated oral vaccine derived from a Vibrio cholerae O1 El Tor Inaba strain by a series of deletions and modifications, including deletion of the entire CT genetic element. Peru-15 is also a stable, motility-defective strain and is unable to recombine with homologous DNA. We wished to determine whether a single oral dose of Peru-15 was safe and immunogenic and whether it would provide significant protection against moderate and severe diarrhea in a randomized, double-blind, placebo-controlled human volunteer cholera challenge model. A total of 59 volunteers were randomly allocated to groups to receive either 2 × 108 CFU of reconstituted, lyophilized Peru-15 vaccine diluted in CeraVacx buffer or placebo (CeraVacx buffer alone). Approximately 3 months after vaccination, 36 of these volunteers were challenged with approximately 105 CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Among vaccinees, 98% showed at least a fourfold increase in vibriocidal antibody titers. After challenge, 5 (42%) of the 12 placebo recipients and none (0%) of the 24 vaccinees had moderate or severe diarrhea (≥3,000 g of diarrheal stool) (P = 0.002; protective efficacy, 100%; lower one-sided 95% confidence limit, 75%). A total of 7 (58%) of the 12 placebo recipients and 1 (4%) of the 24 vaccinees had any diarrhea (P < 0.001; protective efficacy, 93%; lower one-sided 95% confidence limit, 62%). The total number of diarrheal stools, weight of diarrheal stools, incidence of fever, and peak stool V. cholerae excretion among vaccinees were all significantly lower than in placebo recipients. Peru-15 is a well-tolerated and immunogenic oral cholera vaccine that affords protective efficacy against life-threatening cholera diarrhea in a human volunteer challenge model. This vaccine may therefore be a safe and effective tool to prevent cholera in travelers and is a strong candidate for further evaluation to prevent cholera

  10. A Single Mutation at PB1 Residue 319 Dramatically Increases the Safety of PR8 Live Attenuated Influenza Vaccine in a Murine Model without Compromising Vaccine Efficacy

    PubMed Central

    Cox, Andrew

    2015-01-01

    The live attenuated influenza vaccine (LAIV) is preferentially recommended for use in most children yet remains unsafe for the groups most at risk. Here we have improved the safety of a mouse-adapted live attenuated influenza vaccine containing the same attenuating amino acid mutations as in human LAIV by adding an additional mutation at PB1 residue 319. This results in a vaccine with a 20-fold decrease in protective efficacy and a 10,000-fold increase in safety. PMID:26676793

  11. Itchy, Scaly Skin? Living with Psoriasis

    MedlinePlus

    ... exit disclaimer . Subscribe Itchy, Scaly Skin? Living With Psoriasis The thick, red, scaly skin of psoriasis can ... Diet Itchy, Scaly Skin? Wise Choices Links Treating Psoriasis Doctors often use a trial-and-error approach ...

  12. Assisted Living

    MedlinePlus

    ... but they don't need full-time nursing care. Some assisted living facilities are part of retirement ... change. Assisted living costs less than nursing home care. It is still fairly expensive. Older people or ...

  13. Light Phenomena over the ESO Observatories II: Red Sprites

    NASA Astrophysics Data System (ADS)

    Horálek, P.; Christensen, L. L.; Bór, J.; Setvák, M.

    2016-03-01

    A rare atmospheric phenomenon, known as red sprites, was observed and captured on camera from the La Silla Observatory. This event signalled the first time that these extremely short-lived flashes of red light, originating in the Earth’s upper atmosphere, were photographed from a major astronomical observatory. Further images of red sprites from the La Silla Paranal Observatory sites are presented and the nature of red sprites is discussed.

  14. Red Dwarfs and the End of the Main Sequence

    NASA Astrophysics Data System (ADS)

    Adams, F. C.; Graves, G. J. M.; Laughlin, G.

    2004-12-01

    This paper celebrates the contributions of Peter Bodenheimer to our understanding of stellar evolution by focusing on the long term development of red dwarf stars. We show that these diminutive stellar objects remain convective over most of their lives, they continue to burn hydrogen for trillions of years, and they do not experience red giant phases in their old age. Instead, red dwarfs turn into blue dwarfs and finally white dwarfs. This work shows (in part) why larger stars do become red giants.

  15. Modeling and simulation of Red Teaming. Part 1, Why Red Team M&S?

    SciTech Connect

    Skroch, Michael J.

    2009-11-01

    Red teams that address complex systems have rarely taken advantage of Modeling and Simulation (M&S) in a way that reproduces most or all of a red-blue team exchange within a computer. Chess programs, starting with IBM's Deep Blue, outperform humans in that red-blue interaction, so why shouldn't we think computers can outperform traditional red teams now or in the future? This and future position papers will explore possible ways to use M&S to augment or replace traditional red teams in some situations, the features Red Team M&S should possess, how one might connect live and simulated red teams, and existing tools in this domain.

  16. Jupiter' high-latitude storms: a Little Red Spot tracked through a Jovian year

    NASA Astrophysics Data System (ADS)

    Rogers, J. H.; Adamoli, G.; Mettig, H.-J.

    2011-02-01

    As well as the Great Red Spot, Jupiter sometimes presents one or more Little Red Spots (LRSs) in various latitudes. An LRS is often seen in the North North Temperate Zone (NNTZ), but the frequency and properties of these ovals have never been studied in detail. Here we review all our records of the red, white, and methane-bright anticyclonic ovals in the NNTZ. There is a simple conclusion: A single LRS, which we name NN-LRS-1, has persisted from 1993 to 2009. It has varied in colour between red and off-white, but has been methane-bright throughout these years. This now ranks among the most long-lived spots ever recorded on the planet. There is always at least one other oval in this latitude. This was a second methane-bright LRS from 1994-1997, and there was also a smaller LRS in 2006. The other ovals are all white; two have lasted for four years or more and were sometimes methane-bright; others have had shorter lives and were not. These results suggest that red colour, and the high-level haze that accompanies it, are correlated with the size and longevity of the oval. All these ovals have variable speeds, alternating irregularly between slow (~ 1°/month in System II longitude) and fast (~ 12°/month). The latitudes of the ovals range from 40 to 41.5°N, and correlate closely with their instantaneous speeds. However the larger, longer-lived ovals (especially the LRS) are centred systematically further south than the smaller white ovals, because they distort the retrograding jetstream more deeply. Similar behaviour in other domains on the planet explains how ovals of different sizes move with a single slow current while also being sensitive to the zonal speed gradient. An oval at 60°S shows very similar behaviour and has probably existed since 1994 or earlier.

  17. The effect of a single injection of nitroxynil at 20 mg/kg live mass in the treatment of parafilaria bovicola infestations in cattle.

    PubMed

    Wellington, A C; van Schalkwyk, L

    1982-06-01

    The efficacy of nitroxynil administered once by subcutaneous injection at a dosage regimen of 20 mg/kg live mass was evaluated against natural infestations of parafilaria bovicola in cattle. Trial animals were slaughtered 14 weeks after treatment. Treatment reduced the number of bleeding points by 97,8%, eosinophil-positive carcass lesions by 85,7% and eosinophil-positive lesion area by 92,8%, compared with controls. PMID:7120274

  18. Assisted Living

    MedlinePlus

    ... it, too. Back to top What is the Cost for Assisted Living? Although assisted living costs less than nursing home care, it is still ... of services an older person chooses, the price costs can range from less than $25,000 a ...

  19. Validation of method for enhanced production of red-shifted bioluminescent photons in vivo

    NASA Astrophysics Data System (ADS)

    Dragavon, Joe; Blazquez, Samantha; Rogers, Kelly L.; Samson, Chelsea; Tournebize, Régis; Shorte, Spencer

    2011-02-01

    Bioluminescence Imaging (BLI) is an increasingly useful and applicable technique that allows for the non-invasive observation of biological events in intact living organisms, ranging from single cells to small rodents. Though the photon production occurs within the host, significant exposure times can be necessary due to the low photon flux compared to fluorescence imaging. The optical absorption spectrum of haemoglobin strongly overlaps most bioluminescent emission spectra, greatly attenuating the total detectable photons in animal models. We have developed and validated a technique that is able to red-shift the bioluminescent photons to the more desirable optical region of > 650 nm, a region of minimal absorbance by hemoglobin. This red-shift occurs by using bioluminescence as an internal light source capable of exciting a fluorophore, such as a fluorescent protein or a quantum dot, that emits in the red. Interestingly, in the absence of an absorber, this excitation can occur over substantial distances (microns to centimeters), far exceeding distances associated to, and thereby precluding, resonance energy transfer phenomena. We show this novel technique yields a substantial increase in the number of red photons for in vitro and ex vivo conditions, suggesting eventually utility for in vivo studies on, for example, intact living mice.

  20. A single design strategy for dual sensitive pH probe with a suitable range to map pH in living cells

    PubMed Central

    Yu, Kang-Kang; Hou, Ji-Ting; Li, Kun; Yao, Qian; Yang, Jin; Wu, Ming-Yu; Xie, Yong-Mei; Yu, Xiao-Qi

    2015-01-01

    Due to the lack of a proper imaging approach, a veracious pH map of normal and abnormal cell is still rare. In this work, we presented a rhodamine-salicylaldehyde combination (Rh-SA2) as a novel pH probe, which has dual sensitive units for both acidic and basic environment. This dual sensitive probe acts like a chameleon in living cells and offers the doubling guarantees for endocellular pH mapping. Moreover, a quantitative measurement of cellular pH changes was allowed and the endocellular pH values under drug-associated stimuli were also investigated. PMID:26486180

  1. Assisted Living

    MedlinePlus

    ... a resident's needs depends as much on the philosophy and services of the assisted living facility as ... the facility provide a written statement of its philosophy of care? Visit each facility more than once, ...

  2. Assisted Living

    MedlinePlus

    ... premises. Adult foster care has the advantages of maintaining frail older adults in a more home-like ... pay to live in these communities, though some facilities have beds for skilled care that are funded ...

  3. Living Laboratories

    ERIC Educational Resources Information Center

    Mules, B. R.

    1976-01-01

    Presented is a review of various methods of keeping live animals, including scorpions, spiders, crabs, crayfish, shrimp, ants, fish, mice, and birds, as well as plants as a school science project/display. (SL)

  4. Healthy Living

    MedlinePlus

    ... Environment Kids Health Kids Environment Kids Health Topics Environment & Health Healthy Living Pollution Reduce, Reuse, Recycle Science – How It Works The Natural World Games Brainteasers Puzzles Riddles Songs Activities Be ...

  5. Assisted Living

    MedlinePlus

    ... are part of retirement communities. Others are near nursing homes, so a person can move easily if needs change. Assisted living costs less than nursing home care. It is still fairly expensive. Older people ...

  6. Assisted Living

    MedlinePlus

    ... Recreational activities Security Transportation How to Choose a Facility A good match between a facility and a resident's needs depends as much on the philosophy and services of the assisted living facility as it does on the quality of care. ...

  7. The Lived Experiences of Single Hispanic Mothers Raising Gang-Affiliated Male Youth Released from Texas Juvenile Justice Department State Facilities: A Phenomenological Study

    ERIC Educational Resources Information Center

    Rodriguez-Almendarez, Ruby

    2013-01-01

    Purpose of the Study: The purpose of this transcendental phenomenological study (Moustakas, 1994) was to describe the experiences that single Hispanic mothers of gang-affiliated male juveniles face during their sons' reentry process after being released from a Texas Juvenile Justice Department state facility. Methods: After an extensive…

  8. Girl Talk: A Qualitative Study of Girls Talking about the Meaning of Their Lives in an Urban Single-Sex Elementary School

    ERIC Educational Resources Information Center

    Ridenour, Carolyn S.; Hassell Hughes, Sheila

    2016-01-01

    The suburban-urban achievement gap (diminishing until the 1980s) has stopped its narrowing trend, and single-sex schools are proliferating as a reform model, especially in urban areas. In this study researchers interviewed eight elementary school girls (in an all-girls school) three times over 2 years, and the resulting 23 transcripts were…

  9. Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation

    PubMed Central

    Mendenhall, Alexander R.; Tedesco, Patricia M.; Sands, Bryan; Johnson, Thomas E.; Brent, Roger

    2015-01-01

    In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, “classical” multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex

  10. Characterizing warfare in red teaming.

    PubMed

    Yang, Ang; Abbass, Hussein A; Sarker, Ruhul

    2006-04-01

    Red teaming is the process of studying a problem by anticipating adversary behaviors. When done in simulations, the behavior space is divided into two groups; one controlled by the red team which represents the set of adversary behaviors or bad guys, while the other is controlled by the blue team which represents the set of defenders or good guys. Through red teaming, analysts can learn about the future by forward prediction of scenarios. More recently, defense has been looking at evolutionary computation methods in red teaming. The fitness function in these systems is highly stochastic, where a single configuration can result in multiple different outcomes. Operational, tactical and strategic decisions can be made based on the findings of the evolutionary method in use. Therefore, there is an urgent need for understanding the nature of these problems and the role of the stochastic fitness to gain insight into the possible performance of different methods. This paper presents a first attempt at characterizing the search space difficulties in red teaming to shed light on the expected performance of the evolutionary method in stochastic environments. PMID:16604725

  11. The interaction of 193 nm excimer laser radiation with single-crystal zinc oxide: Generation of long lived highly excited particles with evidence of Zn Rydberg formation

    SciTech Connect

    Khan, Enamul H.; Langford, S. C.; Dickinson, J. T.; Boatner, L. A.

