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Sample records for live single red

  1. [Instant effect of temperature on the oxygen carrying capacity of single living intact red blood cell].

    PubMed

    Yao, Cheng-can; Li, Xiao-kun; Huang, Yao-xiong

    2005-04-01

    The instant effect of temperature on the absorption spectra of the hemoglobin in single living intact red blood cells was investigated, by employing a highly sensitive fast multi-channel micro-spectrophotometer system to perform non-invasive, in situ, real time measurements on the cells. It was found that both the heights and position of the specific peaks in the absorption spectra of intercellular hemoglobin were changed with temperature, indicating that the oxygen carrying capacity of red blood cells varies with temperature. The correlations of the structure and concentration as well as the function of hemoglobin, and the molecular mechanism were also discussed.

  2. Raman Tweezers Spectroscopy of Live, Single Red and White Blood Cells

    PubMed Central

    Bankapur, Aseefhali; Zachariah, Elsa; Chidangil, Santhosh; Valiathan, Manna; Mathur, Deepak

    2010-01-01

    An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC) and white blood cells (WBC) under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip. PMID:20454686

  3. Living with a Single Parent

    MedlinePlus

    ... Happens in the Operating Room? Living With a Single Parent KidsHealth > For Kids > Living With a Single Parent ... single parents can be a great idea, too. Single Parents and Work Single parents are often working parents ...

  4. Living with a Single Parent

    MedlinePlus

    ... Video: Getting an X-ray Living With a Single Parent KidsHealth > For Kids > Living With a Single Parent ... single parents can be a great idea, too. Single Parents and Work Single parents are often working parents ...

  5. 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.

  6. 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

  7. Red photoluminescence of living systems at the room temperature : measurements and results

    NASA Astrophysics Data System (ADS)

    Kudryashova, I. S.; Rud, V. Yu; Shpunt, V. Ch; Rud, Yu V.; Glinushkin, A. P.

    2016-08-01

    Presents results of a study of the red luminescence of living plants at room temperature. The analysis of obtained results allows to conclude that the photoluminescence spectra for green leaves in all cases represent the two closely spaced bands.

  8. The application of KillerRed for acute protein inactivation in living cells

    PubMed Central

    Jarvela, Timothy S.; Linstedt, Adam D.

    2017-01-01

    Generating loss of protein function is a powerful investigatory tool particularly if carried out at a physiologically relevant timescale in a live-cell fluorescent imaging experiment. KillerRed mediated chromophore assisted light inactivation (CALI) uses genetic encoding for specificity and light for acute inactivation that can also be spatially restricted. This unit provides protocols for setting up and carrying out properly controlled KillerRed experiments during live-cell imaging of cultured cells. PMID:24984963

  9. 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

  10. 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

  11. Tracking single molecules at work in living cells.

    PubMed

    Kusumi, Akihiro; Tsunoyama, Taka A; Hirosawa, Kohichiro M; Kasai, Rinshi S; Fujiwara, Takahiro K

    2014-07-01

    Methods for imaging and tracking single molecules conjugated with fluorescent probes, called single-molecule tracking (SMT), are now providing researchers with the unprecedented ability to directly observe molecular behaviors and interactions in living cells. Current SMT methods are achieving almost the ultimate spatial precision and time resolution for tracking single molecules, determined by the currently available dyes. In cells, various molecular interactions and reactions occur as stochastic and probabilistic processes. SMT provides an ideal way to directly track these processes by observing individual molecules at work in living cells, leading to totally new views of the biochemical and molecular processes used by cells whether in signal transduction, gene regulation or formation and disintegration of macromolecular complexes. Here we review SMT methods, summarize the recent results obtained by SMT, including related superresolution microscopy data, and describe the special concerns when SMT applications are shifted from the in vitro paradigms to living cells.

  12. Single-Molecule mRNA Detection in Live Yeast

    PubMed Central

    Lenstra, Tineke L.

    2016-01-01

    Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub-second temporal resolution and nanometer spatial resolution. Following transcription in single cells indicates that gene activity is heterogeneous between cells and also exhibits random variability over time even within single cells. Studies of mRNAs in yeast can take advantage of the powerful genetics available in this model organism and allow mechanistic questions to be addressed. In this chapter, we describe an approach for visualizing mRNA and transcription in live yeast cells. The method is based on binding of fluorescently labeled MS2 and PP7 coat proteins to stem loops sequences that are introduced into the gene of interest. We give detailed protocols for the construction of the necessary yeast strains, for image acquisition, and for validation. PMID:27110320

  13. 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.

  14. 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

  15. 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

  16. Flexibility of single microvilli on live neutrophils and lymphocytes

    NASA Astrophysics Data System (ADS)

    Yao, Da-Kang; Shao, Jin-Yu

    2007-08-01

    We measured the flexural stiffness of single microvilli on live human neutrophils and lymphocytes using 40-nm fluorescent beads. The beads were bound to the tips of the microvilli by anti- L -selectin antibodies. Digital bead images were acquired with an exposure time of 3s at high magnification. Using a Gaussian point spread function, we obtained an analytical expression that relates the image profile to the flexural stiffness. We found that the flexural stiffnesses were 7 and 4pN/μm for single microvilli on human neutrophils and lymphocytes, respectively. We also verified with live cells that 75% of neutrophil L -selectin and 72% of lymphocyte L -selectin were on the microvillus tips. Our results indicate that the leukocyte microvilli in contact with the endothelium or other surfaces will bend easily under physiological shear stresses.

  17. 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.

  18. Monitoring dopamine release from single living vesicles with nanoelectrodes.

    PubMed

    Wu, Wen-Zhan; Huang, Wei-Hua; Wang, Wei; Wang, Zong-Li; Cheng, Jie-Ke; Xu, Tao; Zhang, Rong-Ying; Chen, Yu; Liu, Jie

    2005-06-29

    Carbon fiber nanoelectrodes (tip diameter = ca. 100 nm) have been first used to monitor real-time dopamine release from single living vesicles of single rat pheochromocytoma (PC12) cells. The experiments show that active and inactive release sites exist on the surface of cells, and the spatial distributions have been differentiated even in the same active release zone. It is first demonstrated that multiple vesicles can sequentially release dopamine at the same site of the cell surface, which possibly plays the main role in the dopamine release from PC12 cells.

  19. 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)

  20. 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.

  1. Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

    PubMed

    Drobizhev, Mikhail; Stoltzfus, Caleb; Topol, Igor; Collins, Jack; Wicks, Geoffrey; Mikhaylov, Alexander; Barnett, Lauren; Hughes, Thomas E; Rebane, Aleksander

    2014-08-07

    Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

  2. 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

  3. 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.

  4. ARMCOM Red Team Role in The Single Integrated Development Test Cycle

    DTIC Science & Technology

    1975-11-01

    USADACS Tdv*c* Ubrtry RIA-76-U146 5 0712 01001238 0 AOA0ivi& ARMCOM RED TEAM ROLE IN THE SINGLE INTEGRATED DEVELOPMENT TEST CYCLE THOMAS N...TITLE (and Subtitle) ARMCOM Red Team Role in The Single Integrated Development Test Cycle. 5. TYPE OF REPORT & PERIOD COVERED Note...per year. In addition to the Red Team function, the Systems Analysis Directorate will also function in the Blue Team role to support the developer

  5. Single-molecule imaging in live cell using gold nanoparticles.

    PubMed

    Leduc, Cécile; Si, Satyabrata; Gautier, Jérémie J; Gao, Zhenghong; Shibu, Edakkattuparambil S; Gautreau, Alexis; Giannone, Grégory; Cognet, Laurent; Lounis, Brahim

    2015-01-01

    Optimal single particle tracking experiments in live cells requires small and photostable probes, which do not modify the behavior of the molecule of interest. Current fluorescence-based microscopy of single molecules and nanoparticles is often limited by bleaching and blinking or by the probe size. As an alternative, we present in this chapter the synthesis of a small and highly specific gold nanoprobe whose detection is based on its absorption properties. We first present a protocol to synthesize 5-nm-diameter gold nanoparticles and functionalize them with a nanobody, a single-domain antibody from camelid, targeting the widespread green fluorescent protein (GFP)-tagged proteins with a high affinity. Then we describe how to detect and track these individual gold nanoparticles in live cell using photothermal imaging microscopy. The combination of a probe with small size, perfect photostability, high specificity, and versatility through the vast existing library of GFP-proteins, with a highly sensitive detection technique enables long-term tracking of proteins with minimal hindrance in confined and crowded environments such as intracellular space.

  6. 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

  7. Single-Molecule Imaging of RNA Splicing in Live Cells.

    PubMed

    Rino, José; Martin, Robert M; Carvalho, Célia; de Jesus, Ana C; Carmo-Fonseca, Maria

    2015-01-01

    Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Here, we describe a strategy that permits direct measurements of intron dynamics in single pre-mRNA molecules in live cells. This approach reveals that splicing can occur much faster than previously proposed and opens new avenues for studying how kinetic mechanisms impact on RNA biogenesis.

  8. 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.

  9. Analysis of gene expression in single live neurons.

    PubMed Central

    Eberwine, J; Yeh, H; Miyashiro, K; Cao, Y; Nair, S; Finnell, R; Zettel, M; Coleman, P

    1992-01-01

    We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in greater than 10(6)-fold amplification of the original starting material, which is adequate for analysis--e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease. Images PMID:1557406

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Microspectrofluorometric analysis of drug phototoxicity in single living cells

    NASA Astrophysics Data System (ADS)

    Morliere, Patrice; Santus, Rene C.; Maziere, J. C.; Geze, Marc; Bazin, M.; Kohen, Elli

    1993-03-01

    The study of primary photobiological processes on the basis of structure-activity relationship is important for a better understanding of drug phototoxicity. An ideal approach for the understanding of the phototoxic response is provided by the study of drugs purposely used in photochemotherapeuties for which the determination of primary photochemical targets is a prerequisite for the investigation of the phototherapeutic action. For instance, in the so-called 'photodynamic therapy' of cancers, the photodynamic properties of porphyrins more or less specifically localized in tumors are responsible for their photocytotoxicity. Microfluorometry and particularly microspectrofluorometry are powerful non invasive techniques for carrying out quantitative photobiological investigations in real time in single living cells. This approach allows one to monitor the drug localization, to follow the drug fate, and to study photosensitized events in living cells. We illustrate some aspects of such investigations with photofrin II, a mixture of porphyrins currently used in phase III clinical trials, and other porphyrins including protoporphyrin which is encountered in genetic and drug-induced cutaneous porphyrias. To demonstrate the usefulness of microspectrofluorometry in such studies, we present data on the photosensitizer localization, on the photosensitizer photobleaching, and on structural or functional photosensitized damage to organelles.

  15. Laser-induced microlesion of single dendrites in living mice

    NASA Astrophysics Data System (ADS)

    Sacconi, L.; Panteri, R.; Masi, A.; Diana, G.; Buffelli, M.; Keller, F.; Pavone, F. S.

    2007-02-01

    Recently, two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex in living mice. In this work we study the in vivo spine rearrangements after an acute and selective damage. For this purpose, we have used a near-IR femtosecond pulsed laser to combine two-photon microscopy imaging with microdissection operation on fluorescently-labeled neurons. Three-dimensional reconstructions of dendrites expressing fluorescence protein have been performed in the cortex of YFP-H and GFP-M transgenic living mice. Afterwards, single dendrites have been laser-dissected irradiating the structure with a high femtosecond laser energy dose. By using a chronically implanted glass window we performed long-term imaging in the area of the dissected dendrite. We will show that laser ablation can be performed with micrometric precision and without visible collateral damage to nearby neuronal structures. Also, we will evidence the morphological changes of the dendritic branches and dendritic spines after this specific perturbation inside the intact neuronal network. Laser microdissection of selected structures of the neuronal branching in vivo represents a promising tool for neurobiological research.

  16. Safety of red ginseng oil for single oral administration in Sprague–Dawley rats

    PubMed Central

    Bak, Min-Ji; Kim, Kyu-Bong; Jun, Mira; Jeong, Woo-Sik

    2013-01-01

    The single oral administration of red ginseng oil (5000 mg/kg) to Sprague–Dawley rats induced no changes in behavioral patterns, clinical signs, and body weight, and hepatotoxicity parameters such as aspartate aminotransferase and alanine aminotransferase for 14 d. Therefore, these results suggest that the red ginseng oil is safe and nontoxic acutely. PMID:24558315

  17. Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test.

    PubMed

    Su, Yan; Gao, Lijun; Ma, Qiang; Zhou, Lishe; Qin, Liangyi; Han, Lihong; Qin, Wenbin

    2010-09-01

    To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A(2), HbA was first shown to bind with HbA(2) in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA(2) outside the RBC was shown by cross electrophoresis. The starch-agarose gel electrophoresis of hemolysate, RBCs, freeze-thawed RBCs and the supernatant of freeze-thawed RBCs showed that the interaction between HbA and HbA(2) was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA(2) in RBCs (RBC HbA-HbA(2)) and hemolysate (hemolysate HbA-HbA(2)) were isolated from the starch-agarose gel and separated by 5-12% SDS-PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA-HbA(2) but not in hemolysate HbA-HbA(2). Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α-globin, δ-globin and β-globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.

  18. 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.

  19. 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

  20. Bilateral microphthalmia and aphakia associated with multiple eye abnormalities in a free-living European red deer calf (Cervus elaphus).

    PubMed

    Mutinelli, Franco; Vercelli, Antonella; Carminato, Antonio; Luchesa, Lucio; Pasolli, Claudio; Cova, Mariapia; Marchioro, Wendy; Melchiotti, Erica; Vascellari, Marta

    2012-04-01

    A free-living European red deer calf (Cervus elaphus) was euthanized due to bilateral microphthalmia. Lens was missing, replaced by proliferating squamous epithelial cells; hyperplastic squamous cells, sebaceous and mucinous glands were observed within the cornea with the characteristics of inclusion cyst. Findings were consistent with congenital microphthalmia/aphakia, with multiple eye abnormalities.

  1. 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

  2. Design and Synthesis of Single Nanoparticle Optical Biosensors for Imaging and Characterization of Single Receptor Molecules on Single Living Cells

    PubMed Central

    Huang, Tao; Nallathamby, Prakash D.; Gillet, Daniel; Nancy Xu, Xiao-Hong

    2008-01-01

    At the cellular level, a small number of protein molecules (receptors) can induce significant cellular responses, emphasizing the importance of molecular detection of trace amounts of protein on single living cells. In this study, we designed and synthesized silver (Ag) nanoparticle biosensors (AgMMUA-IgG) by functionalizing (11.6 ± 3.5) nm Ag nanoparticles with a mixed monolayer of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (MCH) (1:3 mole ratio), and covalently conjugating IgG with MUA on the nanoparticle surface. We found that the nanoparticle biosensors preserve their biological activity and photostability and can be utilized to quantitatively detect individual receptor molecules (T-ZZ), map the distribution of receptors (0.21–0.37 molecule/µm2), and measure their binding affinity and kinetics at concentrations below their dissociation constant, on single living cells in real time over hrs. The dynamic range of detection is 0–50 molecules per cell. We also found that the binding rate (2–27 molecules/min) is highly dependent upon the coverage of receptors on living cells and their ligand concentration. The binding association and dissociation rate constants and affinity constant are k1 = (9.0 ± 2.6) × 103 M−1s−1, k−1 = (3.0 ± 0.4) × 10−4 s−1, and KB = (4.3 ± 1.1) × 107 M−1, respectively. PMID:17867652

  3. 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.

  4. 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

  5. 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.

  6. Red antenna states of Photosystem I trimers from Arthrospira platensis revealed by single-molecule spectroscopy.

    PubMed

    Brecht, Marc; Hussels, Martin; Schlodder, Eberhard; Karapetyan, Navassard V

    2012-03-01

    Single-molecule fluorescence spectroscopy at 1.4K was used to investigate the spectral properties of red (long-wavelength) chlorophylls in trimeric Photosystem I (PSI) complexes from the cyanobacterium Arthrospira platensis. Three distinct red antenna states could be identified in the fluorescence spectra of single PSI trimers from A. platensis in the presence of oxidized P700. Two of them are responsible for broad emission bands centered at 726 and 760nm. These bands are similar to those found in bulk fluorescence spectra measured at cryogenic temperatures. The broad fluorescence bands at ≅726 and ≅760nm belong to individual emitters that are broadened by strong electron-phonon coupling giving rise to a large Stokes-shift of about 20nm and rapid spectral diffusion. An almost perpendicular orientation of the transition dipole moments of F726 and F760 has to be assumed because direct excitation energy transfer does not occur between F726 and F760. For the first time a third red state assigned to the pool absorbing around 708nm could be detected by its zero-phonon lines. The center of the zero-phonon line distribution is found at ≅714nm. The spectral properties of the three red antenna states show a high similarity to the red antenna states found in trimeric PSI of Thermosynechoccocus elongatus. Based on these findings a similar organization of the red antenna states in PSI of these two cyanobacteria is discussed.

  7. 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,…

  8. 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.

  9. 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

  10. Red-IR stimulated luminescence in K-feldspar: Single or multiple trap origin?

    NASA Astrophysics Data System (ADS)

    Thalbitzer Andersen, Martin; Jain, Mayank; Tidemand-Lichtenberg, Peter

    2012-08-01

    We investigate on the origins of the infra-red stimulated luminescence (IRSL) signals in 3 potassium feldspars based on IR-red spectroscopy (˜700-1050 nm) using a fiber-coupled tunable Ti:Sapphire laser, in combination with different thermal and optical (pre)treatments of the samples. We also measure dose-response curves with different wavelengths and at different stimulation temperatures so as to be able to distinguish between traps based on their electron trapping cross-sections. Our data suggest that the dosimetric signals, IRSL, and the post IR-IRSL in K-feldspars arise from a single electron trapping centre.

  11. Intimate Partner Violence: The Lived Experience of Single Women.

    PubMed

    Thomas, Laura; Scott-Tilley, Donna

    2017-03-01

    Research in intimate partner violence has focused on married, cohabiting, adolescents, or college aged women. The experience of intimate partner violence by single women has not been studied separately from other groups of women. An interpretive phenomenological approach was used with feminist inquiry to gain insight into the experience of intimate partner violence by single women. The overarching theme was control and manipulation by the abuser. Subthemes included not feeling safe, poor communication skills, and caretaking. Nurses need to be aware of the occurrence of intimate partner violence in male and female partnered relationships to provide comprehensive and nonjudgmental care.

  12. Living-unrelated kidney donation: a single-center experience.

    PubMed

    Peters, T G; Jones, K W; Walker, G W; Charlton, R K; Antonucci, L E; Repper, S M; Hunter, R D

    1999-02-01

    For 140 consecutive renal transplants performed from January 1995 to October 1997, 25 (18%) were from living-unrelated donors (15 women, 10 men, aged 25-63, mean 43 yr). All donors had pre-transplant imaging evaluation of renal anatomy following renal function assessment (minimal creatinine clearance 75 cm3/min). Admission to the hospital on the day of donation preceded nephrectomy under general anesthesia using an anterior flank, extra-retroperitoneal approach (no rib resection). Post-operative epidural pain control was used for all but 1 donor. The 25 kidney donors were hospitalized for 2 (n = 1), 3 (n = 12), 4 (n = 7), or 5-8 d (n = 5) (average 3.9 d) and had a mean hospitalization charge of $15,501 (range $10,808-$29,579). One intra-operative hemorrhage required transfusion; 1 late neural-related pain syndrome required outpatient wound exploration. Two kidneys were lost: a husband recipient from repetitive acute rejections at 3 months; a friend recipient from chronic rejection at 2.5 yr; both await cadaver transplant. The other 23 kidneys are functioning with a mean serum creatinine of 1.8 (range 1.0-3.3) at 3-36 months (patient survival 100%; graft survival 92%). While most donors were spouses (8 husbands and 10 wives), friends, distant cousins, in-laws, and adoptive relatives did well as donors and recipients. Transplantation may increase by 20% or more at centers which encourage broad application of living donor nephrectomy.

  13. 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…

  14. 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

  15. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

    PubMed Central

    Demeautis, Claire; Sipieter, François; Roul, Julien; Chapuis, Catherine; Padilla-Parra, Sergi; Riquet, Franck B.; Tramier, Marc

    2017-01-01

    Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM. PMID:28106114

  16. 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

  17. Single-cell census of mechanosensitive channels in living bacteria.

    PubMed

    Bialecka-Fornal, Maja; Lee, Heun Jin; DeBerg, Hannah A; Gandhi, Chris S; Phillips, Rob

    2012-01-01

    Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering.

  18. 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.

  19. "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…

  20. Single nanoparticle photothermal tracking (SNaPT) of 5-nm gold beads in live cells.

    PubMed

    Lasne, David; Blab, Gerhard A; Berciaud, Stéphane; Heine, Martin; Groc, Laurent; Choquet, Daniel; Cognet, Laurent; Lounis, Brahim

    2006-12-15

    Tracking individual nano-objects in live cells during arbitrary long times is a ubiquitous need in modern biology. We present here a method for tracking individual 5-nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against single fluorescent molecule tracking in live COS7 cells on subsecond timescales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. Single Nanoparticle Photothermal Tracking has the unique potential to record arbitrary long trajectory of membrane proteins using nonfluorescent nanometer-sized labels.

  1. 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."

  2. 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.

  3. 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.

  4. 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

  5. Live single cell functional phenotyping in droplet nano-liter reactors.

    PubMed

    Konry, Tania; Golberg, Alexander; Yarmush, Martin

    2013-11-11

    While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surface and secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

  6. Live single cell functional phenotyping in droplet nano-liter reactors

    NASA Astrophysics Data System (ADS)

    Konry, Tania; Golberg, Alexander; Yarmush, Martin

    2013-11-01

    While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

  7. Live single cell functional phenotyping in droplet nano-liter reactors

    PubMed Central

    Konry, Tania; Golberg, Alexander; Yarmush, Martin

    2013-01-01

    While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function. PMID:24212247

  8. Imaging Single-mRNA Localization and Translation in Live Neurons

    PubMed Central

    Lee, Byung Hun; Bae, Seong-Woo; Shim, Jaeyoun Jay; Park, Sung Young; Park, Hye Yoon

    2016-01-01

    Local protein synthesis mediates precise spatio-temporal regulation of gene expression for neuronal functions such as long-term plasticity, axon guidance and regeneration. To reveal the underlying mechanisms of local translation, it is crucial to understand mRNA transport, localization and translation in live neurons. Among various techniques for mRNA analysis, fluorescence microscopy has been widely used as the most direct method to study localization of mRNA. Live-cell imaging of single RNA molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein (mRNP) complexes in neurons. Here, we review recent advances in the study of mRNA localization and translation in live neurons using novel techniques for single-RNA imaging. PMID:28030897

  9. 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

  10. Optimized single-number quantity for rating the airborne sound insulation of constructions: Living sounds.

    PubMed

    Virjonen, Petra; Hongisto, Valtteri; Oliva, David

    2016-12-01

    ISO 717-1 [(1996). International Organization for Standardization, Geneva, Switzerland] and ASTM 413 [(2010). American Society for Testing and Materials International] define various single-number quantities (SNQs) that are commonly used to rate objectively airborne sound insulation of constructions. Recent psychoacoustic evidence suggests that none of them is appropriate for a wide range of living sound stimuli. The purpose of the study was to develop an alternative compromising SNQ for the frequency range 50-5000 Hz that explains well the annoyance caused by various airborne living sounds transmitted from the neighboring dwelling. Optimal reference spectra for different living sounds were found by mathematical optimization. Experimental data from a psychoacoustic laboratory study [Hongisto, Oliva, and Keränen (2014). Acta Acust. Acust. 100, 848-863] were utilized. The subjects (n = 59) had evaluated the disturbance of living sounds that were electrically filtered to mimic transmission through commonly used wall structures. To find a high-performing reference spectrum for living sounds in general, the optimized reference spectra were averaged over all sound types. The resulting SNQ was called Rw + Copt. The related reference spectrum deviates significantly from the reference spectrum for living activities, C50-5000, below 315 Hz. The suggested SNQ correlates better with the subjective disturbance caused by living sounds than any of the present standardized SNQs of ISO 717-1 or ASTM 413.