    2014-08-28

    In past studies, we have observed copious emissions of ionic and atomic Zn from single-crystal ZnO accompanying irradiation of single-crystal ZnO with 193-nm excimer laser irradiation at fluences below the onset of optical breakdown. The Zn{sup +} and ground state Zn° are studied using time-of-flight techniques and are mass selected using a quadrupole mass spectrometer. Simultaneously, we have observed emitted particles that are detectable with a Channeltron electron multiplier but cannot be mass selected. It is a reasonable hypothesis that these particles correspond to a neutral atom or molecule in highly excited long lived states. We provide strong evidence that they correspond to high lying Rydberg states of atomic Zn. We propose a production mechanism involving laser excitation via a two photon resonance excitation of Zn°.

  12. A single recombinant anti-RhD IgG prevents RhD immunization: association of RhD-positive red blood cell clearance rate with polymorphisms in the FcgammaRIIA and FcgammaIIIA genes.

    PubMed

    Miescher, Sylvia; Spycher, Martin O; Amstutz, Hanspeter; De Haas, Masja; Kleijer, Marion; Kalus, Ulrich J; Radtke, Hartmut; Hubsch, Alphonse; Andresen, Irmgard; Martin, Roland M; Bichler, Johann

    2004-06-01

    A single recombinant immunoglobulin G1 (IgG1) anti-RhD antibody (MonoRho) was compared with a currently used polyclonal anti-RhD product (Rhophylac) in a phase 1 study for safety, efficacy of Rhesus D (RhD)-positive red blood cell (RBC) clearance, and prevention of RhD immunization in RhD-negative men challenged with 15 mL RhD-positive RBCs. Both the polyclonal product and recombinant anti-RhD effectively cleared RhD-positive RBCs after intravenous and intramuscular injection. The recombinant anti-RhD demonstrated a slower clearance rate compared with the polyclonal anti-RhD. There was no dose response, and there was considerable variation among subjects who received the same dose of recombinant anti-RhD. Interestingly, RhD-positive RBC clearance rates were strongly associated with Fcgamma receptor IIA (FcgammaRIIA) and FcgammaIIIA but not with FcgammaIIIB polymorphisms. Subjects homozygous for FcgammaRIIA-131H or FcgammaRIIIA-158V allotypes showed a faster clearance rate compared with both the heterozygote and the corresponding alternative homozygote allotypes. A similar but less marked trend was seen for the polyclonal anti-RhD. Despite the variation in clearance rates there was no evidence of anti-RhD alloantibodies in any of the subjects at +6 months after the RBC challenge. PMID:14976055

  13. The detection of cancer in living tissue with single-cell precision and the development of a system for targeted drug delivery to cancer

    NASA Astrophysics Data System (ADS)

    Fields, Adam; Pi, Sean; Ramek, Alex; Bernheim, Taylor; Fields, Jessica; Pernodet, Nadine; Rafailovich, Miriam

    2007-03-01

    The development of innovations in the field of cancer diagnostics is imperative to improve the early identification of malignant cells within the human body. Two novel techniques are presented for the detection of cancer cells in living tissue. First, shear modulation force microscopy (SMFM) was employed to measure cell mechanics of normal and cancer cells in separate and mixed tissue cultures. We found that the moduli of normal keratinocytes were twice as high as the moduli of SCC cancerous keratinocytes, and that the cancer cells were unambiguously identifiable from a mixture of both kinds of cells. Second, confocal microscopy and the BIAcore 2000 were used to demonstrate the preferential adhesion of glass micro-beads impregnated with fluorescent dye to the membranes of cancer cells as compared to those of normal cells. In addition to their use as a cancer detection system, these hollow and porous beads present a model system for targeted drug delivery in the treatment of cancer.

  14. Photoacoustics of individual live cells and particles

    NASA Astrophysics Data System (ADS)

    Kudryashov, Sergey I.; Allen, Susan D.; Galanzha, Ekaterina I.; Galitovskaya, E.; Zharov, Vladimir P.

    2006-02-01

    The photoacoustic (PA) technique has been employed to a number of new biomedical applications based of highly sensitive detection of laser-induced acoustic waves from individual live cells and single absorbing micro-particles or clusters of nanoparticles. These applications involve both linear and non-linear thermoacoustic phenomena initiated by focused nanosecond single laser pulse and detected with a fast PZT-ceramic acoustic transducer. Particularly, we present the following experimental results: 1) monitoring of linear and non-linear PA responses from red blood cells in suspensions in vitro; 2) detection of PA responses from breast cancer cell targeted with gold nanoparticles; 3) PA study of linear and non-linear interaction of laser with colored polystyrene micro-particles as model single absorbers; 4) monitoring of PA responses from moving absorbers in flow in vitro (PA flow cytometry in vitro); 5) recording of PA responses from blood flow in vivo on rat mesentery as animal model (PA flow cytometery in vivo); and 6) monitoring of sedimentation kinetics of particles and cells. The obtained results demonstrate the high sensitivity, low background, simple detection principle, easy data acquisition, and straightforward interpretation of the PA data.

  15. Safety and immunogenicity of single dose live attenuated varicella vaccine (VR 795 Oka strain) in healthy Indian children: a randomized controlled study.

    PubMed

    Mitra, Monjori; Faridi, Mma; Ghosh, Apurba; Shah, Nitin; Shah, Raju; Chaterjee, Suparna; Narang, Manish; Bhattacharya, Nisha; Bhat, Gandhali; Choudhury, Harish; Kadhe, Ganesh; Mane, Amey; Roy, Sucheta

    2015-01-01

    Varicella, an acute viral systemic infection that may cause lifelong latent infection with the potential for causing clinical reactivation, may be prevented by immunization. The present study was an open label, randomized, controlled, phase III, multicentre trial, conducted to evaluate and compare the safety, tolerability and immunogenicity of a freeze dried live attenuated Oka strain Varicella Vaccine (VR 795 Oka strain) with Varilrix (Oka-RIT strain) in children. A total of 268 healthy Indian children aged 12 months to 12 y with baseline VZV IgG antibody (<100 mIU/ mL) were enrolled, and 256 children completed the study. The extent of rise of VZV IgG antibody titer assessed as 3-fold and 4-fold rise from baseline was found to be significantly higher (89.1% and 85.2%) in the test group as compared to control group (73.4% and 61.7%). The post-vaccination GMT of the test group was significantly higher (112.5 mIU/mL) as compared with the control group (67.8 mIU/mL) (P < 0.001). The seroconversion rate considering the 5 gp ELISA units/ml equivalent to 10mIU/ml were similar in the control (96.5%) and the test (98.3%) groups. The adverse events were not different in the control and test groups (P > 0.05). The test live attenuated vaccine was found to be highly immunogenic, safe and comparable to Varilrix used in control arm. PMID:25692656

  16. Red microchip VECSEL array

    NASA Astrophysics Data System (ADS)

    Hastie, Jennifer E.; Morton, Lynne G.; Calvez, Stephane; Dawson, Martin D.; Leinonen, Tomi; Pessa, Markus; Gibson, Graham; Padgett, Miles J.

    2005-09-01

    We report an InGaP/AlInGaP/GaAs microchip vertical-external-cavity surface emitting laser operating directly at red wavelengths and demonstrate its potential for array-format operation. Optical pumping with up to 3.3W at 532nm produced a maximum output power of 330mW at 675nm, in a single circularly-symmetric beam with M2<2. Simultaneous pumping with three separate input beams, generated using a diffractive optical element, achieved lasing from three discrete areas of the same chip. Output power of ~95mW per beam was obtained from this 3x1 array, each beam having a Gaussian intensity profile with M2<1.2. In a further development, a spatial light modulator allowed computer control over the orientation and separation of the pump beams, and hence dynamic control over the configuration of the VECSEL array.

  17. Red microchip VECSEL array.

    PubMed

    Hastie, Jennifer; Morton, Lynne; Calvez, Stephane; Dawson, Martin; Leinonen, Tomi; Pessa, Markus; Gibson, Graham; Padgett, Miles

    2005-09-01

    We report an InGaP/AlInGaP/GaAs microchip vertical-external-cavity surface emitting laser operating directly at red wavelengths and demonstrate its potential for array-format operation. Optical pumping with up to 3.3W at 532nm produced a maximum output power of 330mW at 675nm, in a single circularly-symmetric beam with M2<2. Simultaneous pumping with three separate input beams, generated using a diffractive optical element, achieved lasing from three discrete areas of the same chip. Output power of ~95mW per beam was obtained from this 3x1 array, each beam having a Gaussian intensity profile with M2<1.2. In a further development, a spatial light modulator allowed computer control over the orientation and separation of the pump beams, and hence dynamic control over the configuration of the VECSEL array. PMID:19498743

  18. Detection of the free living amoeba Naegleria fowleri by using conventional and real-time PCR based on a single copy DNA sequence.

    PubMed

    Régoudis, Estelle; Pélandakis, Michel

    2016-02-01

    The amoeba-flagellate Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM). This thermophilic species occurs worldwide and tends to proliferate in warm aquatic environment. The PAM cases remain rare but this infection is mostly fatal. Here, we describe a single copy region which has been cloned and sequenced, and was used for both conventional and real-time PCR. Targeting a single-copy DNA sequence allows to directly quantify the N. fowleri cells. The real-time PCR results give a detection limit of 1 copy per reaction with high reproducibility without the need of a Taqman probe. This procedure is of interest as compared to other procedures which are mostly based on the detection of multi-copy DNA associated with a Taqman probe. PMID:26688582

  19. The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces

    PubMed Central

    Arnfinnsdottir, Nina Bjørk; Ottesen, Vegar; Lale, Rahmi; Sletmoen, Marit

    2015-01-01

    In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis. PMID:26039378

  20. Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics

    PubMed Central

    Lo, Camden Yeung-Wah; Chen, Sijie; Creed, Sarah Jayne; Kang, Miaomiao; Zhao, Na; Tang, Ben Zhong; Elgass, Kirstin Diana

    2016-01-01

    Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology. PMID:27492961

  1. Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics.

    PubMed

    Lo, Camden Yeung-Wah; Chen, Sijie; Creed, Sarah Jayne; Kang, Miaomiao; Zhao, Na; Tang, Ben Zhong; Elgass, Kirstin Diana

    2016-01-01

    Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology. PMID:27492961

  2. Dramatic Change in Jupiter's Great Red Spot

    NASA Technical Reports Server (NTRS)

    Simon, A. A.; Wong, M. H.; Rogers, J. H.; Orton, G. S.; de Pater, I.; Asay-Davis, X.; Carlson, R. W.; Marcus, P. S.

    2015-01-01

    Jupiter's Great Red Spot (GRS) is one of its most distinct and enduring features, having been continuously observed since the 1800's. It currently spans the smallest latitude and longitude size ever recorded. Here we show analyses of 2014 Hubble spectral imaging data to study the color, structure and internal dynamics of this long-live storm.

  3. Live visualizations of single isolated tubulin protein self-assembly via tunneling current: effect of electromagnetic pumping during spontaneous growth of microtubule

    NASA Astrophysics Data System (ADS)

    Sahu, Satyajit; Ghosh, Subrata; Fujita, Daisuke; Bandyopadhyay, Anirban

    2014-12-01

    As we bring tubulin protein molecules one by one into the vicinity, they self-assemble and entire event we capture live via quantum tunneling. We observe how these molecules form a linear chain and then chains self-assemble into 2D sheet, an essential for microtubule, --fundamental nano-tube in a cellular life form. Even without using GTP, or any chemical reaction, but applying particular ac signal using specially designed antenna around atomic sharp tip we could carry out the self-assembly, however, if there is no electromagnetic pumping, no self-assembly is observed. In order to verify this atomic scale observation, we have built an artificial cell-like environment with nano-scale engineering and repeated spontaneous growth of tubulin protein to its complex with and without electromagnetic signal. We used 64 combinations of plant, animal and fungi tubulins and several doping molecules used as drug, and repeatedly observed that the long reported common frequency region where protein folds mechanically and its structures vibrate electromagnetically. Under pumping, the growth process exhibits a unique organized behavior unprecedented otherwise. Thus, ``common frequency point'' is proposed as a tool to regulate protein complex related diseases in the future.

  4. Healthy Living

    MedlinePlus

    ... health. Some you cannot control, such as your genetic makeup or your age. But you can make changes to your lifestyle. By taking steps toward healthy living, you can help reduce your risk of heart disease, cancer, stroke and other serious diseases: Get ...

  5. Retiring Lives

    ERIC Educational Resources Information Center

    Carnell, Eileen, Ed.; Lodge, Caroline, Ed.

    2009-01-01

    "Retiring Lives" presents fourteen personal real life stories from people at various stages of retiring. Each author recounts their own story about retiring, bringing together many aspects of the experiences: the social, psychological and practical. These inspirational and illustrated stories will encourage the reader to hold up these experiences…

  6. Learn & Live.

    ERIC Educational Resources Information Center

    Burness, Patty, Ed.; Snider, William, Ed.