  11. 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.

  12. 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.

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

    DOE PAGES

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; ...

    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

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

    PubMed

    Lenn, Tchern; Leake, Mark C

    2012-06-01

    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.

  15. 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.

  16. Direct metabolomics for plant cells by live single-cell mass spectrometry.

    PubMed

    Fujii, Takashi; Matsuda, Shuichi; Tejedor, Mónica Lorenzo; Esaki, Tsuyoshi; Sakane, Iwao; Mizuno, Hajime; Tsuyama, Naohiro; Masujima, Tsutomu

    2015-09-01

    Live single-cell mass spectrometry (live MS) provides a mass spectrum that shows thousands of metabolite peaks from a single live plant cell within minutes. By using an optical microscope, a cell is chosen for analysis and a metal-coated nanospray microcapillary tip is used to remove the cell's contents. After adding a microliter of ionization solvent to the opposite end of the tip, the trapped contents are directly fed into the mass spectrometer by applying a high voltage between the tip and the inlet port of the spectrometer to induce nanospray ionization. Proteins are not detected because of insufficient sensitivity. Metabolite peaks are identified by exact mass or tandem mass spectrometry (MS/MS) analysis, and isomers can be separated by combining live MS with ion-mobility separation. By using this approach, spectra can be acquired in 10 min. In combination with metabolic maps and/or molecular databases, the data can be annotated into metabolic pathways; the data analysis takes 30 min to 4 h, depending on the MS/MS data availability from databases. This method enables the analysis of a number of metabolites from a single cell with rapid sampling at sub-attomolar-level sensitivity.

  17. Four-Dimensional Spatial Nanometry of Single Particles in Living Cells Using Polarized Quantum Rods

    PubMed Central

    Watanabe, Tomonobu M.; Fujii, Fumihiko; Jin, Takashi; Umemoto, Eiji; Miyasaka, Masayuki; Fujita, Hideaki; Yanagida, Toshio

    2013-01-01

    Single particle tracking is widely used to study protein movement with high spatiotemporal resolution both in vitro and in cells. Quantum dots, which are semiconductor nanoparticles, have recently been employed in single particle tracking because of their intense and stable fluorescence. Although single particles inside cells have been tracked in three spatial dimensions (X, Y, Z), measurement of the angular orientation of a molecule being tracked would significantly enhance our understanding of the molecule’s function. In this study, we synthesized highly polarized, rod-shaped quantum dots (Qrods) and developed a coating method that optimizes the Qrods for biological imaging. We describe a Qrod-based single particle tracking technique that blends optical nanometry with nanomaterial science to simultaneously measure the three-dimensional and angular movements of molecules. Using Qrods, we spatially tracked a membrane receptor in living cells in four dimensions with precision close to the single-digit range in nanometers and degrees. PMID:23931303

  18. Tracking living decapod larvae: mass staining of eggs with neutral red prior to hatching.

    PubMed

    Øresland, V; Horobin, R W

    2012-04-01

    Mass staining of decapod females carrying eggs, with subsequent identification of hatched larvae in the environment, is a research tool with great potential for field ecologists wishing to track the movements of larvae. For this to be achieved, however, numerous requirements must be met. These include adequate dye solubility, short staining time, dye penetration through different tissues, dye retention within the organism, absence of toxic and behavioral effects, low visibility to predators of stained larvae, no loss of staining owing to preservatives and low cost. The dye, neutral red, appears to meet most of these requirements. This dye was used in aliquots of 0.7 g/770 ml seawater applied to the females of Norway lobster (Nephrops norvegicus) and European lobster (Homarus gammarus) for 10 min. This procedure stained lobster eggs and embryos so that hatched larvae could be distinguished easily by fluorescence microscopy from larvae that hatched from unstained eggs. Stained larvae that were preserved in 4% formaldehyde in seawater were still stained after 1 year. Larvae should not come in contact with ethanol, because it extracts the dye rapidly.

  19. Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells

    PubMed Central

    Gebhardt, J Christof M; Suter, David M; Roy, Rahul; Zhao, Ziqing W; Chapman, Alec R; Basu, Srinjan; Maniatis, Tom; Xie, X Sunney

    2013-01-01

    Imaging single fluorescent proteins in living mammalian cells is challenging due to out-of-focus fluorescence excitation by common microscopy schemes. We report the development of a novel fluorescence microscopy method, reflected light sheet microscopy (RLSM), which allows selective plane illumination throughout the nucleus of living mammalian cells, for reducing out-of-focus fluorescence signal. Generation of a thin light sheet parallel to the imaging plane and close to the sample surface is achieved by reflecting an elliptical laser beam incident from the top by 45° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 Hz time resolution. We demonstrate the sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determine the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor (ER), enabling us to resolve different modes of DNA binding of GR. Finally, we demonstrate two-color single molecule imaging by observing the spatio-temporal co-localization of two different protein pairs. The combination of our single molecule measurements and statistical analysis reveals dynamic properties of transcription factors in live mammalian cells. PMID:23524394

  20. 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.

  1. Red Mud Catalytic Pyrolysis of Pinyon Juniper and Single-Stage Hydrotreatment of Oils

    SciTech Connect

    Agblevor, Foster A.; Elliott, Douglas C.; Santosa, Daniel M.; Olarte, Mariefel V.; Burton, Sarah D.; Swita, Marie; Beis, Sedat H.; Christian, Kyle; Sargent, Brandon

    2016-10-20

    Pinyon juniper biomass feedstocks, which cover a large acreage of rangeland in the western United States, are being eradicated and, therefore, considered as a convenient biomass feedstock for biofuel production. Pinyon juniper whole biomass (wood, bark, and leaves) were pyrolyzed in a pilot-scale bubbling fluidized-bed reactor at 450 °C, and the noncondensable gases were recycled to fluidize the reactor. Red mud was used as the in situ catalyst for the pyrolysis of the pinyon juniper biomass. The pyrolysis products were condensed in three stages, and products were analyzed for physicochemical properties. The condenser oil formed two phases with the aqueous fraction, whereas the electrostatic precipitator oils formed a single phase. The oil pH was 3.3; the higher heating value (HHV) was 28 MJ/kg; and the viscosity was less than 100 cP. There was a direct correlation between the viscosity of the oils and the alcohol/ether content of the oils, and this was also related to the aging rate of the oils. The catalytic pyrolysis oils were hydrotreated in a continuous single-stage benchtop hydrotreater to produce hydrocarbon fuels with a density of 0.80$-$0.82 cm3/g. The hydrotreater ran continuously for over 300 h with no significant catalyst deactivation or coke formation. This is the first time that such a long single-stage hydrotreatment has been demonstrated on biomass catalytic pyrolysis oils.

  2. Gas Exchange and Phytoluminography of Single Red Kidney Bean Leaves during Periods of Induced Stomatal Oscillations

    PubMed Central

    Ellenson, James L.; Raba, Richard M.

    1983-01-01

    This report examines the capabilities of a new approach to the study of gas exchange and electron transport properties of single, intact leaves. The method combines conventional aspects of analysis with an image intensification system that records the spatial distribution of delayed light emission (DLE) over single leaf surfaces. The combined system was used to investigate physiological perturbations induced by exposure of single leaves of Phaseolus vulgaris cv `California Light Red' to a combination of SO2 (0.5 microliters per liter) and ozone (0.1 microliters per liter). Exposure of one-half of a leaf to this combination induced DLE and stomatal oscillations, but only in the half of the leaf exposed to the combined gases. Examination of phytoluminographs taken during these oscillations revealed distinct leaf patches where the greatest changes in DLE intensity occurred. This phenomenon is interpreted to be evidence that control of stomatal activity of intact plant leaves occurs within discrete leaf areas defined within the vascular network. Images Fig. 6 PMID:16662989

  3. 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.

  4. Potential for a live red seabream iridovirus (RSIV) vaccine in rock bream Oplegnathus fasciatus at a low rearing temperature.

    PubMed

    Oh, So-Young; Oh, Myung-Joo; Nishizawa, Toyohiko

    2014-01-09

    Serious economic losses have occurred in fingerlings and market-sized rock bream Oplegnathus fasciatus in Korea due to red seabream iridovirus (RSIV) infection. We demonstrated previously that viral multiplication in fish is downregulated by maintaining fish at far from optimum temperatures at the onset of disease. We applied this concept to develop a live RSIV vaccine in rock bream. Mortalities in rock bream that were inoculated with RSIV and reared at 21-30°C were ≥90%, whereas no mortality was observed in fish that received an RSIV inoculation and were reared at ≤18°C. RSIV kinetics revealed that RSIV multiplied rapidly in fish reared at 24.3±1.3°C, and achieved the critical level for rock bream (approximately 10(9.0) genomes/mg) within 28 days. In contrast, the RSIV genome was detected on day 10 in fish that received an RSIV inoculation at 15.5°C, and peaked on day 28 at 10(5.91±0.54) genomes/mg, then decreasing gradually, and were then maintained under the detection level beginning on day 84 after RSIV inoculation. Furthermore, the fish surviving the RSIV infection at low rearing temperature were strongly protected from re-challenge with homologous RSIV; the threshold level of RSIV for rock bream to mount a protective immune response was ≤10(5.4) genomes/mg. Cohabitation experiments revealed that the spread of RSIV from rock bream vaccinated with a live RSIV could be low if it is limited to fish in the late stage (≥84 days of elapse) after vaccination. Thus, it was concluded that when rock bream are reared at ≤18°C and inoculated with RSIV, the survivors can mount a protective immune response against RSIV, suggesting a positive effect of a live RSIV vaccine for rock bream.

  5. Reduced dyes enhance single-molecule localization density for live superresolution imaging.

    PubMed

    Carlini, Lina; Benke, Alexander; Reymond, Luc; Lukinavičius, Gražvydas; Manley, Suliana

    2014-03-17

    Cell-permeable rhodamine dyes are reductively quenched by NaBH4 into a non-fluorescent leuco-rhodamine form. Quenching is reversible, and their fluorescence is recovered when the dyes are oxidized. In living cells, oxidation occurs spontaneously, and can result in up to ten-fold higher densities of single molecule localizations, and more photons per localization as compared with unmodified dyes. These two parameters directly impact the achievable resolution, and we see a significant improvement in the quality of live-cell point-localization super-resolution images taken with reduced dyes. These improvements carry over to increase the density of trajectories for single-molecule tracking experiments.

  6. Three-Dimensional Tracking of Single Granules in Living PC-12 Cells Employing TIRFM and WFFM.

    PubMed

    Xiong, Jun; Li, Dongdong; Zhu, Dan; Qu, Anlian

    2005-01-01

    A comparative study was carried out on evaluating the performance of total internal reflection fluorescence microscopy (TIRFM) and deconvolution wide-field fluorescence microscopy (WFFM) in tracking single secretory granules. Both techniques have been applied to follow the three-dimensional mobility of single secretory granules in living neuroendocrine PC-12 cells. Both techniques return the similar result that most acridine orange-labeled granules were found to travel in random and caged diffusion, and only a small fraction of granules traveled in directed diffusion. Furthermore, the size and 3-D diffusion coefficient of secretory granules, obtained by these two imaging techniques, yield the same value. Together, our results demonstrate the potential of the combination TIRFM and WFFM in tracking long-termed motion of granules throughout live whole cells.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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).

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

    PubMed

    Dang, Cuong; Lee, Joonhee; Breen, Craig; Steckel, Jonathan S; Coe-Sullivan, Seth; Nurmikko, Arto

    2012-04-29

    Colloidal quantum dots exhibit efficient photoluminescence with widely tunable bandgaps as a result of quantum confinement effects. Such quantum dots are emerging as an appealing complement to epitaxial semiconductor laser materials, which are ubiquitous and technologically mature, but unable to cover the full visible spectrum (red, green and blue; RGB). However, the requirement for high colloidal-quantum-dot packing density, and losses due to non-radiative multiexcitonic Auger recombination, have hindered the development of lasers based on colloidal quantum dots. Here, we engineer CdSe/ZnCdS core/shell colloidal quantum dots with aromatic ligands, which form densely packed films exhibiting optical gain across the visible spectrum with less than one exciton per colloidal quantum dot on average. This single-exciton gain allows the films to reach the threshold of amplified spontaneous emission at very low optical pump energy densities of 90 µJ cm(-2), more than one order of magnitude better than previously reported values. We leverage the low-threshold gain of these nanocomposite films to produce the first colloidal-quantum-dot vertical-cavity surface-emitting lasers (CQD-VCSEL). Our results represent a significant step towards full-colour single-material lasers.

  13. 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.

  14. Blurring the boundaries of space: shaping nursing lives at the Red Cross outposts in Ontario, 1922-1945.

    PubMed

    Elliott, Jayne

    2004-01-01

    Historians and other scholars interested in the history of hospitals have investigated the links between medical architecture and the organization of space with the evolution of modern medicine. The transformation over time in the architectural for of medical institutions has tended to reflect developments in medical science and therapeutic efficiency as well as elements in the broader social climate. Some authors, however, have argued for the agency of structure and spatial organization, to consider that they are not just containers with which human activities take place, but which also actively construct or constitute social practices and relations. Most studies of this nature have centred on large medical buildings especially in urban areas, and have examined the impact of architectural arrangement in relation to administrators and architects, physicians and patients. Fe have considered the interconnections of form and space with nurses, despite the prominence of institutional nursing labour since the late 19th-century. The following discussion begins an exploration of these concepts within the rural environment. Between 1922 and 1984, the Ontario Division of the Canadian Red Cross Society administered an outpost program in which it operated small hospitals and nursing stations in isolated communities throughout the northern reaches of the province. This article will focus primarily o n the one-nurse stations that the Division managed during the interwar years and the nurses that it hired to staff them. The interior spatial organization of these outposts, which led in particular to their multiple functions as tiny hospitals, community health centres and nurses' homes, not only shaped both the professional practice and the social or private lives of the Red Cross nurses but also contributed to the diffusion of contemporary precepts in health and medical care throughout a remote population.

  15. 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.

  16. Multicolor imaging of hydrogen peroxide level in living and apoptotic cells by a single fluorescent probe.

    PubMed

    Wen, Ying; Xue, Fengfeng; Lan, Haichuang; Li, Zhenhua; Xiao, Shuzhang; Yi, Tao

    2017-05-15

    To understand the entangled relationship between reactive oxygen species (ROS) and apoptosis, there is urgent need for simultaneous dynamic monitoring of these two important biological events. In this study, we have developed a fluorescent probe, pep4-NP1, which can simultaneously detect H2O2 and caspase 3, the respective markers of ROS and apoptosis. The probe contains a H2O2 fluorescence reporter (NP1) and Cy5 fluorescent chromophore connected by a caspase 3 specific recognition peptide. The detecting strategy was realized through a controllable fluorescence resonance energy transfer (FRET) process between NP1 and Cy5 of pep4-NP1, after reaction with H2O2, which was verified by molecular calculation and in vitro spectral studies. In the absent of caspase 3, the accumulation of H2O2 induces red fluorescence of pep4-NP1 centered at 663nm in living cells due to the existence of FRET. In contrast, FRET is inhibited in apoptotic cells due to cleavage of the peptide spacer of pep4-NP1 by over-expressed caspase 3. Consequently, green fluorescence (555nm) predominated when labelling production of H2O2 in apoptotic cells. Moreover, Pep4-NP1 shows excellent selectivity towards H2O2 and caspase 3 on their respective reaction sites. Therefore, pep4-NP1 can distinguish endogenously generated H2O2 between living cells and apoptotic cells with different fluorescence wavelengths, providing additional information on the ROS production pathways.

  17. Studying the mechanism of CD47-SIRPα interactions on red blood cells by single molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Pan, Yangang; Wang, Feng; Liu, Yanhou; Jiang, Junguang; Yang, Yong-Guang; Wang, Hongda

    2014-08-01

    The interaction forces and binding kinetics between SIRPα and CD47 were investigated by single-molecule force spectroscopy (SMFS) on both fresh and experimentally aged human red blood cells (hRBCs). We found that CD47 experienced a conformation change after oxidation, which influenced the interaction force and the position of the energy barrier between SIRPα and CD47. Our results are significant for understanding the mechanism of phagocytosis of red blood cells at the single molecule level.The interaction forces and binding kinetics between SIRPα and CD47 were investigated by single-molecule force spectroscopy (SMFS) on both fresh and experimentally aged human red blood cells (hRBCs). We found that CD47 experienced a conformation change after oxidation, which influenced the interaction force and the position of the energy barrier between SIRPα and CD47. Our results are significant for understanding the mechanism of phagocytosis of red blood cells at the single molecule level. Electronic supplementary information (ESI) available: Experimental section. See DOI: 10.1039/c4nr02889a

  18. 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

  19. 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

  20. The pregnancy rate and live birth rate after kidney transplantation: a single-center experience.

    PubMed

    Fontana, I; Santori, G; Fazio, F; Valente, U

    2012-09-01

    Kidney transplantation is the treatment of choice for end-stage renal disease (ESRD). Kidney transplantation recipients live longer and have better quality of life than patients on dialysis. Hypothalamic gonadal dysfunction in females who have ESRD may be reversed within the first few months after kidney transplantation, such as the ability to have children. Despite thousands of successful pregnancies in transplantation recipients, there is limited information about it. In this study, we evaluated the pregnancy rates and live birth rates in women (n = 133) who underwent kidney transplantation in our center from 1983 to 2010. Recipients of a second kidney transplantation and recipients of multiorgan transplantations were excluded. We observed 33 pregnancies with 11 live births (33.3%), 12 spontaneous abortions (36.36%), and 10 therapeutic abortions (30.3%). The pregnancy rate was 18%. The live birth rate was 33.3%. Therapeutic abortions were 36.3%, and the pregnancies resulting in fetal loss were 30.3%. The pregnancies were identified in 32 women. The majority of women (n = 32; 96.9%) had a single pregnancy, whereas 1 woman (3.1%) had two pregnancies. In our series, the pregnancy rates for kidney transplantation recipients were markedly lower and decreased more rapidly than those reported in the general population.

  1. Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy

    PubMed Central

    Greiss, Ferdinand; Deligiannaki, Myrto; Jung, Christophe; Gaul, Ulrike; Braun, Dieter

    2016-01-01

    In multicellular organisms, single-fluorophore imaging is obstructed by high background. To achieve a signal/noise ratio conducive to single-molecule imaging, we adapted reflected light-sheet microscopy (RLSM) to image highly opaque late-stage Drosophila embryos. Alignment steps were modified by means of commercially available microprisms attached to standard coverslips. We imaged a member of the septate-junction complex that was used to outline the three-dimensional epidermal structures of Drosophila embryos. Furthermore, we show freely diffusing single 10 kDa Dextran molecules conjugated to one to two Alexa647 dyes inside living embryos. We demonstrate that Dextran diffuses quickly (∼6.4 μm2/s) in free space and obeys directional movement within the epidermal tissue (∼0.1 μm2/s). Our single-particle-tracking results are supplemented by imaging the endosomal marker Rab5-GFP and by earlier reports on the spreading of morphogens and vesicles in multicellular organisms. The single-molecule results suggest that RLSM will be helpful in studying single molecules or complexes in multicellular organisms. PMID:26910430

  2. Comparable clinical outcomes and live births after single vitrified-warmed and fresh blastocyst transfer.

    PubMed

    Feng, Guixue; Zhang, Bo; Zhou, Hong; Shu, Jinhui; Gan, Xianyou; Wu, Fangrong; Deng, Xihe

    2012-11-01

    Selective single-blastocyst transfer (SBT) in fresh cycles has been effective in reducing multiple pregnancies. However, we do not know whether this successful strategy of fresh transfer cycles is suitable for cryopreserved cycles. The present study was undertaken to evaluate the feasibility and value of SBT in vitrified-warmed cycles. Clinical pregnancy rate (CPR) was similar with vitrified and fresh SBT (46.61% versus 52.15% respectively). Of the pregnant patients, monozygotic twin, miscarriage and ectopic pregnancy rates were similar with vitrified and fresh SBT. For the newborns, no significant difference was observed in live birth, low birthweight, premature delivery and birth defects rates between vitrified and fresh SBT. With respect to the quality of transferred blastocysts (from BB to AA), a similar CPR and miscarriage rate was obtained for both vitrified and fresh SBT when a similar blastocyst cohort graded ≥ 3BB was transferred. The data show that vitrified SBT is an effective means of reducing multiple pregnancy and that comparable clinical outcomes and live births are achieved if single blastocysts graded ≥ 3BB are transferred for both vitrified and fresh SBT. These data should encourage clinics to evaluate their embryo transfer policy and adopt vitrified SBT as everyday practice. Selective single-blastocyst transfer in fresh cycles has been an effective method to reduce the multiple pregnancies. However, due to a lack of adequate studies, we do not know whether this successful strategy in fresh transfer cycles is suitable in cryopreserved cycles. The present study was undertaken to explore the feasibility and value of single-blastocyst transfer in vitrified-warmed cycles. We found that single-blastocyst transfer in vitrified-warmed cycles is an effective means of reducing multiple pregnancy, and comparable clinical outcomes and live births were achieved if single blastocysts graded ≥ 3BB were transferred for both vitrified-warmed and fresh

  3. Rapid measurement of molecular transport and interaction inside living cells using single plane illumination.

    PubMed

    Hedde, Per Niklas; Stakic, Milka; Gratton, Enrico

    2014-11-14

    The ability to measure biomolecular dynamics within cells and tissues is very important to understand fundamental physiological processes including cell adhesion, signalling, movement, division or metabolism. Usually, such information is obtained using particle tracking methods or single point fluctuation spectroscopy. We show that image mean square displacement analysis, applied to single plane illumination microscopy data, is a faster and more efficient way of unravelling rapid, three-dimensional molecular transport and interaction within living cells. From a stack of camera images recorded in seconds, the type of dynamics such as free diffusion, flow or binding can be identified and quantified without being limited by current camera frame rates. Also, light exposure levels are very low and the image mean square displacement method does not require calibration of the microscope point spread function. To demonstrate the advantages of our approach, we quantified the dynamics of several different proteins in the cyto- and nucleoplasm of living cells. For example, from a single measurement, we were able to determine the diffusion coefficient of free clathrin molecules as well as the transport velocity of clathrin-coated vesicles involved in endocytosis. Used in conjunction with dual view detection, we further show how protein-protein interactions can be quantified.

  4. Rapid Measurement of Molecular Transport and Interaction inside Living Cells Using Single Plane Illumination

    PubMed Central

    Hedde, Per Niklas; Stakic, Milka; Gratton, Enrico

    2014-01-01

    The ability to measure biomolecular dynamics within cells and tissues is very important to understand fundamental physiological processes including cell adhesion, signalling, movement, division or metabolism. Usually, such information is obtained using particle tracking methods or single point fluctuation spectroscopy. We show that image mean square displacement analysis, applied to single plane illumination microscopy data, is a faster and more efficient way of unravelling rapid, three-dimensional molecular transport and interaction within living cells. From a stack of camera images recorded in seconds, the type of dynamics such as free diffusion, flow or binding can be identified and quantified without being limited by current camera frame rates. Also, light exposure levels are very low and the image mean square displacement method does not require calibration of the microscope point spread function. To demonstrate the advantages of our approach, we quantified the dynamics of several different proteins in the cyto- and nucleoplasm of living cells. For example, from a single measurement, we were able to determine the diffusion coefficient of free clathrin molecules as well as the transport velocity of clathrin-coated vesicles involved in endocytosis. Used in conjunction with dual view detection, we further show how protein-protein interactions can be quantified. PMID:25394360

  5. Wolf-Rayet, Yellow and Red Supergiant in the single massive stars perspective

    NASA Astrophysics Data System (ADS)

    Georgy, Cyril; Hirschi, R.; Ekstrom, S.; Meynet, G.