    Along with a companion documentary video, "Learn & Live," this resource manual focuses on innovative schools around the country that are integrating technology and involving parents, business, and the community. Ten chapters are divided into four sections. In Section 1, "Students," two chapters look at learning and assessment. The two chapters in…

  7. Outdoor Living.

    ERIC Educational Resources Information Center

    Cotter, Kathy

    Course objectives and learning activities are contained in this curriculum guide for a 16-week home economics course which teaches cooking and sewing skills applicable to outdoor living. The course goals include increasing male enrollment in the home economics program, developing students' self-confidence and ability to work in groups, and…

  8. Successful treatment of small-for-size syndrome in adult-to-adult living-related liver transplantation: single center series.

    PubMed

    Gruttadauria, Salvatore; Mandala', Lucio; Miraglia, Roberto; Caruso, Settimo; Minervini, Marta Ida; Biondo, Domenico; Volpes, Riccardo; Vizzini, Giovanni; Marsh, J Wallis; Luca, Angelo; Marcos, Amadeo; Gridelli, Bruno

    2007-01-01

    The portal hyperperfusion, or small-for-size syndrome (SFSS), is a widely recognized clinical complication that may occur after segmental liver transplantation. Several surgical strategies have been proposed to reduce portal blood inflow and portal pressure after partial liver transplantation. In particular, splenic artery ligation and splenectomy have been used without a firm hemodynamic basis for these procedures. Our group recently demonstrated that, in patients with cirrhosis and portal hypertension, the occlusion of the splenic artery causes a significant reduction in the portal pressure gradient, which is directly related to the spleen volume and indirectly related to the liver volume. This concept is at the center of our strategy for performing early splenic artery embolization (SAE) for the treatment of SFSS after living-related liver transplantation (LRLT). Six patients developed small-for-size syndrome, defined as: onset within the first week after LRLT of progressive hyperbilirubinemia without mechanical cause; marked cholestasis; centrilobular sinusoidal dilatation and hepatocyte atrophy at liver biopsy; and refractory ascites in the absence of vascular complications. All six patients who underwent SAE rapidly improved their clinical condition, with an evident decrease in the value of bilirubin in the serum, in the production of ascites, and improvement in condition of pancytopenia. Coagulopathy expressed by the international normalized ratio value (INR) was not a reliable early marker of SFSS in this series; in fact a slight improvement in the result of this test was already present immediately after LRLT and before SAE. Because splenic flow clearly contributes to portal hyperperfusion, an early SAE can relieve the partial graft from the deleterious effect of this portal overflow. PMID:17988271

  9. ATTIRE: Albumin To prevenT Infection in chronic liveR failurE: study protocol for a single-arm feasibility trial

    PubMed Central

    Muirhead, Nicola; Shabir, Zainib; PH De Maeyer, Roel; Maini, Alexander AN; W Gilroy, Derek; J O'Brien, Alastair

    2016-01-01

    Introduction Circulating prostaglandin E2 levels are elevated in acutely decompensated cirrhosis and have been shown to contribute to immune suppression. Albumin binds and inactivates this hormone. Human albumin solution could thus be repurposed as an immune restorative drug in these patients. This feasibility study aims to determine whether it is possible and safe to restore serum albumin to >30 g/L and maintain it at this level in patients admitted with acute decompensated cirrhosis using repeated 20% human albumin infusions according to daily serum albumin levels. Methods and analysis Albumin To prevenT Infection in chronic liveR failurE (ATTIRE) stage 1 is a multicentre, open label dose feasibility trial. Patients with acutely decompensated cirrhosis admitted to hospital with a serum albumin of <30 g/L are eligible, subject to exclusion criteria. Daily intravenous human albumin solution will be infused, according to serum albumin levels, for up to 14 days or discharge in all patients. The primary end point is daily serum albumin levels for the duration of the treatment period and the secondary end point is plasma-induced macrophage dysfunction. The trial will recruit 80 patients. Outcomes will be used to assist with study design for an 866 patient randomised controlled trial at more than 30 sites across the UK. Ethics and dissemination Research ethics approval was given by the London-Brent research ethics committee (ref: 15/LO/0104). The clinical trials authorisation was issued by the medicines and healthcare products regulatory agency (ref: 20363/0350/001-0001). Results Will be disseminated through peer reviewed journals and international conferences. Recruitment of the first participant occurred on 26/05/2015. Trial registration number The trial is registered with the European Medicines Agency (EudraCT 2014-002300-24) and has been adopted by the NIHR (ISRCTN 14174793). This manuscript refers to V.4.0 of the protocol; Pre-results. PMID:26810999

  10. Shape anisotropy induces rotations in optically trapped red blood cells

    NASA Astrophysics Data System (ADS)

    Bambardekar, Kapil; Dharmadhikari, Jayashree A.; Dharmadhikari, Aditya K.; Yamada, Toshihoro; Kato, Tsuyoshi; Kono, Hirohiko; Fujimura, Yuichi; Sharma, Shobhona; Mathur, Deepak

    2010-07-01

    A combined experimental and theoretical study is carried out to probe the rotational behavior of red blood cells (RBCs) in a single beam optical trap. We induce shape changes in RBCs by altering the properties of the suspension medium in which live cells float. We find that certain shape anisotropies result in the rotation of optically trapped cells. Indeed, even normal (healthy) RBCs can be made to rotate using linearly polarized trapping light by altering the osmotic stress the cells are subjected to. Hyperosmotic stress is found to induce shape anisotropies. We also probe the effect of the medium's viscosity on cell rotation. The observed rotations are modeled using a Langevin-type equation of motion that takes into account frictional forces that are generated as RBCs rotate in the medium. We observe good correlation between our measured data and calculated results.

  11. Unique and protective contributions of parenting and classroom processes to the adjustment of African American children living in single-parent families.

    PubMed

    Brody, Gene H; Dorsey, Shannon; Forehand, Rex; Armistead, Lisa

    2002-01-01

    The unique contributions that parenting processes (high levels of monitoring with a supportive, involved mother-child relationship) and classroom processes (high levels of organization, rule clarity, and student involvement) make to children's self-regulation and adjustment were examined with a sample of 277 single-parent African American families. A multi-informant design involving mothers, teachers, and 7- to 15-year-old children was used. Structural equation modeling indicated that parenting and classroom processes contributed uniquely to children's adjustment through the children's development of self-regulation. Additional analyses suggested that classroom processes can serve a protective-stabilizing function when parenting processes are compromised, and vice versa. Further research is needed to examine processes in both family and school contexts that promote child competence and resilience. PMID:14717257

  12. Safety and immunogenicity of single-dose live oral cholera vaccine strain CVD 103-HgR, prepared from new master and working cell banks.

    PubMed

    Chen, Wilbur H; Greenberg, Richard N; Pasetti, Marcela F; Livio, Sofie; Lock, Michael; Gurwith, Marc; Levine, Myron M

    2014-01-01

    Currently, no cholera vaccine is available for persons traveling from the United States to areas of high cholera transmission and who for reasons of occupation or host factors are at increased risk for development of the disease. A single-dose oral cholera vaccine with a rapid onset of protection would be particularly useful for such travelers and might also be an adjunct control measure for cholera outbreaks. The attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR harbors a 94% deletion of the cholera toxin A subunit gene (ctxA) and has a mercury resistance gene inserted in the gene encoding hemolysin A. We undertook a phase I randomized placebo-controlled two-site trial to assess the safety and immunogenicity of a preliminary formulation of CVD 103-HgR prepared from new master and working cell banks. Healthy young adults were randomized (5:1 vaccinees to placebo recipients) to receive a single oral dose of ∼4.4 × 10(8) CFU of vaccine or a placebo. Blood serum vibriocidal and cholera toxin-specific IgG antibodies were measured before and 10, 14, and 28 days following vaccination or placebo. Excretion of the vaccine strain in the stool was assessed during the first week postvaccination. A total of 66 subjects were enrolled, comprising 55 vaccinees and 11 placebo recipients. The vaccine was well tolerated. The overall vibriocidal and anti-cholera toxin seroconversion rates were 89% and 57%, respectively. CVD 103-HgR is undergoing renewed manufacture for licensure in the United States under the auspices of PaxVax. Our data mimic those from previous commercial formulations that elicited vibriocidal antibody seroconversion (a correlate of protection) in ∼90% of vaccinees. (This study has been registered at ClinicalTrials.gov under registration no. NCT01585181.). PMID:24173028

  13. Red blood cell production

    MedlinePlus

    ... cells are an important element of blood. Their job is to transport oxygen to the body’s tissues in exchange for carbon dioxide, which is carried to and eliminated by the lungs. Red blood cells are formed in the red bone marrow of bones. Stem cells in the red bone marrow called hemocytoblasts ...

  14. RED-LETTER DAYS

    EPA Science Inventory

    The word "red-letter" is an adjective meaning "of special significance." It's origin is from the practice of marking Christian holy days in red letters on calendars. The "red-letter days" to which I refer occurred while I was a graduate student of ...

  15. International Federation of Red Cross and Red Crescent Societies

    MedlinePlus

    ... dignity and resilience Geneva, 14 September 2016 – The International Federation of Red Cross and Red Crescent Societies... ... News Contact us Sitemap Go to top The International Federation of Red Cross and Red Crescent Societies ( ...

  16. Stark shift of the absorption spectra in Ge/Ge1-xSnx/Ge type-I single QW cell for mid-wavelength infra-red modulators

    NASA Astrophysics Data System (ADS)

    Yahyaoui, N.; Sfina, N.; Lazzari, J.-L.; Bournel, A.; Said, M.

    2015-09-01

    For mid-wavelength infra-red (MWIR) modulation or detection applications, we propose α-Sn rich Ge/Ge1-xSnx/Ge a type-I single quantum wells (SQW) partially strain compensated on Ge1-ySny relaxed layers grown onto (0 0 1)-oriented Ge substrate. Such elementary cells with W-like potential profiles of conduction and valence bands have been modeled by solving the one-dimensional Schrödinger equation under an applied external electrical field. First, strain effects on electrons, heavy holes (hh) and light holes (lh) energy bands for strained/relaxed Ge1-xSnx/Ge1-ySny heterointerfaces are investigated using the model-solid theory in the whole ranges (0 ⩽ x, y ⩽ 1) of Sn compositions. From the obtained band-discontinuities, band gaps and effective masses, Ge1-ySny/Ge/Ge0.80Sn0.20/Ge/Ge1-ySny cells are computed as a function of the Ge0.80Sn0.20 well width for three compositions of the Ge1-ySny buffer layer (y = 0.05, 0.07 and 0.09) in order to get the optimum quantum confinement of electrons and holes levels while keeping a reasonable amount of averaged strain in the cell. The electric field effect on the absorption spectra is given. An absorption coefficient in the 6× to 3 × 103 cm-1 range is reasonably obtained for a SQW at room temperature with a rather large Stark shift of the direct transition between 0.46 and 0.38 eV (i.e., λ = 3.26-2.70 μm) at large external fields (50 kV/cm). These characteristics are attractive for the design of MWIR optical modulators.

  17. A single base deletion in the Tfm androgen receptor gene creates a short-lived messenger RNA that directs internal translation initiation.

    PubMed Central

    Gaspar, M L; Meo, T; Bourgarel, P; Guenet, J L; Tosi, M

    1991-01-01

    Testosterone-resistant male mice hemizygous for the X-chromosome-linked mutant gene Tfm express detectable but severely reduced levels of androgen receptor mRNA, amounting to about 10% of the level found in normal male littermates. No structural abnormality could be identified in the coding region of the messenger by a series of RNase-protection assays. However, cell-free translation of RNAs transcribed in vitro from enzymatically amplified overlapping segments of exon 1 revealed a truncated receptor protein and helped to localize the site of premature termination. Sequence analysis of the relevant DNA segment disclosed that deletion of a single nucleotide in the hexacytidine stretch at position 1107-1112 alters the reading frame of the messenger and introduces 41 missense amino acids before a premature termination codon at position 1235-1237. Separately initiated carboxyl-terminal polypeptides are synthesized in vitro, starting probably at the in-frame AUG codon 1507-1509, which lies in a favorable context for translation initiation, and at the non-AUG codon 1144-1146. Transcriptional impairments of the Tfm gene were ruled out by a quantitative analysis of enzymatically amplified nuclear RNA precursors. No other change could be identified by sequencing the complete coding region of Tfm cDNA. The finding of the unsuspected termination codon and the evidence of internally initiated carboxyl-terminal polypeptides reconcile previous conclusions and account for all known phenotypic properties of the mutation. Images PMID:1924321

  18. Evaluation of the Single-Item Self-Rating Adherence Scale for Use in Routine Clinical Care of People Living with HIV

    PubMed Central

    Feldman, BJ; Fredericksen, RJ; Crane, PK; Safren, SA; Mugavero, MJ; Willig, James H; Simoni, JM; Wilson, IB; Saag, MS; Kitahata, MM; Crane, HM

    2012-01-01

    The Self-Rating Scale Item (SRSI) is a single-item self-report adherence measure that uses adjectives in a 5-point Likert scale, from “very poor” to “excellent,” to describe medication adherence over the past 4 weeks. This study investigated the SRSI in 2,399 HIV-infected patients in routine care at two outpatient primary HIV clinics. Correlations between the SRSI and four commonly used adherence items ranged from 0.37–0.64. Correlations of adherence barriers, such as depression and substance use, were comparable across all adherence items. General estimating equations suggested the SRSI is as good as or better than other adherence items (p’s <0.001 vs. <0.001–0.99) at predicting adherence-related clinical outcomes, such as HIV viral load and CD4+ cell count. These results and the SRSI’s low patient burden suggest its routine use could be helpful for assessing adherence in clinical care and should be more widespread, particularly where more complex instruments may be impractical. PMID:23108721

  19. [Pseudo-scabies transmitted by red fox].

    PubMed

    Birk, R W; Tebbe, B; Schein, E; Zouboulis, C C; Orfanos, C E

    1999-02-01

    Pseudoscabies, i.e. infestation of human skin with animal mites may occasionally occur and should be considered in the differential diagnosis of pruritic and papular skin disease. We report here on a 52-year-old woman with pseudoscabies or canine scabies (Sarcoptes scabiei var. canis), transmitted by indirect contact with a red fox in the urban area of Berlin. Red foxes may live in unhabited areas of metropolitan large cities, i.e. in garages, car wrecks and cellars. Full remission of the prolonged and pruritic rush was seen after topical administration of lindane together with systemic corticosteroids. PMID:10097956

  20. Identification of Cyanobacteriochromes Detecting Far-Red Light.

    PubMed

    Rockwell, Nathan C; Martin, Shelley S; Lagarias, J Clark

    2016-07-19

    The opacity of mammalian tissue to visible light and the strong attenuation of infrared light by water at ≥900 nm have contributed to growing interest in the development of far-red and near-infrared absorbing tools for visualizing and actuating responses within live cells. Here we report the discovery of cyanobacteriochromes (CBCRs) responsive to light in this far-red window. CBCRs are linear tetrapyrrole (bilin)-based light sensors distantly related to plant phytochrome sensors. Our studies reveal far-red (λmax = 725-755 nm)/orange (λmax = 590-600 nm) and far-red/red (λmax = 615-685 nm) photoswitches that are small (<200 amino acids) and can be genetically reconstituted in living cells. Phylogenetic analysis and characterization of additional CBCRs demonstrated that far-red/orange CBCRs evolved after a complex transition from green/red CBCRs known for regulating complementary chromatic acclimation. Incorporation of different bilin chromophores demonstrated that tuning mechanisms responsible for red-shifted chromophore absorption act at the A-, B-, and/or C-rings, whereas photoisomerization occurs at the D-ring. Two such proteins exhibited detectable fluorescence extending well into the near-infrared region. This work extends the spectral window of CBCRs to the edge of the infrared, raising the possibility of using CBCRs in synthetic biology applications in the far-red region of the spectrum. PMID:27295035

  1. The phylogeny of the red panda (Ailurus fulgens): evidence from the hindlimb.

    PubMed

    Fisher, Rebecca E; Adrian, Brent; Elrod, Clay; Hicks, Michelle

    2008-11-01

    The red panda (Ailurus fulgens) is an endangered carnivore living in the temperate forests of the Himalayas and southern China. The phylogeny of the red panda has been the subject of much debate. Morphological and molecular studies have supported a wide range of possible relationships, including close ties to procyonids, ursids, mustelids, and mephitids. This study provides additional morphological data, including muscle maps, for Ailurus. The hindlimbs of four cadavers from the National Zoological Park were dissected. Red pandas retain a number of muscles lost in other carnivore groups, including muscles and tendons related to their robust and weight-bearing hallux. Three features, including a single-bellied m. sartorius, a proximal insertion for m. abductor digiti V, and an absent m. articularis coxae, are found in all terrestrial arctoids, including Ailurus. In addition, red pandas are similar to ursids and canids in lacking a caudal belly of m. semitendinosus, while they resemble procyonids and mustelids in the degree of fusion observed between mm. gluteus medius and piriformis. Furthermore, Ailurus and procyonids are characterized by numerous subdivisions within the adductor compartment, while red pandas and raccoons share a variable m. semimembranosus, composed of one, two, or three bellies. Lastly, a deep plantar muscle inserting onto the metatarsophalangeal joint of the hallux is described for Ailurus. This muscle has not been previously described and is given the name m. flexor hallucis profundus. Additional dissections of the forelimb and axial musculature of red pandas may shed further light on the phylogeny of this species. In addition, the muscle maps presented here offer a valuable resource for interpreting the functional anatomy of fossil ailurids. PMID:19014366

  2. The phylogeny of the red panda (Ailurus fulgens): evidence from the hindlimb

    PubMed Central

    Fisher, Rebecca E; Adrian, Brent; Elrod, Clay; Hicks, Michelle

    2008-01-01

    The red panda (Ailurus fulgens) is an endangered carnivore living in the temperate forests of the Himalayas and southern China. The phylogeny of the red panda has been the subject of much debate. Morphological and molecular studies have supported a wide range of possible relationships, including close ties to procyonids, ursids, mustelids, and mephitids. This study provides additional morphological data, including muscle maps, for Ailurus. The hindlimbs of four cadavers from the National Zoological Park were dissected. Red pandas retain a number of muscles lost in other carnivore groups, including muscles and tendons related to their robust and weight-bearing hallux. Three features, including a single-bellied m. sartorius, a proximal insertion for m. abductor digiti V, and an absent m. articularis coxae, are found in all terrestrial arctoids, including Ailurus. In addition, red pandas are similar to ursids and canids in lacking a caudal belly of m. semitendinosus, while they resemble procyonids and mustelids in the degree of fusion observed between mm. gluteus medius and piriformis. Furthermore, Ailurus and procyonids are characterized by numerous subdivisions within the adductor compartment, while red pandas and raccoons share a variable m. semimembranosus, composed of one, two, or three bellies. Lastly, a deep plantar muscle inserting onto the metatarsophalangeal joint of the hallux is described for Ailurus. This muscle has not been previously described and is given the name m. flexor hallucis profundus. Additional dissections of the forelimb and axial musculature of red pandas may shed further light on the phylogeny of this species. In addition, the muscle maps presented here offer a valuable resource for interpreting the functional anatomy of fossil ailurids. PMID:19014366

  3. Interpretation of the Jupiter Red Spot, 1

    NASA Technical Reports Server (NTRS)

    Kuiper, G. P.

    1973-01-01

    With the aid of 8900 micron CH4 filter photographs it is shown that the Red Spot is normally the highest cloud feature in the Jovian atmosphere, located squarely on the South Tropical Zone at 22 deg. S. The Red Spot is a rising column, obviously having its own energy source; it spreads outward at the top level, in anticyclonic rotation. It is concluded that the large and persistent Jovian cloud masses, of which the Red Spot is the largest, are major organized storm arrays embedded in a near stagnant atmosphere, each probably possessing numerous short-lived rising hot columns driven by released latent heat; and covered by a horizontally-expanding gigantic cirrus cloud system, in anti-cyclonic rotation. The meteorological theory of Organized Cumulus Convection is applied to the Red Spot and the White Ovals. Evidence exists that the array of hot towers under the Red Spot is eccentric. The 90-day and the occassional 5-8 year fluctuations in the motion of the Red Spot may be due to processes analogous to those found in the Earth's Tropical Convergence. The 90-day oscillation has been observed for 8 years.

  4. Household Structure and Living Conditions in Nigeria

    ERIC Educational Resources Information Center

    Mberu, Blessing Uchenna

    2007-01-01

    Data on 7,632 households from the 1999 Nigeria Demographic and Health Survey are used to examine household structure and living conditions in Nigeria. The study finds significant disadvantage in living conditions of single-adult, female- and single-adult, male-headed households relative to two-parent households. Extended households show no…

  5. Fluorescence enhancement of single-phase red-blue emitting Ba3MgSi2O8:Eu2+,Mn2+ phosphors via Dy3+ addition for plant cultivation

    NASA Astrophysics Data System (ADS)

    Liu, Ling-Yun; Wang, Da-Jian; Mao, Zhi-Yong; Liu, Yan-Hua; Li, Xue-Zheng; Lu, Qi-Fei

    2009-01-01

    Fluorescence enhancement of red and blue concurrently emitting Ba3MgSi2O8:Eu2+,Mn2+ phosphors for plant cultivation has been investigated by Dy3+ addition. The Ba3MgSi2O8:Eu2+,Mn2+,Dy3+(BMS-EMD) phosphors have two-color emissions at the wavelength peak values of 437 nm and 620 nm at the excitation of 350 nm. The two emission bands are coincident with the absorption spectrum for photosynthesis of plants. An obvious enhancement effect has been observed upon addition of Dy3+ with amount of 0.03 mol%, in which the intensities of both blue and red bands reach a maximum. The origin of red and blue emission bands is analysed. The photochromic parameters of the samples at the nearly UV excitation are tested. This fluoresence enhancement is of great significance for special solid state lighting equipment used in plant cultivation.

  6. Hidden values in bauxite residue (red mud): Recovery of metals

    SciTech Connect

    Liu, Yanju; Naidu, Ravi

    2014-12-15

    Highlights: • Current iron recovery techniques using red mud are depicted. • Advantages and disadvantages exist in different recovering processes. • Economic and environmental friendly integrated usage of red mud is promising. - Abstract: Bauxite residue (red mud) is a hazardous waste generated from alumina refining industries. Unless managed properly, red mud poses significant risks to the local environment due to its extreme alkalinity and its potential impacts on surface and ground water quality. The ever-increasing generation of red mud poses significant challenges to the aluminium industries from management perspectives given the low proportion that are currently being utilized beneficially. Red mud, in most cases, contains elevated concentrations of iron in addition to aluminium, titanium, sodium and valuable rare earth elements. Given the scarcity of iron supply globally, the iron content of red mud has attracted increasing research interest. This paper presents a critical overview of the current techniques employed for iron recovery from red mud. Information on the recovery of other valuable metals is also reviewed to provide an insight into the full potential usage of red mud as an economic resource rather than a waste. Traditional hydrometallurgy and pyrometallurgy are being investigated continuously. However, in this review several new techniques are introduced that consider the process of iron recovery from red mud. An integrated process which can achieve multiple additional values from red mud is much preferred over the single process methods. The information provided here should help to improve the future management and utilization of red mud.

  7. Deciphering and dating the red panda's ancestry and early adaptive radiation of Musteloidea.

    PubMed

    Sato, Jun J; Wolsan, Mieczyslaw; Minami, Shinji; Hosoda, Tetsuji; Sinaga, Martua H; Hiyama, Kozue; Yamaguchi, Yasunori; Suzuki, Hitoshi

    2009-12-01

    Few species have been of more disputed affinities than the red or lesser panda (Ailurus fulgens), an endangered endemic Southeast Asian vegetarian member of the placental mammalian order Carnivora. This peculiar carnivoran has mostly been classified with raccoons (Procyonidae) or bears (Ursidae), grouped with the giant panda (Ailuropoda melanoleuca) in their own family, or considered a separate lineage of equivocal ancestry. Recent molecular studies have indicated a close affinity of the red panda to a clade of procyonids and mustelids (weasels, otters, martens, badgers, and allies), but have failed to unambiguously resolve the position of this species relative to mephitids (skunks and stink badgers). We examined the relationship of the red panda to other extant species of the carnivoran suborder Caniformia using a set of concatenated approximately 5.5-kb sequences from protein-coding exons of five nuclear genes. Bayesian, maximum likelihood, and parsimony phylogenetic analyses strongly supported the red panda as the closest living relative of a clade containing Procyonidae and Mustelidae to the exclusion of Mephitidae. These three families together with the red panda (which is classified here as a single extant species of a distinct family, Ailuridae) compose the superfamily Musteloidea, a clade strongly supported by all our phylogenetic analyses as sister to the monophyletic Pinnipedia (seals, sea lions, walruses). The approximately unbiased, Kishino-Hasegawa, and Templeton topology tests rejected (P<0.05) each of all possible alternative hypotheses about the relationships among the red panda and mephitids, procyonids, and mustelids. We also estimated divergence times for the red panda's lineage and ones of other caniform taxa, as well as the ages of the first appearance datums for the crown and total clades of musteloids and the total clades of the red panda, mephitids, procyonids, and mustelids. Bayesian relaxed molecular-clock analysis using combined

  8. Anterior Chamber Live Loa loa: Case Report.

    PubMed

    Kagmeni, G; Cheuteu, R; Bilong, Y; Wiedemann, P

    2016-01-01

    We reported a case of unusual intraocular Loa loa in a 27-year-old patient who presented with painful red eye. Biomicroscopy revealed a living and active adult worm in the anterior chamber of the right eye. After surgical extraction under local anesthesia, parasitological identification confirmed L. loa filariasis. PMID:27441005

  9. Anterior Chamber Live Loa loa: Case Report

    PubMed Central

    Kagmeni, G.; Cheuteu, R.; Bilong, Y.; Wiedemann, P.

    2016-01-01

    We reported a case of unusual intraocular Loa loa in a 27-year-old patient who presented with painful red eye. Biomicroscopy revealed a living and active adult worm in the anterior chamber of the right eye. After surgical extraction under local anesthesia, parasitological identification confirmed L. loa filariasis. PMID:27441005

  10. Red Clover Breeding Progress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover (Trifolium pratense L.) is an important forage legume grown on approximately 4 million hectares worldwide. It has a long and varied history in agriculture. Active breeding efforts began at the end of the 19th century. Since this time significant improvement in red clover cultivar for a...