    2013-06-01

    Rotation and mass loss are the key ingredients determining the fate of single massive stars. In recent years, a large effort has been made to compute whole grids of stellar models at different metallicities, including or not the effects of rotation, with the Geneva evolution code. In this talk, I will focus on the evolved stages of massive star evolution (red and yellow supergiants, Wolf-Rayet stars), in the framework of these new grids of models. I will highlight the effects of rotation and mass loss on the post-main sequence evolution of massive stars at solar and lower metallicity. In particular, I will discuss their impact on the maximum mass for a star to end its life as a RSG (leading to a type IIP supernova), on the possibility for a star to finish as a YSG, and on the initial mass ranges leading to various WR star subtypes. I will then compare the results predicted by our code with observed populations of evolved massive stars, bringing constraints on our computations, as well as some indications on the binary star fraction needed to reproduce them.

  6. Particle-based modeling effect of shape transform of single sickle red blood cells

    NASA Astrophysics Data System (ADS)

    Yang, Jun; Karniadakis, George; Dao, Ming

    2016-11-01

    Sickle red blood cells often exhibit various sickled shapes as well as higher shear and bending stiffness. To study the membrane biomechanical properties related to cell morphology, we employed multiscale coarse grain models based on dissipative particle dynamics (DPD). Through the proper orthogonal decomposition (POD) we analyst the membrane fluctuation of a single cell which probe the membrane mechanical properties. In this work, the membrane mechanics alteration caused by cell volume and surface area variation are tested. We verified that with same ratio of surface area and volume, volume differences will not affect the membrane fluctuation. We also found that by expanding the whole cell the membrane fluctuation performance does not change. To further quantify the pure shape effects, we generate cells with different aspect ratio of major axis and minor axis at which membrane exhibit different fluctuation indicating the mechanical properties divergence. Through the spatial-temporal autocorrelation of membrane fluctuations characteristics, the membrane bending stiffness and shear modulus are carefully calibrated against QPI experimental data.

  7. Adsorption of neutral red and malachite green onto grapefruit peel in single and binary systems.

    PubMed

    Zou, Weihua; Gao, Shuaipeng; Zou, Xiuli; Bai, Hongjuan

    2013-05-01

    This study characterized the properties of NaOH-modified grapefruit peel (MGP) and investigated its adsorption properties, specifically the adsorption of the synthetic dyes neutral red (NR) and malachite green (MG) onto MGP, in single and binary systems by means of batch techniques. The adsorption equilibrium data of NR onto MGP fit well with both the Langmuir and Koble-Corrigan models, while the Koble-Corrigan and Dubinin-Radushkevich models seemed to agree better with MG adsorption. The maximum equilibrium quantities of NR and MG from the Langmuir model were 640.3 and 314.9 mg/g at 298 K, respectively. The Elovich model was a better fit with the kinetic process, which suggested that ion exchange was one of the main mechanisms at work. The thermodynamic parameters of adsorption systems indicated spontaneous and endothermic processes. In the binary system experiments, NR and MG exhibited competitive adsorption. The quantity of MG adsorbed was more strongly influenced by NR, due to the higher affinity of MGP for the latter.

  8. The Indigenous Red Ribbon Storytelling Study: What does it mean for Indigenous peoples living with HIV and a substance use disorder to access antiretroviral therapy in Saskatchewan?

    PubMed

    Nowgesic, Earl; Meili, Ryan; Stack, Sandra; Myers, Ted

    2015-01-01

    Indigenous peoples living with HIV are less likely than non-Indigenous peoples living with HIV to access antiretroviral therapy; however, there is not enough contextual information surrounding this issue. The Indigenous Red Ribbon Storytelling Study was conducted in part to examine how Indigenous peoples living with HIV construct and understand their experiences accessing antiretroviral therapy. Our study design was critical Indigenous qualitative research, using the Behavioral Model of Health Services Use and community-based participatory research approaches. The study was conducted in partnership with Indigenous and non-Indigenous organizations. Study participants were adults from two Canadian cities. The study methods included 20 individual and two Indigenous sharing circle interviews, six participant observation sessions, a short survey and thematic analysis. Accessing antiretroviral therapy within the context of living with a substance use disorder was an overarching theme. Indigenous peoples living with HIV felt they had to choose between living with their active substance use disorder and accessing antiretroviral therapy. They felt misunderstood as a person living with a substance use disorder and often felt coerced into using antiretroviral therapy. Despite these challenges, they persevered as Indigenous peoples living with HIV and a substance use disorder. Further research on antiretroviral therapy access among Indigenous peoples living with HIV and a substance use disorder, particularly from the perspective of health service providers, is needed.

  9. 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.

  10. 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

  11. The lived experiences of single Taiwanese mothers being resilient after divorce.

    PubMed

    Hong, Rei-Mei; Welch, Anthony

    2013-01-01

    The lived experiences of being resilient as described by 13 single Taiwanese mothers after divorce was the focus of this study. A descriptive phenomenological approach to inquiry was the theoretical framework underpinning the study. Information was gathered through two in-depth face-to-face digitally recorded interviews with each participant. Each of the participants had suffered from depression. For the analysis of the participants' transcripts of interview the authors used Colaizzi's method. Four themes emerged from the analysis process: having faith in God, bending with the ebb and flow of daily life, finding strength in the support and friendship of others, and new found freedom and hope for the future. Findings of this study have the potential to enhance understanding of the mental health needs of single mothers and their children in the provision of holistic health care delivery.

  12. 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.

  13. Hepatic cavernous hemangioma: diagnosis with /sup 99m/Tc-labeled red cells and single-photon emission CT

    SciTech Connect

    Brodsky, R.I.; Friedman, A.C.; Maurer, A.H.; Radecki, P.D.; Caroline, D.F.

    1987-01-01

    During the performance of high-resolution real-time abdominal sonography, small echogenic hepatic masses are frequently discovered. A second imaging test to confirm the suspected diagnosis of hemangioma is often required. Planar labeled red-cell imaging will often not detect hemangiomas smaller than 3 cm. We studied 14 patients with labeled red-cell scintigraphy and single-photon emission CT (SPECT). Six hemangiomas were diagnosed by SPECT that would have been missed by planar imaging alone. All six were smaller than 2.5 cm. With the addition of SPECT, labeled red-cell scintigraphy has specificity and sensitivity that make it at least as reliable as dynamic CT for the noninvasive diagnosis of hepatic cavernous hemangioma.

  14. Regulation of RNA polymerase II activation by histone acetylation in single living cells.

    PubMed

    Stasevich, Timothy J; Hayashi-Takanaka, Yoko; Sato, Yuko; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Sakata-Sogawa, Kumiko; Tokunaga, Makio; Nagase, Takahiro; Nozaki, Naohito; McNally, James G; Kimura, Hiroshi

    2014-12-11

    In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

  15. 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.

  16. Perception of color emotions for single colors in red-green defective observers

    PubMed Central

    Inoue, Takaaki

    2016-01-01

    It is estimated that inherited red-green color deficiency, which involves both the protan and deutan deficiency types, is common in men. For red-green defective observers, some reddish colors appear desaturated and brownish, unlike those seen by normal observers. Despite its prevalence, few studies have investigated the effects that red-green color deficiency has on the psychological properties of colors (color emotions). The current study investigated the influence of red-green color deficiency on the following six color emotions: cleanliness, freshness, hardness, preference, warmth, and weight. Specifically, this study aimed to: (1) reveal differences between normal and red-green defective observers in rating patterns of six color emotions; (2) examine differences in color emotions related to the three cardinal channels in human color vision; and (3) explore relationships between color emotions and color naming behavior. Thirteen men and 10 women with normal vision and 13 men who were red-green defective performed both a color naming task and an emotion rating task with 32 colors from the Berkeley Color Project (BCP). Results revealed noticeable differences in the cleanliness and hardness ratings between the normal vision observers, particularly in women, and red-green defective observers, which appeared mainly for colors in the orange to cyan range, and in the preference and warmth ratings for colors with cyan and purple hues. Similarly, naming errors also mainly occurred in the cyan colors. A regression analysis that included the three cone-contrasts (i.e., red-green, blue-yellow, and luminance) as predictors significantly accounted for variability in color emotion ratings for the red-green defective observers as much as the normal individuals. Expressly, for warmth ratings, the weight of the red-green opponent channel was significantly lower in color defective observers than in normal participants. In addition, the analyses for individual warmth ratings in

  17. Perception of color emotions for single colors in red-green defective observers.

    PubMed

    Sato, Keiko; Inoue, Takaaki

    2016-01-01

    It is estimated that inherited red-green color deficiency, which involves both the protan and deutan deficiency types, is common in men. For red-green defective observers, some reddish colors appear desaturated and brownish, unlike those seen by normal observers. Despite its prevalence, few studies have investigated the effects that red-green color deficiency has on the psychological properties of colors (color emotions). The current study investigated the influence of red-green color deficiency on the following six color emotions: cleanliness, freshness, hardness, preference, warmth, and weight. Specifically, this study aimed to: (1) reveal differences between normal and red-green defective observers in rating patterns of six color emotions; (2) examine differences in color emotions related to the three cardinal channels in human color vision; and (3) explore relationships between color emotions and color naming behavior. Thirteen men and 10 women with normal vision and 13 men who were red-green defective performed both a color naming task and an emotion rating task with 32 colors from the Berkeley Color Project (BCP). Results revealed noticeable differences in the cleanliness and hardness ratings between the normal vision observers, particularly in women, and red-green defective observers, which appeared mainly for colors in the orange to cyan range, and in the preference and warmth ratings for colors with cyan and purple hues. Similarly, naming errors also mainly occurred in the cyan colors. A regression analysis that included the three cone-contrasts (i.e., red-green, blue-yellow, and luminance) as predictors significantly accounted for variability in color emotion ratings for the red-green defective observers as much as the normal individuals. Expressly, for warmth ratings, the weight of the red-green opponent channel was significantly lower in color defective observers than in normal participants. In addition, the analyses for individual warmth ratings in

  18. 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

    2016-09-06

    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.

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

    PubMed Central

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

    2016-01-01

    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

  20. 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

  1. 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

  2. Living with an Old Red Dwarf: X-ray-UV Emissions of Kapteyn’s Star - Effects of X-UV radiation on Habitable Zone Planets hosted by old Red Dwarf Stars

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

    Red dwarfs (dM) stars make up over 75% of the local stellar population and a significant fraction (~40-50%) are older than the Sun. Because of the high frequency of red dwarfs and their longevity (> 50 Gyr), there is a greater possibility of more advanced life in red dwarf-exoplanet systems. MEarths, UVES, SDSS-III, and the upcoming TESS mission are some surveys that are targeting red dwarfs in the search for hosted potentially habitalble planets. As part of Villanova's 'Living with a Red Dwarf' program, we have obtained HST-COS Ultraviolet spectra (1150-3000A) and Chandra X-ray observations of Kapteyn's star (GJ 191; M1 V, V = 8.85 mag , d = 12.76 +/- 0.05 ly). Kapyteyn's Star is important for the study of old red dwarfs because it is the nearest (Pop II) halo star with a radial velocity of +245.2 km/s and an estimated age of 11.2 +/-0.9 Gyrs. Recently Kapteyn's Star was found to host two super-Earth mass planets - one of these is orbiting inside the star's Habitable Zone (Anglada-Escude' 2014: MNRAS 443, L89). In our program, Kapteyn's star is the oldest red dwarf and as such serves as an anchor for our age, rotation, and activity relations. The spectra obtained from HST/COS provide one of the cleanest measurements of the important HI Lyman-alpha 1215.6 A emission flux for red dwarfs. This is due to the large Doppler shift from the high radial velocity, separating the stellar Ly-alpha emission from by the Ly-alpha ISM and local geo-coronal sources. These observations further provide calibrations at the old age/low rotation/low activity extremes for our relations. As the nearest and brightest old red dwarf star, Kapteyn's Star also provides insights into its magnetic properties to investigae coronal x-ray and UV emission for the large population of old, slowly rotating red dwarf stars. Kapteyn's star also serves as a proxy for the numerous metal-poor old disk - Pop II M dwarfs by providing information about X-UV emissions. This information is crucial for

  3. 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

  4. Manipulation and Motion of Organelles and Single Molecules in Living Cells.

    PubMed

    Norregaard, Kamilla; Metzler, Ralf; Ritter, Christine M; Berg-Sørensen, Kirstine; Oddershede, Lene B

    2017-03-08

    The biomolecule is among the most important building blocks of biological systems, and a full understanding of its function forms the scaffold for describing the mechanisms of higher order structures as organelles and cells. Force is a fundamental regulatory mechanism of biomolecular interactions driving many cellular processes. The forces on a molecular scale are exactly in the range that can be manipulated and probed with single molecule force spectroscopy. The natural environment of a biomolecule is inside a living cell, hence, this is the most relevant environment for probing their function. In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theoretical tools for analyzing single molecule data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly used force spectroscopy techniques, namely optical tweezers, magnetic tweezers, and atomic force microscopy, are described in detail, and their strength and limitations related to in vivo experiments are discussed. Finally, recent exciting discoveries within the field of in vivo manipulation and dynamics of single molecule and organelles are reviewed.

  5. Three-dimensional tracking of single secretory granules in live PC12 cells.

    PubMed

    Li, Dongdong; Xiong, Jun; Qu, Anlian; Xu, Tao

    2004-09-01

    Deconvolution wide-field fluorescence microscopy and single-particle tracking were used to study the three-dimensional mobility of single secretory granules in live PC12 cells. Acridine orange-labeled granules were found to travel primarily in random and caged diffusion, whereas only a small fraction of granules traveled in directed fashion. High K(+) stimulation increased significantly the percentage of granules traveling in directed fashion. By dividing granules into the near-membrane group (within 1 microm from the plasma membrane) and cytosolic group, we have revealed significant differences between these two groups of granules in their mobility. The mobility of these two groups of granules is also differentially affected by disruption of F-actin, suggesting different mechanisms are involved in the motion of the two groups of granules. Our results demonstrate that combined deconvolution and single-particle tracking may find its application in three-dimensional tracking of long-term motion of granules and elucidating the underlying mechanisms.

  6. 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.

  7. Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells

    PubMed Central

    Lee, Timothy K.; Meng, Kevin; Shi, Handuo; Huang, Kerwyn Casey

    2016-01-01

    The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis. PMID:27774981

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Presynaptic structure of Aplysia single live neuron by atomic force and confocal laser scanning microscope.

    PubMed

    Park, Aee-Young; Chae, Yeon-Su; Lee, Seung-Hee; Kaang, Bong-Kiun; Lee, Seonghoon

    2013-05-02

    The structural and functional plasticity of Aplysia mechanosensory presynaptic neurons has been studied in relation with the mechanism underlying learning and memory. Long-term facilitation (LTF), which is a well-known cellular model for long-term memory in Aplysia, is accompanied by new synaptic structural growth or change. We developed a combined atomic force microscope and confocal laser scanning microscope (AFM-CLSM) system integrated with a MATLAB routine for image processing to concurrently obtain high-resolution 3-dimensional (3D) outer-surface morphological images and 3D interior fluorescence images. With our combined AFM-CLSM system, volumetric changes in the presynaptic structures (varicosities) of Aplysia live sensory-motor neuron cocultures were observed. The spatial distribution of synaptic vesicle molecules in the preexisting varicosities was monitored together with a volumetric change in the varicosities. Our combined AFM-CLSM system is successfully adapted for measuring learning-related structural changes and the movement of synaptic molecules in the single live neuron through interaction force and fluorescence imaging.

  13. 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

  14. 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.

  15. Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells

    PubMed Central

    Hoornweg, Tabitha E.; van Duijl-Richter, Mareike K. S.; Ayala Nuñez, Nilda V.; Albulescu, Irina C.; van Hemert, Martijn J.

    2016-01-01

    ABSTRACT Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in Africa, Asia, and the Americas. The cell entry pathway hijacked by CHIKV to infect a cell has been studied previously using inhibitory compounds. There has been some debate on the mechanism by which CHIKV enters the cell: several studies suggest that CHIKV enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. Here we applied live-cell microscopy and monitored the cell entry behavior of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain detailed insight into the dynamic events that occur during CHIKV entry. We observed that almost all particles fused within 20 min after addition to the cells. Of the particles that fused, the vast majority first colocalized with clathrin. The average time from initial colocalization with clathrin to the moment of membrane fusion was 1.7 min, highlighting the rapidity of the cell entry process of CHIKV. Furthermore, these results show that the virus spends a relatively long time searching for a receptor. Membrane fusion was observed predominantly from within Rab5-positive endosomes and often occurred within 40 s after delivery to endosomes. Furthermore, we confirmed that a valine at position 226 of the E1 protein enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV enters cells via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes. IMPORTANCE Since its reemergence in 2004, chikungunya virus (CHIKV) has spread rapidly around the world, leading to millions of infections. CHIKV often causes chikungunya fever, a self-limiting febrile illness with severe arthralgia. Currently, no vaccine or specific antiviral treatment against CHIKV is available. A potential antiviral strategy is to interfere with the cell

  16. 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.

  17. Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli

    PubMed Central

    Di Paolo, Diana; Afanzar, Oshri; Armitage, Judith P.; Berry, Richard M.

    2016-01-01

    For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time. This article is part of the themed issue ‘The new bacteriology’. PMID:27672145

  18. 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.

  19. Live attenuated B. pertussis as a single-dose nasal vaccine against whooping cough.

    PubMed

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

    2006-07-01

    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.

  20. Raman spectroscopy of a single living cell in environmentally stressed conditions

    NASA Astrophysics Data System (ADS)

    Singh, Gajendra P.; Creely, Caitriona; Volpe, Giovanni; Grotsch, Helga; Petrov, Dmitri

    2005-08-01

    Living cells initiate a stress response in order to survive environmentally stressful conditions. We monitored changes in the Raman spectra of an optically trapped Saccharomyces cerevisiae yeast cell under normal and hyperosmotic stress conditions. When the yeast cells were challenged with a high concentration of glucose so as to exert hyperosmotic stress, it was shown that two chemical substances - glycerol and ethanol - could be monitored in real time in a single cell. The volume of the detection area of our confocal microspectrometer is approximately 1 fL. The average quantities of detected glycerol and ethanol are about 300 attomol and 700 attomol respectively. This amounts to the detection of approximately 108 glycerol molecules and 4 X 108 ethanol molecules after 36 min of hyper osmotic stress. Besides this, we also optically trapped a single yeast cell for up to three hours under normal conditions and monitored the changes in the Raman spectra during the lag phase of its growth and the G1 phase of its cell cycle. During the lag phase the cell synthesises new proteins and the observed behavior of the peaks corresponding to these proteins as well as those of RNA served as a sensitive indicator of the adaptation of the cell to its changed environment. The changes observed in the Raman spectra of a trapped yeast cell in the late G1 phase or the beginning of S phase corresponded to the growth of a bud.

  1. SIDT2 mediates gymnosis, the uptake of naked single-stranded oligonucleotides into living cells.

    PubMed

    Takahashi, Masayuki; Contu, Viorica Raluca; Kabuta, Chihana; Hase, Katsunori; Fujiwara, Yuuki; Wada, Keiji; Kabuta, Tomohiro

    2017-03-09

    Single-stranded oligonucleotides (ssOligos) are efficiently taken up by living cells without the use of transfection reagents. This phenomenon called 'gymnosis' enables the sequence-specific silencing of target genes in various types of cells. Several antisense ssOligos are used for the treatment of human diseases. However, the molecular mechanism underlying the uptake of naked ssOligos into cells remains to be elucidated. Here, we show that systemic RNA interference deficient-1 (SID-1) transmembrane family 2 (SIDT2), a mammalian ortholog of the Caenorhabditis elegans double-stranded RNA channel SID-1, mediates gymnosis. We show that the uptake of naked ssOligos into cells is significantly downregulated by knockdown of SIDT2. Furthermore, knockdown of SIDT2 inhibited the effect of antisense RNA mediated by gymnosis. Overexpression of SIDT2 enhanced the uptake of naked ssOligos into cells, while a single amino acid mutation in SIDT2 abolished this effect. Our findings highlight the mechanism of extra- and intracellular RNA transport and may contribute to the further development of nucleic acid-based therapies.

  2. 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

  3. 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

  4. 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.

  5. In silico model-driven assessment of the effects of single nucleotide polymorphisms (SNPs) on human red blood cell metabolism.

    PubMed

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

    2002-11-01

    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.

  6. Efficient frequency downconversion at the single photon level from the red spectral range to the telecommunications C-band.

    PubMed

    Zaske, Sebastian; Lenhard, Andreas; Becher, Christoph

    2011-06-20

    We report on single photon frequency downconversion from the red part of the spectrum (738 nm) to the telecommunications C-band. By mixing attenuated laser pulses with an average photon number per pulse < 1 with a strong continuous light field at 1403 nm in a periodically poled Zn:LiNbO3 ridge waveguide an internal conversion efficiency of ∼ 73% is achieved. We further investigate the noise properties of the process by measuring the output spectrum. Our results indicate that by narrow spectral filtering a quantum interface should be feasible which bridges the wavelength gap between quantum emitters like color centers in diamond emitting in the red part of the spectrum and low-loss fiber-optic telecommunications wavelengths.

  7. Mandibular osteomyelitis in red deer (Cervus elaphus hispanicus) and fallow deer (Dama dama): occurrence and associated factors in free-living populations in southern Spain.

    PubMed

    Azorit, Concepción; Oya, Antonia; Tellado, Sierra; Carrasco, Rafael; Moro, Javier

    2012-01-01

    The prevalence of mandibular osteomyelitis, which results in a condition called lumpy jaw, and factors associated with its occurrence were investigated in syntopic free-living populations of red deer (Cervus elaphus hispanicus) and fallow deer (Dama dama) in Spain. The study material consisted of 3,586 mandibles from 2,548 red deer and 1,038 fallow deer shot during sport hunting, herd management culls, and programs for population control between 1988 and 1997 (period 1) and 2002 and 2009 (period 2) in eastern Sierra Morena, southern Spain. Disease prevalence ranged from 0.36% to 10.91% among age groups. Older animals were significantly more likely to be affected than younger ones. Red deer stags had higher prevalence than other groups. There was a significantly higher prevalence in period 1, probably associated with differences in climatic and population conditions. High population densities of female red deer contributed significantly to occurrence of disease. Intensive herd management and poor environmental conditions were considered risk factors that increased susceptibility to disease. The study of this affliction could be useful for monitoring general herd welfare and ecologic changes in Mediterranean ecosystems.

  8. A practical approach for the detection of DNA nanostructures in single live human cells by fluorescence microscopy.

    PubMed

    Bergamini, C; Angelini, P; Rhoden, K J; Porcelli, A M; Fato, R; Zuccheri, G

    2014-05-15

    In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, cell cycle stage, make the cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. In this paper, we describe the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with epifluorescence microscopy. We report that HeLa cells internalize these nanostructures spontaneously with a higher efficiency with respect to single-stranded or double-stranded oligonucleotides. Our findings suggest that DNA tetrahedra could serve as a platform for the realization of a series of multifunctional intracellular biosensors for the analysis of single live cells.

  9. Orientations between Red Antenna States of Photosystem I Monomers from Thermosynechococcus elongatus Revealed by Single-Molecule Spectroscopy.

    PubMed

    Skandary, Sepideh; Konrad, Alexander; Hussels, Martin; Meixner, Alfred J; Brecht, Marc

    2015-10-29

    Single-molecule spectroscopy at low temperature was used to study the spectral properties, heterogeneities, and spectral dynamics of the chlorophyll a (Chl a) molecules responsible for the fluorescence emission of photosystem I monomers (PS I-M) from the cyanobacterium Thermosynechococcus elongatus. The fluorescence spectra of single PS I-M are dominated by several red-shifted chlorophyll a molecules named C708 and C719. The emission spectra show broad spectral distributions and several zero-phonon lines (ZPLs). Compared with the spectra of the single PS I trimers, some contributions are missing due to the lower number of C719 Chl's in monomers. Polarization-dependent measurements show an almost perpendicular orientation between the emitters corresponding to C708 and C719. These contributions can be assigned to chlorophyll dimers B18B19, B31B32, and B32B33.