  11. Cobb's Red Cabbage Indicator.

    ERIC Educational Resources Information Center

    Cobb, Vicki

    1998-01-01

    Describes the use of an indicator made from the pigment in red cabbage. Cabbage is grated then soaked in water. When the water is a strong red, the cabbage is strained out. The cabbage-juice indicator is then used to test for acids and bases. Includes a list of good foods to test for acidity and alkalinity. (PVD)

  12. 7X performance results - final report : ASCI Red vs Red Storm.

    SciTech Connect

    Dinge, Dennis C.; Davis, Michael E.; Haskell, Karen H.; Ballance, Robert A.; Gardiner, Thomas Anthony; Stevenson, Joel O.; Noe, John P.

    2011-04-01

    The goal of the 7X performance testing was to assure Sandia National Laboratories, Cray Inc., and the Department of Energy that Red Storm would achieve its performance requirements which were defined as a comparison between ASCI Red and Red Storm. Our approach was to identify one or more problems for each application in the 7X suite, run those problems at multiple processor sizes in the capability computing range, and compare the results between ASCI Red and Red Storm. The first part of this report describes the two computer systems, the applications in the 7X suite, the test problems, and the results of the performance tests on ASCI Red and Red Storm. During the course of the testing on Red Storm, we had the opportunity to run the test problems in both single-core mode and dual-core mode and the second part of this report describes those results. Finally, we reflect on lessons learned in undertaking a major head-to-head benchmark comparison.

  13. ISS Live!

    NASA Technical Reports Server (NTRS)

    Price, Jennifer; Harris, Philip; Hochstetler, Bruce; Guerra, Mark; Mendez, Israel; Healy, Matthew; Khan, Ahmed

    2013-01-01

    International Space Station Live! (ISSLive!) is a Web application that uses a proprietary commercial technology called Lightstreamer to push data across the Internet using the standard http port (port 80). ISSLive! uses the push technology to display real-time telemetry and mission timeline data from the space station in any common Web browser or Internet- enabled mobile device. ISSLive! is designed to fill a unique niche in the education and outreach areas by providing access to real-time space station data without a physical presence in the mission control center. The technology conforms to Internet standards, supports the throughput needed for real-time space station data, and is flexible enough to work on a large number of Internet-enabled devices. ISSLive! consists of two custom components: (1) a series of data adapters that resides server-side in the mission control center at Johnson Space Center, and (2) a set of public html that renders the data pushed from the data adapters. A third component, the Lightstreamer server, is commercially available from a third party and acts as an intermediary between custom components (1) and (2). Lightstreamer also provides proprietary software libraries that are required to use the custom components. At the time of this reporting, this is the first usage of Web-based, push streaming technology in the aerospace industry.

  14. Live Virus Smallpox Vaccine

    MedlinePlus

    ... Index SMALLPOX FACT SHEET The Live Virus Smallpox Vaccine The vaccinia virus is the "live virus" used ... cannot cause smallpox. What is a "live virus" vaccine? A "live virus" vaccine is a vaccine that ...

  15. Aspergillosis in a red-crowned crane

    USGS Publications Warehouse

    Stroud, R.K.; Duncan, R.M.

    1983-01-01

    An unusual form of pulmonary aspergillosis in a red-crowned crane (Grus japonensis) is described in this report. The major lesion is unique because it closely resembles a lesion referred to as an aspergilloma. An aspergilloma is a single large granulomatous lesion that resembles a tumor and is caused by fungi of the genus Aspergillus.

  16. Blood Banking in Living Droplets

    PubMed Central

    Shao, Lei; Zhang, Xiaohui; Xu, Feng; Song, YoungSeok; Keles, Hasan Onur; Matloff, Laura; Markel, Jordan; Demirci, Utkan

    2011-01-01

    Blood banking has a broad public health impact influencing millions of lives daily. It could potentially benefit from emerging biopreservation technologies. However, although vitrification has shown advantages over traditional cryopreservation techniques, it has not been incorporated into transfusion medicine mainly due to throughput challenges. Here, we present a scalable method that can vitrify red blood cells in microdroplets. This approach enables the vitrification of large volumes of blood in a short amount of time, and makes it a viable and scalable biotechnology tool for blood cryopreservation. PMID:21412411

  17. Living Nanomachines

    NASA Astrophysics Data System (ADS)

    Carlier, M.-F.; Helfer, E.; Wade, R.; Haraux, F.

    The living cell is a kind of factory on the microscopic scale, in which an assembly of modular machines carries out, in a spatially and temporally coordinated way, a whole range of activities internal to the cell, including the synthesis of substances essential to its survival, intracellular traffic, waste disposal, and cell division, but also activities related to intercellular communication and exchanges with the outside world, i.e., the ability of the cell to change shape, to move within a tissue, or to organise its own defence against attack by pathogens, injury, and so on. These nanomachines are made up of macromolecular assemblies with varying degrees of complexity, forged by evolution, within which work is done as a result of changes in interactions between proteins, or between proteins and nucleic acids, or between proteins and membrane components. All these cell components measure a few nanometers across, so the mechanical activity of these nanomachines all happens on the nanometric scale. The directional nature of the work carried out by biological nanomachines is associated with a dissipation of energy. As examples of protein assemblies, one could mention the proteasome, which is responsible for the degradation of proteins, and linear molecular motors such as actomyosin, responsible for muscle contraction, the dynein-microtubule system, responsible for flagellar motility, and the kinesin-microtubule system, responsible for transport of vesicles, which transform chemical energy into motion. Nucleic acid-protein assemblies include the ribosome, responsible for synthesising proteins, polymerases, helicases, elongation factors, and the machinery of DNA replication and repair; the mitotic spindle is an integrated system involving several of these activities which drive chromosome segregation. The machinery coupling membranes and proteins includes systems involved in the energy metabolism, such as the ATP synthase rotary motor, signalling cascades, endocytosis

  18. Spectral Tuning of Deep Red Cone Pigments†

    PubMed Central

    Amora, Tabitha L.; Ramos, Lavoisier S.; Galan, Jhenny F.; Birge, Robert R.

    2008-01-01

    Visual pigments are G-protein-coupled receptors that provide a critical interface between organisms and their external environment. Natural selection has generated vertebrate pigments that absorb light from the far-UV (360 nm) to the deep red (630 nm) while using a single chromophore, in either the A1 (11-cis-retinal) or A2 (11-cis-3,4-dehydroretinal) form. The fact that a single chromophore can be manipulated to have an absorption maximum across such an extended spectral region is remarkable. The mechanisms of wavelength regulation remain to be fully revealed, and one of the least well-understood mechanisms is that associated with the deep red pigments. We investigate theoretically the hypothesis that deep red cone pigments select a 6-s-trans conformation of the retinal chromophore ring geometry. This conformation is in contrast to the 6-s-cis ring geometry observed in rhodopsin and, through model chromophore studies, the vast majority of visual pigments. Nomographic spectral analysis of 294 A1 and A2 cone pigment literature absorption maxima indicates that the selection of a 6-s-trans geometry red shifts M/LWS A1 pigments by ~1500 cm−1 (~50 nm) and A2 pigments by ~2700 cm−1 (~100 nm). The homology models of seven cone pigments indicate that the deep red cone pigments select 6-s-trans chromophore conformations primarily via electrostatic steering. Our results reveal that the generation of a 6-s-trans conformation not only achieves a significant red shift but also provides enhanced stability of the chromophore within the deep red cone pigment binding sites. PMID:18370404

  19. Fluorogenic Probes for Multicolor Imaging in Living Cells.

    PubMed

    Lukinavičius, Gražvydas; Reymond, Luc; Umezawa, Keitaro; Sallin, Olivier; D'Este, Elisa; Göttfert, Fabian; Ta, Haisen; Hell, Stefan W; Urano, Yasuteru; Johnsson, Kai

    2016-08-01

    Here we present a far-red, silicon-rhodamine-based fluorophore (SiR700) for live-cell multicolor imaging. SiR700 has excitation and emission maxima at 690 and 715 nm, respectively. SiR700-based probes for F-actin, microtubules, lysosomes, and SNAP-tag are fluorogenic, cell-permeable, and compatible with superresolution microscopy. In conjunction with probes based on the previously introduced carboxy-SiR650, SiR700-based probes permit multicolor live-cell superresolution microscopy in the far-red, thus significantly expanding our capacity for imaging living cells. PMID:27420907

  20. Living with Gout

    MedlinePlus Videos and Cool Tools

    ... purines -- those foods include liver, seafood and red meat. Historically gout has been seen as a disease ... affluent -- those who could afford large quantities of meats and red wine. The popular comic image was ...

  1. Living with Heart Disease

    MedlinePlus

    ... followup visits. Rate This Content: NEXT >> Featured Video All of Our Stories Are Red: Yaskary's Story 04/ ... part of the National Institutes of Health (NIH). All of Our Stories Are Red: Eileen's Story 04/ ...

  2. Educationally and Economically Disadvantaged Children. Joint Hearing Reviewing Available Education Opportunities for Disadvantaged Children, Focusing on Those Children Living in Poverty, Those from Single-Parent Families, and Those Whose Parents Are Teenagers, before the Committee on Labor and Human Resources, United States Senate and the Committee on Education and Labor. House of Representatives, One Hundredth Congress, First Session.

    ERIC Educational Resources Information Center

    Congress of the U.S., Washington, DC. House Committee on Education and Labor.

    This joint hearing before the Senate Committee on Labor and Human Resources and the House Committee on Education and Labor focused on educationally and economically disadvantaged children. Of special concern were children living in poverty, those from single parent families, and those whose parents are teenagers. Educational programs are needed to…

  3. Red Bull Stratos Presentation

    NASA Video Gallery

    Red Bull Stratos High Performance Director Andy Walshe & Technical Project Director Art Thompson share the Stratos story with JSC. Supported by a team of experts, Felix Baumgartner reached 128,100 ...

  4. Jupiter's Great Red Spot

    NASA Technical Reports Server (NTRS)

    1996-01-01

    This view of Jupiter's Great Red Spot is a mosaic of two images taken by the Galileo spacecraft. The image was created using two filters, violet and near-infrared, at each of two camera positions. The Great Red Spot is a storm in Jupiter's atmosphere and is at least 300 years-old. Winds blow counterclockwise around the Great Red Spot at about 400 kilometers per hour (250 miles per hour). The size of the storm is more than one Earth diameter (13,000 kilometers or 8,000 miles) in the north-south direction and more than two Earth diameters in the east-west direction. In this oblique view, where the Great Red Spot is shown on the planet's limb, it appears longer in the north-south direction. The image was taken on June 26, 1996.

    The Galileo mission is managed by NASA's Jet Propulsion Laboratory.

  5. Red blood cell production

    MedlinePlus Videos and Cool Tools

    ... or another. Red blood cells are an important element of blood. Their job is to transport oxygen ... hemocytoblasts give rise to all of the formed elements in blood. If a hemocytoblast commits to becoming ...

  6. Characterization of MSB Synapses in Dissociated Hippocampal Culture with Simultaneous Pre- and Postsynaptic Live Microscopy

    PubMed Central

    Reilly, James E.; Hanson, Hugo H.; Fernández-Monreal, Mónica; Hof, Patrick R.; Phillips, Greg R.

    2011-01-01

    Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our live microscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity. PMID:22028887

  7. Whence the red panda?

    PubMed

    Flynn, J J; Nedbal, M A; Dragoo, J W; Honeycutt, R L

    2000-11-01

    The evolutionary history of the red panda (Ailurus fulgens) plays a pivotal role in the higher-level phylogeny of the "bear-like" arctoid carnivoran mammals. Characters from morphology and molecules have provided inconsistent evidence for placement of the red panda. Whereas it certainly is an arctoid, there has been major controversy about whether it should be placed with the bears (ursids), ursids plus pinnipeds (seals, sea lions, walrus), raccoons (procyonids), musteloids (raccoons plus weasels, skunks, otters, and badgers [mustelids]), or as a monotypic lineage of uncertain phylogenetic affinities. Nucleotide sequence data from three mitochondrial genes and one nuclear intron were analyzed, with more complete taxonomic sampling of relevant taxa (arctoids) than previously available in analyses of primary molecular data, to clarify the phylogenetic relationships of the red panda to other arctoid carnivorans. This study provides detailed phylogenetic analyses (both parsimony and maximum-likelihood) of primary character data for arctoid carnivorans, including bootstrap and decay indices for all arctoid nodes, and three statistical tests of alternative phylogenetic hypotheses for the placement of the red panda. Combined phylogenetic analyses reject the hypotheses that the red panda is most closely related to the bears (ursids) or to the raccoons (procyonids). Rather, evidence from nucleotide sequences strongly support placement of the red panda within a broad Musteloidea (sensu lato) clade, including three major lineages (the red panda, the skunks [mephitids], and a clearly monophyletic clade of procyonids plus mustelids [sensu stricto, excluding skunks]). Within the Musteloidea, interrelationships of the three major lineages are unclear and probably are best considered an unresolved trichotomy. These data provide compelling evidence for the relationships of the red panda and demonstrate that small taxonomic sample sizes can result in misleading or possibly erroneous

  8. Bioavailability and Biokinetics of Anthocyanins From Red Grape Juice and Red Wine

    PubMed Central

    Bitsch, Roland

    2004-01-01

    In a comparative study, 9 healthy volunteers ingested a single oral dose of 400 mL red grape juice or red wine with dose-adjusted anthocyanin content (283.5 mg or 279.6 mg, resp) in crossover. The content of anthocyanin glucosides was detected in plasma and urinary excretion. Additionally, the plasmatic antioxidant activity was assessed after intake. Based on the plasma content, biokinetic criteria of the single anthocyanins were calculated, such as AUC, cmax, tmax, and the elimination rate t1/2. The urinary excretion of total anthocyanins differed significantly and amounted to 0.18% (red wine) and 0.23% (red grape juice) of the administered dose. Additionally, the plasmatic antioxidant activity increased to higher levels after juice ingestion compared to wine. The intestinal absorption of the anthocyanins of red grape juice seemed to be improved compared to red wine, suggesting a possible synergistic effect of the glucose content of the juice. The improved absorption resulted in an enhanced plasmatic bioactivity. PMID:15577192

  9. Child Living Arrangements by Race and Income: A Supplementary Analysis.

    ERIC Educational Resources Information Center

    Primus, Wendell E.

    This supplementary analysis to "Declining Share of Children Lived with Single Mothers in the Late 1990s" employs an alternative methodology to provide a clearer picture of changes in living arrangements within different income groups. The original study concluded that children were significantly less likely to live with single mothers in 2000 than…

  10. mEosFP-Based Green-to-Red Photoconvertible Subcellular Probes for Plants1[W][OA

    PubMed Central

    Mathur, Jaideep; Radhamony, Resmi; Sinclair, Alison M.; Donoso, Ana; Dunn, Natalie; Roach, Elyse; Radford, Devon; S. Mohammad Mohaghegh, P.; Logan, David C.; Kokolic, Ksenija; Mathur, Neeta

    2010-01-01

    Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists’ toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs. PMID:20940350

  11. Hidden values in bauxite residue (red mud): recovery of metals.

    PubMed

    Liu, Yanju; Naidu, Ravi

    2014-12-01

    Bauxite residue (red mud) is a hazardous waste generated from alumina refining industries. Unless managed properly, red mud poses significant risks to the local environment due to its extreme alkalinity and its potential impacts on surface and ground water quality. The ever-increasing generation of red mud poses significant challenges to the aluminium industries from management perspectives given the low proportion that are currently being utilized beneficially. Red mud, in most cases, contains elevated concentrations of iron in addition to aluminium, titanium, sodium and valuable rare earth elements. Given the scarcity of iron supply globally, the iron content of red mud has attracted increasing research interest. This paper presents a critical overview of the current techniques employed for iron recovery from red mud. Information on the recovery of other valuable metals is also reviewed to provide an insight into the full potential usage of red mud as an economic resource rather than a waste. Traditional hydrometallurgy and pyrometallurgy are being investigated continuously. However, in this review several new techniques are introduced that consider the process of iron recovery from red mud. An integrated process which can achieve multiple additional values from red mud is much preferred over the single process methods. The information provided here should help to improve the future management and utilization of red mud. PMID:25269817

  12. Effects of regularly consuming dietary fibre rich soluble cocoa products on bowel habits in healthy subjects: a free-living, two-stage, randomized, crossover, single-blind intervention

    PubMed Central

    2012-01-01

    Background Dietary fibre is both preventive and therapeutic for bowel functional diseases. Soluble cocoa products are good sources of dietary fibre that may be supplemented with this dietary component. This study assessed the effects of regularly consuming two soluble cocoa products (A and B) with different non-starch polysaccharides levels (NSP, 15.1 and 22.0% w/w, respectively) on bowel habits using subjective intestinal function and symptom questionnaires, a daily diary and a faecal marker in healthy individuals. Methods A free-living, two-stage, randomized, crossover, single-blind intervention was carried out in 44 healthy men and women, between 18-55 y old, who had not taken dietary supplements, laxatives, or antibiotics six months before the start of the study. In the four-week-long intervention stages, separated by a three-week-wash-out stage, two servings of A and B, that provided 2.26 vs. 6.60 g/day of NSP respectively, were taken. In each stage, volunteers' diet was recorded using a 72-h food intake report. Results Regularly consuming cocoa A and B increased fibre intake, although only cocoa B significantly increased fibre intake (p < 0.001) with respect to the non-cocoa stage. No changes in body weight were observed in either of the 4 week interventions. With cocoa product B, the number of daily bowel movements increased (p = 0.002), the frequency of having a bowel movement once a day increased (p = 0.009), the time to have a bowel movement was lower (p = 0.016) as well as the feeling of constipation (p = 0.046) without inducing adverse gastrointestinal symptoms, only flatulence increased (p = 0.019). Conclusions Regular consumption of the cocoa products increases dietary fibre intake to recommended levels and product B improves bowel habits. The use of both objective and subjective assessments to evaluate the effects of food on bowel habits is recommended. PMID:22512838

  13. A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry.

    PubMed

    Smith, P J; Wiltshire, M; Davies, S; Patterson, L H; Hoy, T

    1999-10-29

    The deep red fluorescing agent (DRAQ5) is a synthetic anthraquinone with a high affinity for DNA and a high capacity to rapidly enter living cells or stain fixed cells. DRAQ5 is optimally excited by red-light emitting sources and yields a deep red emission spectrum which extends into the low infra-red. DRAQ5 shows excitation at sub-optimal wavelengths including the 488 nm line and the multi-line UV wavelengths emitted by argon-ion lasers. Single beam (488 nm) flow cytometry has been used to demonstrate the utility of DRAQ5-nuclear DNA fluorescence as a discriminating parameter for human leucocytes and lymphoma cells, in combination with fluorochrome-labelled antibodies for the detection of surface antigens and subpopulation recognition. DRAQ5 fluorescence was found to reflect cellular DNA content as evidenced by cell cycle distribution profiles for asynchronous and cell cycle-perturbed populations. Importantly, DRAQ5 can be used in combination with FITC and RPE-labelled antibodies, without the need for fluorescence compensation. PMID:10556697

  14. Contemporary Single Mothers.

    ERIC Educational Resources Information Center

    Eiduson, Bernice T.

    Fifty Caucasian, never-married single mothers aged 18-30, who had opted to keep their babies, were studied longitudinally from the last trimester of pregnancy through the first three years of their children's lives in order to learn the extent to which they had reinterpreted traditional roles and responsibilities and had restructured their lives.…

  15. New red phosphor for near-ultraviolet light-emitting diodes with high color-purity

    SciTech Connect

    Wang, Zhengliang; He, Pei; Wang, Rui; Zhao, Jishou; Gong, Menglian

    2010-02-15

    New red phosphors, Na{sub 5}Eu(MoO{sub 4}){sub 4} doped with boron oxide were prepared by the solid-state reaction. Their structure and photo-luminescent properties were investigated. With the introduction of boron oxide, the red emission intensity of the phosphors under 395 nm excitation is strengthened, with high color-purity (x = 0.673, y = 0.327). The single red light-emitting diode was obtained by combining InGaN chip with the red phosphor, bright red light can be observed by naked eyes from the red light-emitting diodes under a forward bias of 20 mA.

  16. Living with endometriosis

    MedlinePlus

    Pelvic pain - living with endometriosis; Endometrial implant - living with endometriosis; Endometrioma - living with endometriosis ... counter pain relievers can reduce the pain of endometriosis. These include: Ibuprofen (Advil) Naproxen (Aleve) Acetaminophen (Tylenol) ...

  17. Living with an Arrhythmia

    MedlinePlus

    ... from the NHLBI on Twitter. Living With an Arrhythmia Many arrhythmias are harmless. It's common to have an occasional ... heartbeat or mild palpitations . People who have harmless arrhythmias can live healthy lives. They usually don't ...

  18. Living Gluten Free

    MedlinePlus

    ... turn JavaScript on. Feature: Celiac Disease Living Gluten Free Past Issues / Spring 2015 Table of Contents Allowed ... to Live Well with Celiac Disease / Living Gluten-Free Spring 2015 Issue: Volume 10 Number 1 Page ...

  19. Opening Minds: Using Language to Change Lives

    ERIC Educational Resources Information Center

    Johnston, Peter H.

    2012-01-01

    Sometimes a single word changes everything. In his groundbreaking book "Choice Words", Peter Johnston demonstrated how the things teachers say (and don't say) have surprising consequences for the literate lives of students. Now, in "Opening Minds: Using Language to Change Lives", Peter shows how the words teachers choose affect the worlds students…

  20. School on Probation: Teaching That Changes Lives

    ERIC Educational Resources Information Center

    Stoehr, Taylor

    2006-01-01

    For the past dozen years the author has been teaching, along with other volunteers, in a program called "Changing Lives Through Literature," serving the Dorchester District Court of the Commonwealth of Massachusetts. Changing Lives began as a single experiment in New Bedford in 1991 and has spread entirely by word of mouth to a dozen…

  1. In vitro photoacoustic measurement of hemoglobin oxygen saturation using a single pulsed broadband supercontinuum laser source.

    PubMed

    Lee, Changho; Jeon, Mansik; Jeon, Min Yong; Kim, Jeehyun; Kim, Chulhong

    2014-06-20

    We have utilized a single pulsed broadband supercontinuum laser source to photoacoustically sense total hemoglobin concentration (HbT) and oxygen saturation of hemoglobin (SO2) in bloods in vitro. Unlike existing expensive and bulky laser systems typically used for functional photoacoustic imaging (PAI), our laser system is relatively cost-effective and compact. Instead of using two single wavelengths, two wavelength bands were applied to distinguish the concentrations of two different chromophores in the mixture. In addition, we have successfully extracted the total dye concentration and the ratio of the red dye concentration to the total dye concentration in mixed red and blue dye solutions in phantoms. The results indicate that PAI with a cheap and compact fiber based laser source can potentially provide HbT and SO2 in live animals in vivo. PMID:24979418

  2. Reviving red snapper.

    PubMed

    Estabrook, Barry

    2010-01-01

    Red snappers in the Gulf of Mexico once hovered on the brink of extinction, their population having dropped to 2 percent of what had historically swum in the Gulf. But thanks to a recently introduced plan that turns the conventional wisdom of fisheries management on its head, the picture has begun to change. Called Individual Fishing Quotas (IFQs), the new regulations, which give a guaranteed allotment of fish to each participant instead of applying industry-wide quotas, went into effect for Gulf of Mexico Red Snapper (Lutjanus campechanus) in early 2007. The results were immediate and so profound that the Gulf Fishery Management Council voted earlier this year to increase the annual limit on red snapper to nearly 7 million pounds from 5 million. PMID:21542214

  3. Red-based cumulus.

    PubMed

    Gedzelman, Stanley David

    2015-02-01

    Observations and model simulations of cumulus clouds whose bases are tinted red when the Sun is well above the horizon are presented. Conditions for seeing red bases include (1) a red underlying surface (which may consist of dust clouds, as from haboobs) with high albedo, (2) small fractional cloud cover when the Sun is far enough below the zenith for direct sunlight to illuminate much of the surface directly below and around cloud base, (3) optically thick clouds so that the bases are dark, and (4) clouds with bases that are near enough to the observer to appear high in the sky so that the admixture of scattered light from the intervening atmosphere is minimized. PMID:25967822

  4. Chemical toxicity of red cells.

    PubMed Central

    Piomelli, S

    1981-01-01

    Exposure to toxic chemicals may result in alterations of red cell function. In certain cases, the toxic effect requires a genetic predisposition and thus affects only a restricted number of individuals; in other instances, the toxic effect is exerted on the hematopoietic system of every person. Glucose-6-phosphate dehydrogenase deficiency is probably the most widespread genetic disorder. It is observed at highest frequency in populations from subtropical countries as a result of its selective advantage vis à vis falciparum malaria. The gene controlling this enzyme is located on the X-chromosome; thus, the defect is sex-linked. Individuals with a genetic defect of this enzyme are extremely susceptible to hemolysis, when exposed to oxidant drugs (such as certain antimalarials and sulfonamides) because of the inability of their red cells to regenerate NADPH. Lead poisoning result in profound effects on the process of heme synthesis. Among the steps most sensitive to lead toxicity are the enzyme delta-aminolevulinic acid dehydratase and the intramitochondrial step that leads to the incorporation of iron into protoporphyrin. By these mechanisms, in severe lead intoxication there is an accumulation of large amounts of delta-aminolevulinic acid (a compound with inherent neurotoxicity), and there are abnormalities of mitochondrial function in all cells of the body. Individuals living in an industrialized society are unavoidably exposed to some environmental lead. Recent evidence indicates that, even at levels of exposure which do not increase the blood lead level above values presently considered normal, abnormalities of heme synthesis are clearly detectable. PMID:7016524

  5. Common fluorescent proteins for single-molecule localization microscopy

    NASA Astrophysics Data System (ADS)

    Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

    2015-07-01

    Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

  6. Lustre on Red Sky.

    SciTech Connect

    Monk, Stephen Todd; Mervini, Joe

    2010-04-01

    The goals of Lustre on Red Sky are: (1) provide home/projects/scratch Lustre file systems; (2) adhere to the Sun HPC stack; (3) implement software RAID on Sun provided JBODs; and (4) design for easy administration. Conclusions are: (1) software RAID includes additional risks and administration vs. hardware RAID solutions; (2) limited testing of hardware in these configurations make it ill-suited for rapid deployment in a production environment; and (3) Lustre has been a shining star on this machine, Red Sky users are pleased with its performance.