  10. LIVING WITH A RED DWARF: ROTATION AND X-RAY AND ULTRAVIOLET PROPERTIES OF THE HALO POPULATION KAPTEYN’S STAR

    SciTech Connect

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

    2016-04-20

    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{sup −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.

  11. 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.

  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. Outcomes of pediatric living donor kidney transplantation: A single-center experience.

    PubMed

    Pérez-Bertólez, Sonia; Barrero, Rafael; Fijo, Julia; Alonso, Verónica; Ojha, Devicka; Fernández-Hurtado, Miguel Ángel; Martínez, Jerónimo; León, Eduardo; García-Merino, Francisco

    2017-05-01

    Renal transplantation is the treatment of choice for children with ESRD offering advantages of improved survival, growth potential, cognitive development, and quality of life. The aim of our study was to compare the outcomes of LDKT vs DDKT performed in children at a single center. Retrospective chart review of pediatric patients who underwent kidney transplantation from 2005 to 2014 was performed. Ninety-one renal transplants were accomplished, and 31 cases (38.27%) were LDKT, and in 96.7% of the cases, the graft was obtained through laparoscopy. Thirty-four receptors weighted <25 kg. LDKT group had statistically significant lower cold ischemia times than DDKT one. Complication rate was 9.67% for LDKT and 18.33% for DDKT. eGFR was better in LDKT. Patient survival rate was 100% for LDKT and 98.3% for DDKT, and graft survival rate was 96.7% for LDKT and 88.33%-80% for DDKT at a year and 5 years. Our program of pediatric kidney transplantation has achieved optimal patient and graft survival rates with low rate of complications. Living donor pediatric kidney transplants have higher patient and better graft survival rates than deceased donor kidney transplants.

  14. 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

  15. Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain

    NASA Astrophysics Data System (ADS)

    Godin, Antoine G.; Varela, Juan A.; Gao, Zhenghong; Danné, Noémie; Dupuis, Julien P.; Lounis, Brahim; Groc, Laurent; Cognet, Laurent

    2016-11-01

    The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its volume. Signalling molecules, neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep, development and aging and is probably altered in neuropsychiatric and degenerative diseases, as inferred from electron microscopy and macroscopic biophysical investigations. Here we show an approach to directly observe the local ECS structures and rheology in brain tissue using super-resolution imaging. We inject single-walled carbon nanotubes into rat cerebroventricles and follow the near-infrared emission of individual nanotubes as they diffuse inside the ECS for tens of minutes in acute slices. Because of the interplay between the nanotube geometry and the ECS local environment, we can extract information about the dimensions and local viscosity of the ECS. We find a striking diversity of ECS dimensions down to 40 nm, and as well as of local viscosity values. Moreover, by chemically altering the extracellular matrix of the brains of live animals before nanotube injection, we reveal that the rheological properties of the ECS are affected, but these alterations are local and inhomogeneous at the nanoscale.

  16. Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain.

    PubMed

    Godin, Antoine G; Varela, Juan A; Gao, Zhenghong; Danné, Noémie; Dupuis, Julien P; Lounis, Brahim; Groc, Laurent; Cognet, Laurent

    2017-03-01

    The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its volume. Signalling molecules, neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep, development and aging and is probably altered in neuropsychiatric and degenerative diseases, as inferred from electron microscopy and macroscopic biophysical investigations. Here we show an approach to directly observe the local ECS structures and rheology in brain tissue using super-resolution imaging. We inject single-walled carbon nanotubes into rat cerebroventricles and follow the near-infrared emission of individual nanotubes as they diffuse inside the ECS for tens of minutes in acute slices. Because of the interplay between the nanotube geometry and the ECS local environment, we can extract information about the dimensions and local viscosity of the ECS. We find a striking diversity of ECS dimensions down to 40 nm, and as well as of local viscosity values. Moreover, by chemically altering the extracellular matrix of the brains of live animals before nanotube injection, we reveal that the rheological properties of the ECS are affected, but these alterations are local and inhomogeneous at the nanoscale.

  17. Genome Sequences of Polyomaviruses from the Wild-Living Red Colobus (Piliocolobus badius) and Western Chimpanzee (Pan troglodytes verus).

    PubMed

    Ben Salem, Nicole; Leendertz, Fabian H; Ehlers, Bernhard

    2016-10-13

    We identified with PCR and sequencing the full genomes of the recently discovered Pan troglodytes verus polyomavirus 8 and Piliocolobus badius polyomavirus 2 in a western chimpanzee and a western red colobus free-ranging in Taï National Park of Côte d'Ivoire.

  18. FORMING SELF-ASSEMBLED CELL ARRAYS AND MEASURING THE OXYGEN CONSUMPTION RATE OF A SINGLE LIVE CELL.

    PubMed

    Etzkorn, James R; McQuaide, Sarah C; Anderson, Judy B; Meldrum, Deirdre R; Parviz, Babak A

    2009-06-01

    We report a method for forming arrays of live single cells on a chip using polymer micro-traps made of SU8. We have studied the toxicity of the microfabricated structures and the associated environment for two cell lines. We also report a method for measuring the oxygen consumption rate of a single cell using optical interrogation of molecular oxygen sensors placed in micromachined micro-wells by temporarily sealing the cells in the micro-traps. The new techniques presented here add to the collection of tools available for performing "single-cell" biology. A single-cell self-assembly yield of 61% was achieved with oxygen draw down rates of 0.83, 0.82, and 0.71 fmol/minute on three isolated live A549 cells.

  19. Duration of immunity in red wolves (Canis rufus) following vaccination with a modified live parvovirus and canine distemper vaccine.

    PubMed

    Anderson, Kadie; Case, Allison; Woodie, Kathleen; Waddell, William; Reed, Holly H

    2014-09-01

    There is growing information available regarding duration of immunity for core vaccines in both domestic and nondomestic species. Vaccination protocols in nondomestic canids have frequently followed guidelines developed for the domestic dog; however, these protocols can be inappropriate for nondomestic canids such as the African wild dog (Lycaon pictus), leaving some animals susceptible to infectious disease and others at risk for contracting vaccine-induced disease. In this study, red wolves (Canis rufus) were vaccinated against canine distemper virus (CDV) and canine parvovirus (CPV) and vaccination titers were followed annually for 3 yr. One hundred percent of wolves developed and maintained a positive titer to CDV for 3 yr and 96.9% of wolves developed and maintained a positive titer to CPV for 3 yr. Seroconversion for canine adenovirus was sporadic. The results of this study support decreasing the frequency of vaccine administration in the red wolf population to a triennial basis.

  20. Biocompatible photoresistant far-red emitting, fluorescent polymer probes, with near-infrared two-photon absorption, for living cell and zebrafish embryo imaging.

    PubMed

    Adjili, Salim; Favier, Arnaud; Fargier, Guillaume; Thomas, Audrey; Massin, Julien; Monier, Karine; Favard, Cyril; Vanbelle, Christophe; Bruneau, Sylvia; Peyriéras, Nadine; Andraud, Chantal; Muriaux, Delphine; Charreyre, Marie-Thérèse

    2015-04-01

    Exogenous probes with far-red or near-infrared (NIR) two-photon absorption and fluorescence emission are highly desirable for deep tissue imaging while limiting autofluorescence. However, molecular probes exhibiting such properties are often hydrophobic. As an attractive alternative, we synthesized water-soluble polymer probes carrying multiple far-red fluorophores and demonstrated here their potential for live cell and zebrafish embryo imaging. First, at concentrations up to 10 μm, these polymer probes were not cytotoxic. They could efficiently label living HeLa cells, T lymphocytes and neurons at an optimal concentration of 0.5 μm. Moreover, they exhibited a high resistance to photobleaching in usual microscopy conditions. In addition, these polymer probes could be successfully used for in toto labeling and in vivo two-photon microscopy imaging of developing zebrafish embryos, with remarkable properties in terms of biocompatibility, internalization, diffusion, stability and wavelength emission range. The near-infrared two-photon absorption peak at 910 nm is particularly interesting since it does not excite the zebrafish endogenous fluorescence and is likely to enable long-term time-lapse imaging with limited photodamage.

  1. Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP).

    PubMed

    Yang, Sunny Y; Amor, Souheila; Laguerre, Aurélien; Wong, Judy M Y; Monchaud, David

    2016-12-10

    The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

    PubMed

    Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

    2014-12-18

    Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.

  9. Theoretical model for optical oximetry at the capillary level: exploring hemoglobin oxygen saturation through backscattering of single red blood cells

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

    Oxygen saturation (sO2) of red blood cells (RBCs) in capillaries can indirectly assess local tissue oxygenation and metabolic function. For example, the altered retinal oxygenation in diabetic retinopathy and local hypoxia during tumor development in cancer are reflected by abnormal sO2 of local capillary networks. However, it is far from clear whether accurate label-free optical oximetry (i.e., measuring hemoglobin sO2) is feasible from dispersed RBCs at the single capillary level. The sO2-dependent hemoglobin absorption contrast present in optical scattering signal is complicated by geometry-dependent scattering from RBCs. We present a numerical study of backscattering spectra from single RBCs based on the first-order Born approximation, considering practical factors: RBC orientations, size variation, and deformations. We show that the oscillatory spectral behavior of RBC geometries is smoothed by variations in cell size and orientation, resulting in clear sO2-dependent spectral contrast. In addition, this spectral contrast persists with different mean cellular hemoglobin content and different deformations of RBCs. This study shows for the first time the feasibility of, and provides a theoretical model for, label-free optical oximetry at the single capillary level using backscattering-based imaging modalities, challenging the popular view that such measurements are impossible at the single capillary level.

  10. Combining red and blue-detuned optical potentials to form a Lamb-Dicke trap for a single neutral atom.

    PubMed

    He, Xiaodong; Yu, Shi; Xu, Peng; Wang, Jin; Zhan, Mingsheng

    2012-02-13

    We propose and demonstrate a scheme for strong radial confinement of a single 87 Rb atom by a bichromatic far-off resonance optical dipole trap (BFORT). The BFORT is composed of a blue-detuned Laguerre-Gaussian LG01 beam and a red-detuned Gaussian beam. The atomic oscillation frequency measurement shows that the effective trapping dimension is much sharper than that from a diffraction-limited microscopic objective. Theory shows that the added scattering rate due to imposing blue-detuned light is negligible when the temperature of the single atoms is close to ground state temperature. By carrying out sub-Doppler cooling, the mean energy of single atoms trapped in the BFORT is reduced to 15 ± 1 μK. The corresponding mean quantum number of radial vibration n is about 1.65, which satisfies the Lamb-Dicke regime. We conclude that the BFORT is a suitable Lamb-Dicke trap for further cooling a single neutral atom down to the ground state and for further application in quantum information processing.

  11. 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

  12. 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

  13. Barriers to live donor kidney transplants in the pediatric population: A single-center experience.

    PubMed

    Taormina, Shibany P; Galloway, Matthew P; Jain, Amrish

    2017-03-01

    A decrease in live donor pediatric kidney transplants has occurred in the United States. This study investigates barriers that may influence access to live donor kidney transplants in children. Retrospective chart review was conducted for 91 children (69% male, mean age 11.9 years) who underwent pretransplant workup from 2005 to 2015 at an urban pediatric hospital. Fifty-four percent were African American, 32% Caucasian, 8% Arabic, 3% Hispanic, and 3% Others. Government-sponsored insurance (Medicaid/Medicare) was utilized by 73%, and 54% had dual caregivers. Only nine of 68 kidney transplants were live donor transplants. Live donor transplants (11%) were significantly (P=.008) lower than deceased donor transplants (59%) in African Americans. Private insurance was reported by 56% of live donor recipients and 25% of deceased donor recipients. Among live donor recipients, 78% were from dual caregiver families. Caregiver, health-related, financial, and religious/cultural barriers to live donor transplants were reported, several of which may be amenable to positive intervention.