  7. The IOC consensus statement: beyond the Female Athlete Triad--Relative Energy Deficiency in Sport (RED-S).

    PubMed

    Mountjoy, Margo; Sundgot-Borgen, Jorunn; Burke, Louise; Carter, Susan; Constantini, Naama; Lebrun, Constance; Meyer, Nanna; Sherman, Roberta; Steffen, Kathrin; Budgett, Richard; Ljungqvist, Arne

    2014-04-01

    Protecting the health of the athlete is a goal of the International Olympic Committee (IOC). The IOC convened an expert panel to update the 2005 IOC Consensus Statement on the Female Athlete Triad. This Consensus Statement replaces the previous and provides guidelines to guide risk assessment, treatment and return-to-play decisions. The IOC expert working group introduces a broader, more comprehensive term for the condition previously known as 'Female Athlete Triad'. The term 'Relative Energy Deficiency in Sport' (RED-S), points to the complexity involved and the fact that male athletes are also affected. The syndrome of RED-S refers to impaired physiological function including, but not limited to, metabolic rate, menstrual function, bone health, immunity, protein synthesis, cardiovascular health caused by relative energy deficiency. The cause of this syndrome is energy deficiency relative to the balance between dietary energy intake and energy expenditure required for health and activities of daily living, growth and sporting activities. Psychological consequences can either precede RED-S or be the result of RED-S. The clinical phenomenon is not a 'triad' of the three entities of energy availability, menstrual function and bone health, but rather a syndrome that affects many aspects of physiological function, health and athletic performance. This Consensus Statement also recommends practical clinical models for the management of affected athletes. The 'Sport Risk Assessment and Return to Play Model' categorises the syndrome into three groups and translates these classifications into clinical recommendations. PMID:24620037

  8. Automatic red eye correction and its quality metric

    NASA Astrophysics Data System (ADS)

    Safonov, Ilia V.; Rychagov, Michael N.; Kang, KiMin; Kim, Sang Ho

    2008-01-01

    The red eye artifacts are troublesome defect of amateur photos. Correction of red eyes during printing without user intervention and making photos more pleasant for an observer are important tasks. The novel efficient technique of automatic correction of red eyes aimed for photo printers is proposed. This algorithm is independent from face orientation and capable to detect paired red eyes as well as single red eyes. The approach is based on application of 3D tables with typicalness levels for red eyes and human skin tones and directional edge detection filters for processing of redness image. Machine learning is applied for feature selection. For classification of red eye regions a cascade of classifiers including Gentle AdaBoost committee from Classification and Regression Trees (CART) is applied. Retouching stage includes desaturation, darkening and blending with initial image. Several versions of approach implementation using trade-off between detection and correction quality, processing time, memory volume are possible. The numeric quality criterion of automatic red eye correction is proposed. This quality metric is constructed by applying Analytic Hierarchy Process (AHP) for consumer opinions about correction outcomes. Proposed numeric metric helped to choose algorithm parameters via optimization procedure. Experimental results demonstrate high accuracy and efficiency of the proposed algorithm in comparison with existing solutions.

  9. Desiccation sensitivity of white and black (red) oak embryonic axes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genus Quercus is the most important aggregation of hardwoods found on the North American and European continents. Quercus is generally divided into two categories black (red) and white oak groups though a third category of live oaks is sometimes recognized as well. Natural populations of oaks a...

  10. Clover, Red (Trifolium pretense)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover, an important ...

  11. 'Valley Red' Strawberry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    'Valley Red' is a new June-bearing (short-day) strawberry (Fragaria ×ananassa Duchesne ex Rozier) cultivar from the U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS) breeding program in Corvallis, Ore., released in cooperation with the Oregon Agricultural Experiment Station, Th...

  12. 'Vintage' Red Raspberry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    'Vintage' is a new primocane-fruiting red raspberry (Rubus idaeus L.) from the U.S. Dept. of Agriculture–Agricultural Research Service (USDA–ARS) breeding program in Corvallis, OR released in cooperation with the Oregon State Agricultural Experiment Station and the Washington State University Agricu...

  13. Red Cross Swimming Update.

    ERIC Educational Resources Information Center

    Vlasich, Cynthia

    1989-01-01

    Six new aquatic courses, developed by the Red Cross, are described. They are: Infant and Preschool Aquatics, Longfellow's Whale Tales (classroom water safety lessons for K-Six), Basic Water Safety, Emergency Water Safety, Lifeguard Training, and Safety Training for Swim Coaches. (IAH)

  14. Red Emitting VCSEL

    NASA Astrophysics Data System (ADS)

    Jetter, Michael; Roßbach, Robert; Michler, Peter

    This chapter describes the progress in development of vertical-cavity surface-emitting lasers (VCSEL) emitting in the red spectral region around 650 nm for data transmission over polymer optical fibers (POF). First, growth issues of red VCSEL using two different material systems, namely AlGaAs and AlGaInP, are introduced. In particular, the optical and electrical state-of-the-art characteristics as low threshold currents ({≤} 1 mA) and high output powers (several mW) are presented with a special focus on emission wavelength. Also the thermal budget and heat removal in the devices are pointed out with regard to the geometry of the VCSEL. Small-signal modulation response in terms of maximum resonance frequency in dependance on temperature behavior are discussed. Applications of these devices in optical interconnects are described and digital data transmission at data rates up to 2.1 Gbit/s over step-index POF is reported. These properties make red emitting VCSEL perfectly suited for high-speed low power consuming light sources for optical data communication via POF. By introducing InP quantum dots as gain material in red emitting VCSEL nearly temperature independent record low threshold current densities of around 10 A/cm2 could be observed.

  15. 'Saanich' Red Raspberry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    'Saanich' is a new floricane-fruiting red raspberry (Rubus idaeus) cultivar from the breeding program at the Pacific Agri-Food Research Centre (PARC) of Agriculture and Agri-Food Canada, Agassiz, British Columbia. 'Saanich', tested as BC 89-34-41, was selected from a 1989 cross of BC 82-5-161 and BC...

  16. Canadian Red Cross.

    PubMed

    Lavender, Colleen

    2008-01-01

    The Canadian Red Cross is guided by its Fundamental Principles--humanity, impartiality, neutrality, independence, voluntary service, unity, and universality--and organized in a traditional geographic hierarchical structure. Among the characteristics that have contributed to its success are a budgeting process that starts at the local level, measurement of program outcomes, and coordinated fundraising activities at the regional level. PMID:18551842

  17. Live imaging of the lung.

    PubMed

    Looney, Mark R; Bhattacharya, Jahar

    2014-01-01

    Live lung imaging has spanned the discovery of capillaries in the frog lung by Malpighi to the current use of single and multiphoton imaging of intravital and isolated perfused lung preparations incorporating fluorescent molecular probes and transgenic reporter mice. Along the way, much has been learned about the unique microcirculation of the lung, including immune cell migration and the mechanisms by which cells at the alveolar-capillary interface communicate with each other. In this review, we highlight live lung imaging techniques as applied to the role of mitochondria in lung immunity, mechanisms of signal transduction in lung compartments, studies on the composition of alveolar wall liquid, and neutrophil and platelet trafficking in the lung under homeostatic and inflammatory conditions. New applications of live lung imaging and the limitations of current techniques are discussed. PMID:24245941

  18. Live Imaging of the Lung

    PubMed Central

    Looney, Mark R.; Bhattacharya, Jahar

    2015-01-01

    Live lung imaging has spanned the discovery of capillaries in the frog lung by Malpighi to the current use of single and multiphoton imaging of intravital and isolated perfused lung preparations incorporating fluorescent molecular probes and transgenic reporter mice. Along the way, much has been learned about the unique microcirculation of the lung, including immune cell migration and the mechanisms by which cells at the alveolar-capillary interface communicate with each other. In this review, we highlight live lung imaging techniques as applied to the role of mitochondria in lung immunity, mechanisms of signal transduction in lung compartments, studies on the composition of alveolar wall liquid, and neutrophil and platelet trafficking in the lung under homeostatic and inflammatory conditions. New applications of live lung imaging and the limitations of current techniques are discussed. PMID:24245941

  19. Macroparasite community of the Eurasian red squirrel (Sciurus vulgaris): poor species richness and diversity.

    PubMed

    Romeo, Claudia; Pisanu, Benoît; Ferrari, Nicola; Basset, Franck; Tillon, Laurent; Wauters, Lucas A; Martinoli, Adriano; Saino, Nicola; Chapuis, Jean-Louis

    2013-10-01

    The Eurasian red squirrel (Sciurus vulgaris) is the only naturally occurring tree squirrel throughout its range. We aim at improving current knowledge on its macroparasite fauna, expecting that it will have a poor parasite diversity because in species that have no sympatric congeners parasite richness should be lower than in hosts sharing their range with several closely related species, where host-switching events and lateral transmission are promoted. We examined gastro-intestinal helminth and ectoparasite communities (excluding mites) of, respectively, 147 and 311 red squirrel roadkills collected in four biogeographic regions in Italy and France. As expected, the macroparasite fauna was poor: we found five species of nematodes and some unidentified cestodes, three fleas, two sucking lice and two hard ticks. The helminth community was dominated by a single species, the oxyurid Trypanoxyuris (Rodentoxyuris) sciuri (prevalence, 87%; mean abundance, 373 ± 65 worms/host). Its abundance varied among seasons and biogeographic regions and increased with body mass in male hosts while decreased in females. The most prevalent ectoparasites were the flea Ceratophyllus (Monopsyllus) sciurorum (28%), whose presence was affected by season, and the generalist tick Ixodes (Ixodes) ricinus that was found only in France (34%). All the other helminths and arthropod species were rare, with prevalence below 10%. However, the first record of Strongyloides robustus, a common nematode of North American Eastern grey squirrels (S. carolinensis), in two red squirrels living in areas where this alien species co-inhabits, deserves further attention, since low parasite richness could result in native red squirrels being particularly vulnerable to parasite spillover. PMID:23873618

  20. An Examination of the Shared Challenges That Impede Success in the Lives of Single, Multi-Cultural Mothers Enrolled at a Two-Year Community College: A Case Study

    ERIC Educational Resources Information Center

    Gibson, Cheryl

    2012-01-01

    Single mothers struggle with balancing life challenges and childcare necessities while attending college. Consequently, students who are single mothers experience psychological distress. The purpose of this qualitative case study was to examine the common, life and resiliency factors shared by single mothers who are students. The study was…

  1. Holographic analysis on deformation and restoration of malaria-infected red blood cells by antimalarial drug

    NASA Astrophysics Data System (ADS)

    Byeon, Hyeokjun; Ha, Young-Ran; Lee, Sang Joon

    2015-11-01

    Malaria parasites induce morphological, biochemical, and mechanical changes in red blood cells (RBCs). Mechanical variations are closely related to the deformability of individual RBCs. The deformation of various RBCs, including healthy and malaria-infected RBCs (iRBCs), can be directly observed through quantitative phase imaging (QPI). The effects of chloroquine treatment on the mechanical property variation of iRBCs were investigated using time-resolved holographic QPI of single live cells on a millisecond time scale. The deformabilities of healthy RBCs, iRBCs, and drug-treated iRBCs were compared, and the effect of chloroquine on iRBC restoration was experimentally examined. The present results are beneficial to elucidate the dynamic characteristics of iRBCs and the effect of the antimalarial drug on iRBCs.

  2. Red spruce dynamics in an old southern Appalachian forest

    USGS Publications Warehouse

    Busing, R.T.

    2004-01-01

    By the late 1980s the composition and structure of forest stands in the southern Appalachian spruce-fir zone were altered by insect infestations to Fraser fir. The response of red spruce, the sole remaining coniferous forest dominant, to this disturbance was followed over twenty years (1983-2003) in an old spruce-fir forest at Mt. Collins, Great Smoky Mountains National Park. Although diameter growth of canopy red spruce (>30 cm dbh) at six plot sites was considerable (mean 10-yr increment 2.1 cm; 1993-2003), red spruce mortality increased sharply (mean 4% yr-1; 1993-2003). Wind-related mortality of canopy red spruce was substantial after the loss of Fraser fir from the canopy circa 1985 (>70% of the dead spruce had broken or uprooted boles; 1983-2003). Wind damage to red spruce was observed at most plot sites, but it was most pronounced on exposed topographic positions, where canopy gap expansion was extensive. The elevated mortality of red spruce at Mt. Collins was not associated with reduced diameter growth. Altered canopy structure has left large red spruce vulnerable to high winds. With the loss of canopy fir and the subsequent increase in mortality of canopy spruce, total live basal area has declined to about half of its pre-disturbance level.

  3. Electrochemical Red Blood Cell Counting: One at a Time.

    PubMed

    Sepunaru, Lior; Sokolov, Stanislav V; Holter, Jennifer; Young, Neil P; Compton, Richard G

    2016-08-01

    We demonstrate that the concentration of a red blood cell solution under physiological conditions can be determined by electrochemical voltammetry. The magnitude of the oxygen reduction currents produced at an edge-plane pyrolytic graphite electrode was diagnosed analytically at concentrations suitable for a point-of-care test device. The currents could be further enhanced when the solution of red blood cells was exposed to hydrogen peroxide. We show that the enhanced signal can be used to detect red blood cells at a single entity level. The method presented relies on the catalytic activity of red blood cells towards hydrogen peroxide and on surface-induced haemolysis. Each single cell activity is expressed as current spikes decaying within a few seconds back to the background current. The frequency of such current spikes is proportional to the concentration of cells in solution. PMID:27355839

  4. Registration of 'Red Ruby' Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red Ruby’ soft red winter wheat (Triticum aestivum L.) was developed by the Michigan Agricultural Experiment Station and released in 2007 via an exclusive licensing agreement through Michigan State University (MSU) Technologies. Red Ruby was selected from the cross Pioneer ‘2552’/Pioneer ‘2737W’ ma...

  5. A spring aerial census of red foxes in North Dakota

    USGS Publications Warehouse

    Sargeant, A.B.; Pfeifer, W.K.; Allen, S.H.

    1975-01-01

    Systematic aerial searches were flown on transects to locate adult red foxes (Vulpes vulpes), pups, and rearing dens on 559.4 km2 (six townships) in eastern North Dakota during mid-May and mid-June each year from 1969 through 1973 and during mid-April 1969 and early May 1970. The combined sightings of foxes and fox dens from the mid-May and mid-June searches were used to identify individual fox families. The number of fox families was used as the measurement of density. Dens, highly visible during the mid-May searches, were the most reliable family indicator; 84 percent of 270 families identified during the study were represented by dens. Adult foxes second in importance, were most observable during the mid-May searches when 20 to 35 percent of those estimated to be available were sighted. Adult sightings during other search periods ranged from 4 to 17 percent of those available. Pup sightings were the most variable family indicator, but they led to the discovery of some dens. Sources of error for which adjustment factors were determined are: den moves exceeding criterion established for the spacing of dens in a single family, overestimation of the number of fox families living near township boundaries, and the percentage of fox families overlooked during the aerial searches. These adjustment factors appeared to be largely compensatory.

  6. Single-cell label-free photoacoustic flowoxigraphy in vivo

    PubMed Central

    Wang, Lidai; Maslov, Konstantin; Wang, Lihong V.

    2013-01-01

    Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO2) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (CHb), oxygen saturation (sO2), sO2 gradient (), flow speed (vf), and oxygen release rate (rO2), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO2, , vf, and rO2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications. PMID:23536296

  7. The great red flashlight

    NASA Astrophysics Data System (ADS)

    Halbach, Edward A.

    After fifty years of fighting with flashlights which persisted in rolling to the ground, being mislaid, or stashed in a pocket, the author designed a unit which was always on hand and needed no search for the switch. A normally closed switch, internal to the bottom of the flashlight case, is opened by the weight of the unit suspended on a cord about the neck. Lifting the unit with two fingers turns on the red light, while releasing the unit automatically turns it off. A felt covering around the flashlight provides comfort on cold nights. Because this red light would be a welcome tool for other variable star observers, more units were assembled and brought to the AAVSO meeting in Houston for distribution to observers who agreed to give each unit a workout and report on its performance. The author is waiting to hear from these observers.

  8. Great Red Spot (GRS)

    NASA Astrophysics Data System (ADS)

    Murdin, P.

    2000-11-01

    A huge permanent anticyclone in Jupiter's southern hemisphere, visible as a reddish oval at just over 20 °S. The earliest unequivocal observation was by Heinrich Schwabe in 1831 (the often-quoted sighting by Robert Hooke in 1664 now seems to have been of a similar but different spot). The GRS became a striking feature around 1880, when it developed a deep red coloration. It was also prominent in ...

  9. Red giants seismology

    NASA Astrophysics Data System (ADS)

    Mosser, B.; Samadi, R.; Belkacem, K.

    2013-11-01

    The space-borne missions CoRoT and Kepler are indiscreet. With their asteroseismic programs, they tell us what is hidden deep inside the stars. Waves excited just below the stellar surface travel throughout the stellar interior and unveil many secrets: how old is the star, how big, how massive, how fast (or slow) its core is dancing. This paper intends to paparazze the red giants according to the seismic pictures we have from their interiors.

  10. Fragment molecular orbital calculations on red fluorescent proteins (DsRed and mFruits).

    PubMed

    Taguchi, Naoki; Mochizuki, Yuji; Nakano, Tatsuya; Amari, Shinji; Fukuzawa, Kaori; Ishikawa, Takeshi; Sakurai, Minoru; Tanaka, Shigenori

    2009-01-29

    We have performed a series of fragment molecular orbital (FMO) calculations for a family of red fluorescent proteins, DsRed and mFruits. The electronic transition energies were evaluated by the method of configuration interaction singles with perturbative doubles [CIS(D)] including higher-order corrections. The calculated values were in good agreement with the corresponding experimental peak values of spectra. Additionally, the chromophore environment was systematically analyzed in terms of the interaction energies between the pigment moiety and neighboring residues. It was theoretically revealed that the electrostatic interactions play a dominant role in the DsRed chromophore, whereas the color tunings in mFruits are controlled in a more delicate fashion. PMID:19127982

  11. Living with hearing loss

    MedlinePlus

    ... medlineplus.gov/ency/patientinstructions/000360.htm Living with hearing loss To use the sharing features on this page, please enable JavaScript. If you are living with hearing loss, you know that it takes extra effort to ...

  12. Living with Sarcoidosis

    MedlinePlus

    ... page from the NHLBI on Twitter. Living With Sarcoidosis Sarcoidosis has no cure, but you can take ... Content: NEXT >> Featured Video Living With and Managing Sarcoidosis 05/18/2011 This video—presented by the ...

  13. Living with Spina Bifida

    MedlinePlus

    ... Us Information For... Media Policy Makers Living With Spina Bifida Language: English Español (Spanish) Recommend on Facebook Tweet ... of the website provides information about living with spina bifida at different ages. Spina bifida affects the entire ...

  14. Living with Atrial Fibrillation

    MedlinePlus

    ... Topics » Atrial Fibrillation » Living With Atrial Fibrillation Explore Atrial Fibrillation What Is... Types Other Names Causes Who Is at Risk Signs & Symptoms Diagnosis Treatments Prevention Living With Clinical Trials Links Related Topics Arrhythmia ...

  15. Red Spot Movie

    NASA Technical Reports Server (NTRS)

    2000-01-01

    This brief movie shows counterclockwise atmospheric motion around Jupiter's Great Red Spot. The clip was made from blue-filter images taken with the narrow-angle camera on NASA's Cassini spacecraft during seven separate rotations of Jupiter between Oct. 1 and Oct. 5, 2000.

    The clip also shows the eastward and westward motion of the zonal jets, seen as the horizontal stripes flowing in opposite directions. The zonal jets circle the planet. As far as can be determined from both Earth-based and spacecraft measurements, the positions and speeds of the jets have not changed for 100 years. Since Jupiter is a fluid planet without a solid boundary, the jet speeds are measured relative to Jupiter's magnetic field, which rotates, wobbling like a top because of its tilt, every 9 hours 55.5 minutes. The movie shows motions in the magnetic reference frame, so winds to the west correspond to features that are rotating a little slower than the magnetic field, and eastward winds correspond to features rotating a little faster.

    Because the Red Spot is in the southern hemisphere, the direction of motion indicates it is a high-pressure center. Small bright clouds appear suddenly to the west of the Great Red Spot. Scientists suspect these small white features are lightning storms. The storms eventually merge with the Red Spot and surrounding jets, and may be the main energy source for the large-scale features.

    The smallest features in the movie are about 500 kilometers (about 300 miles) across. The spacing of the movie frames in time is not uniform; some consecutive images are separated by two Jupiter rotations, and some by one. The images have been re-projected using a simple cylindrical map projection. They show an area from 50 degrees north of Jupiter's equator to 50 degrees south, extending 100 degrees east-west, about one quarter of Jupiter's circumference.

    Cassini is a cooperative project of NASA, the European Space Agency and the Italian Space Agency. The Jet

  16. Genetic Map-Based Location of the Red Clover (Trifolium pratense L.) Gametophytic Self-incompatibility Locus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover is a hermaphadidic allogamous diploid (2n = 2x = 14) with a homomorphic gametophytic self-incompatibility (GSI) system red clover (Trifolium pratense L.). Red clover GSI has long been studied and it is thought that the genetic control of GSI constitutes a single locus. Although GSI gene...

  17. Fire ant-detecting canines: a complementary method in detecting red imported fire ants.

    PubMed

    Lin, Hui-Min; Chi, Wei-Lien; Lin, Chung-Chi; Tseng, Yu-Ching; Chen, Wang-Ting; Kung, Yu-Ling; Lien, Yi-Yang; Chen, Yang-Yuan

    2011-02-01

    In this investigation, detection dogs are trained and used in identifying red imported fire ants, Solenopsis invicta Buren, and their nests. The methodology could assist in reducing the frequency and scope of chemical treatments for red imported fire ant management and thus reduce labor costs and chemical use as well as improve control and quarantine efficiency. Three dogs previously trained for customs quarantine were retrained to detect the scents of red imported fire ants. After passing tests involving different numbers of live red imported fire ants and three other ant species--Crematogaster rogenhoferi Mayr, Paratrechina longicornis Latreille, and Pheidole megacephala F.--placed in containers, ajoint field survey for red imported fire ant nests by detection dogs and bait traps was conducted to demonstrate their use as a supplement to conventional detection methods. The most significant findings in this report are (1) with 10 or more red imported fire ants in scent containers, the dogs had >98% chance in tracing the red imported fire ant. Upon the introduction of other ant species, the dogs still achieved on average, a 93% correct red imported fire ant indication rate. Moreover, the dogs demonstrated great competence in pinpointing emerging and smaller red imported fire ant nests in red imported fire ant-infested areas that had been previously confirmed by bait trap stations. (2) Along with the bait trap method, we also discovered that approximately 90% of red imported fire ants foraged within a distance of 14 m away from their nests. The results prove detection dogs to be most effective for red imported fire ant control in areas that have been previously treated with pesticides and therefore containing a low density of remaining red imported fire ant nests. Furthermore, as a complement to other red imported fire ant monitoring methods, this strategy will significantly increase the efficacy of red imported fire ant control in cases of individual mount treatment

  18. Living With Lupus

    MedlinePlus

    ... Low blood count Depression, trouble thinking, and/or memory problems. Other signs are mouth sores, unexplained seizures ( ... 8, 10/23, 11/15 Red rash or color change Painful or swollen joints Unexplained fever Chest ...

  19. Construction and characterization of a red-emitting luciferase

    NASA Astrophysics Data System (ADS)

    Eames, Brian F.; Benaron, David A.; Stevenson, David K.; Contag, Christopher H.

    1999-07-01

    Red light is transmitted through live tissue more efficiently than other wavelengths of visible light, thus by red-shifting the emission of bioluminescent reporters, we may enhance their utility for in vivo monitoring of biological processes. Codon changes at positions that may shift the yellow-green emission to red, based on studies of a related luciferase, were introduced into a variant of the North American firefly luciferase. Clones containing the desired mutation were selected based on the introduction of unique restriction enzyme sites and transfected into NIH 3T3 cells. Expression levels were evaluated using an intensified charge coupled device camera. Upon spectral analysis, all mutant luciferases demonstrated red-orange emission. Two emission peaks were detected in each spectrum, each clone with different peak heights at 560 nm and 610 nm. Sequence analyses of the compete coding region of several clones confirmed the presence of the target mutations, although sequence variation was observed at several secondary sites, likely resulting from the infidelity of Taq polymerase used in the mutagenesis protocol. A clone that demonstrated a strong 610 nm peak with a minimum shoulder at 560 nm was selected for use in animals. In summary, a red-shifted mutant of a well-characterized luciferase reporter gene was generated. Red light from this enzyme may both penetrate mammalian tissues to a greater extent and provide a tool for multicolor biological assays.

  20. Red - Take a Closer Look

    PubMed Central

    Buechner, Vanessa L.; Maier, Markus A.; Lichtenfeld, Stephanie; Schwarz, Sascha

    2014-01-01

    Color research has shown that red is associated with avoidance of threat (e.g., failure) or approach of reward (e.g., mating) depending on the context in which it is perceived. In the present study we explored one central cognitive process that might be involved in the context dependency of red associations. According to our theory, red is supposed to highlight the relevance (importance) of a goal-related stimulus and correspondingly intensifies the perceivers’ attentional reaction to it. Angry and happy human compared to non-human facial expressions were used as goal-relevant stimuli. The data indicate that the color red leads to enhanced attentional engagement to angry and happy human facial expressions (compared to neutral ones) - the use of non-human facial expressions does not bias attention. The results are discussed with regard to the idea that red induced attentional biases might explain the red-context effects on motivation. PMID:25254380