  14. Red light, green light: probing single molecules using alternating-laser excitation.

    PubMed

    Santoso, Yusdi; Hwang, Ling Chin; Le Reste, Ludovic; Kapanidis, Achillefs N

    2008-08-01

    Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein-nucleic acid interactions.

  15. Pharmacokinetics of a single dose of voriconazole administered orally with and without food to red-tailed hawks (Buteo jamaicensus).

    PubMed

    Parsley, Ruth A; Tell, Lisa A; Gehring, Ronette

    2017-04-01

    OBJECTIVE To determine the pharmacokinetics of voriconazole administered PO with or without food to red-tailed hawks (Buteo jamaicensus) and whether any observed variability could be explained by measured covariates to inform dose adjustments. ANIMALS 7 adult red-tailed hawks. PROCEDURES In a crossover study design, hawks were randomly assigned to first receive voriconazole (15 mg/kg, PO) injected into a dead mouse (n = 3; fed birds) or without food (4; unfed birds). Sixteen days later, treatments were reversed. Blood samples were collected at various points to measure plasma voriconazole concentrations by ultraperformance liquid chromatography. Pharmacokinetic data were analyzed by noncompartmental methods and fit to a compartmental model through nonlinear mixed-effects regression, with feeding status and body weight investigated as covariates. RESULTS Voriconazole was well absorbed, with quantifiable plasma concentrations up to 24 hours after administration. Mean plasma half-life was approximately 2 hours in fed and unfed birds. Administration of the voriconazole in food delayed absorption, resulting in a significant delay in time to maximum plasma concentration. The final compartmental model included a categorical covariate to account for this lag in absorption as well as body weight as a covariate of total body clearance (relative to unknown bioavailability). CONCLUSIONS AND CLINICAL RELEVANCE A single dose of voriconazole (15 mg/kg) administered PO to red-tailed hawks resulted in mean plasma voriconazole concentrations greater than the targeted value (1 μg/mL). Additional studies with larger sample sizes and multidose regimens are required before the model developed here can be applied in clinical settings.

  16. Single-Beam Acoustic Trapping of Red Blood Cells and Polystyrene Microspheres in Flowing Red Blood Cell Saline and Plasma Suspensions.

    PubMed

    Liu, Hsiao-Chuan; Li, Ying; Chen, Ruimin; Jung, Hayong; Shung, K Kirk

    2017-04-01

    Single-beam acoustic tweezers (SBATs) represent a new technology for particle and cell trapping. The advantages of SBATs are their deep penetration into tissues, reduction of tissue damage and ease of application to in vivo studies. The use of these tools for applications in drug delivery in vivo must meet the following conditions: large penetration depth, strong trapping force and tissue safety. A reasonable penetration depth for SBATs in the development of in vivo applications was established in a previous study conducted in water with zero velocity. However, capturing objects in flowing fluid can provide more meaningful results. In this study, we investigated the capability of SBATs to trap red blood cells (RBCs) and polystyrene microspheres in flowing RBC suspensions. Two different types of RBC suspension were prepared in this work: an RBC phosphate-buffered saline (PBS) suspension and an RBC plasma suspension. The results indicated that SBATs successfully trapped RBCs and polystyrene microspheres in a flowing RBC PBS suspension with an average steady velocity of 1.6 cm/s in a 2-mm-diameter polyimide. Furthermore, SBATs were found able to trap RBCs in a flowing RBC PBS suspension at speeds as high as 7.9 cm/s in a polyimide tube, which is higher than the velocity in capillaries (0.03 cm/s) and approaches the velocity in arterioles and venules. Moreover, the results also indicated that polystyrene microspheres can be trapped in an RBC plasma suspension, where aggregation is observed. This work represents a step forward in using this tool in actual in vivo experimentation.

  17. Faecal egg counts provide a reliable measure of Trichostrongylus tenuis intensities in free-living red grouse Lagopus lagopus scoticus.

    PubMed

    Seivwright, L J; Redpath, S M; Mougeot, F; Watt, L; Hudson, P J

    2004-03-01

    The reliability of different egg counting methods for estimating the intensity of Trichostrongylus tenuis infections in red grouse, Lagopus lagopus scoticus, was investigated in the autumn, when grouse may harbour high parasite intensities. Possible limitations to the use of these methods were also examined. Faecal egg counts were found to accurately estimate T. tenuis worm intensities, at least up to an observed maximum of c. 8000 worms. Two egg counting methods (smear and McMaster) gave consistent results, although the exact relationship with worm intensity differed according to the method used. Faecal egg counts significantly decreased with increasing length of sample storage time, but egg counts were reliable for estimating worm intensity for three weeks. The concentration of eggs in the caecum was also found to reliably estimate worm intensity. However, egg counts from frozen gut samples cannot be used to estimate worm intensities. These results conclude that, despite some limitations, faecal and caecum egg counts provide useful and reliable ways of measuring T. tenuis intensities in red grouse.

  18. Costs of achieving live birth from assisted reproductive technology: a comparison of sequential single and double embryo transfer approaches

    PubMed Central

    Crawford, Sara; Boulet, Sheree L.; Mneimneh, Allison S.; Perkins, Kiran M.; Jamieson, Denise J.; Zhang, Yujia; Kissin, Dmitry M.

    2016-01-01

    Objective To assess treatment and pregnancy/infant-associated medical costs and birth outcomes for assisted reproductive technology (ART) cycles in a subset of patients using elective double embryo (ET) and to project the difference in costs and outcomes had the cycles instead been sequential single ETs (fresh followed by frozen if the fresh ET did not result in live birth). Design Retrospective cohort study using 2012 and 2013 data from the National ART Surveillance System. Setting Infertility treatment centers. Patient(s) Fresh, autologous double ETs performed in 2012 among ART patients younger than 35 years of age with no prior ART use who cryopreserved at least one embryo. Intervention(s) Sequential single and double ETs. Main Outcome Measure(s) Actual live birth rates and estimated ART treatment and pregnancy/infant-associated medical costs for double ET cycles started in 2012 and projected ART treatment and pregnancy/infant-associated medical costs if the double ET cycles had been performed as sequential single ETs. Result(s) The estimated total ART treatment and pregnancy/infant-associated medical costs were $580.9 million for 10,001 double ETs started in 2012. If performed as sequential single ETs, estimated costs would have decreased by $195.0 million to $386.0 million, and live birth rates would have increased from 57.7%–68.0%. Conclusion(s) Sequential single ETs, when clinically appropriate, can reduce total ART treatment and pregnancy/infant-associated medical costs by reducing multiple births without lowering live birth rates. PMID:26604068

  19. 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

  20. 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.

  1. 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.

  2. Evaluation of naked eye single tube red cell osmotic fragility test in detecting beta-thalassemia trait.

    PubMed

    Raghavan, K; Lokeshwar, M R; Birewar, N; Nigam, V; Manglani, M V; Raju, N B

    1991-05-01

    The Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) was applied to 4 groups of subjects: (i) Normal; (ii) Proven beta-thalassemia trait carriers; (iii) Iron deficiency anemia; and (iv) other hemoglobinopathies, to evaluate its effectiveness as a screening test for beta-thalassemia minor. The test was successful in detecting 105/110 subjects with beta-thalassemia trait. The sensitivity of the test was 95.5% and specificity was 87%. The predictive value of the positive test was 70.5% and that of the negative test was 98.3%. NESTROFT was also positive in 9/17 subjects with HbS trait, in 3/3 subjects with HbD trait and in 1/1 subjects with HbE trait. The test proved to be simple, cheap, easy to perform and adaptable for field surveys, coming close to an ideal screening test for beta-thalassemia minor.

  3. 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.

  4. Studying single red blood cells under a tunable external force by combining passive microrheology with Raman spectroscopy.

    PubMed

    Raj, Saurabh; Wojdyla, Michal; Petrov, Dmitri

    2013-04-01

    The dynamic micromechanical and structural properties of single human red blood cells are studied using a combination of dual trap optical tweezers and confocal Raman spectroscopy. Such a combination permits us to show a direct relationship between the rheological properties and chemical structure conformation. The frequency dependence of the complex stiffness of the cells was measured using both one and two probe response functions under identical experimental conditions. Both the microrheology and Raman measurements were performed at different stretching forces applied to the cell. A detailed analysis of the auto- and cross-correlated probe motions allows exploring the local and overall viscoelastic properties of the cells over a controlled range of the deformations. The observed growth of the cell viscoelasticity with stretching was associated with structural changes in the cell membrane monitored via the Raman spectroscopy.

  5. Novel single-cell functional analysis of red blood cells using laser tweezers Raman spectroscopy: application for sickle cell disease.

    PubMed

    Liu, Rui; Mao, Ziliang; Matthews, Dennis L; Li, Chin-Shang; Chan, James W; Satake, Noriko

    2013-07-01

    Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs.

  6. 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.

  7. Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

    PubMed Central

    Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

    2012-01-01

    There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

  8. Mechanical Trap Surface-Enhanced Raman Spectroscopy for Three-Dimensional Surface Molecular Imaging of Single Live Cells.

    PubMed

    Jin, Qianru; Li, Ming; Polat, Beril; Paidi, Santosh K; Dai, Aimee; Zhang, Amy; Pagaduan, Jayson V; Barman, Ishan; Gracias, David H

    2017-03-27

    Reported is a new shell-based spectroscopic platform, named mechanical trap surface-enhanced Raman spectroscopy (MTSERS), for simultaneous capture, profiling, and 3D microscopic mapping of the intrinsic molecular signatures on the membrane of single live cells. By leveraging the functionalization of the inner surfaces of the MTs with plasmonic gold nanostars, and conformal contact of the cell membrane, MTSERS permits excellent signal enhancement, reliably detects molecular signatures, and allows non-perturbative, multiplex 3D surface imaging of analytes, such as lipids and proteins on the surface of single cells. The demonstrated ability underscores the potential of MTSERS to perform 3D spectroscopic microimaging and to furnish biologically interpretable, quantitative, and dynamic molecular maps in live cell populations.

  9. Markers of Decompression Stress of Mass Stranded/Live Caught and Released vs. Single Stranded Marine Mammals

    DTIC Science & Technology

    2014-09-30

    last dive. Aims 2 and 3: We have sampled and analyzed single and mass stranded dolphins (Aim 2) and live- restrained dolphins (Aim 3) for MPs. We...data. Aim 3: In the third year, blood samples from wild-caught dolphins in Sarasota were collected and analyzed for MPs concentrations for Pre...caught dolphins in Sarasota, Florida, were analyzed for MPs. The samples were obtained Pre-, Mid-, and Post-procedure. Whilst earlier analysis of

  10. Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

    PubMed Central

    Haas, Beth L.; Matson, Jyl S.; DiRita, Victor J.; Biteen, Julie S.

    2015-01-01

    Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane. PMID:25123183

  11. Exploring transduction mechanisms of protein transduction domains (PTDs) in living cells utilizing single-quantum dot tracking (SQT) technology.

    PubMed

    Suzuki, Yasuhiro

    2012-01-01

    Specific protein domains known as protein transduction domains (PTDs) can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs), we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP) in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT), to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.

  12. 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.

  13. In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria

    PubMed Central

    Plochowietz, Anne; Farrell, Ian; Smilansky, Zeev; Cooperman, Barry S.; Kapanidis, Achillefs N.

    2017-01-01

    Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of ∼8 μm2/s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes. PMID:27625389

  14. 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.

  15. Impact of Donor Source on the Outcome of Live Donor Kidney Transplantation: A Single Center Experience

    PubMed Central

    Matter, Yasser Elsayed; Nagib, Ayman M; Lotfy, Omar E; Alsayed, Ahmed Maher; Donia, Ahmed F; Refaie, Ayman F; Akl, Ahmed I; Abbas, Mohamed Hamed; Abuelmagd, Mohammed M; Shaeashaa, Hussein A; Shokeir, Ahmed A

    2016-01-01

    Background Renal transplantation is the ideal method for management of end-stage renal disease. The use of living donors for renal transplantation was critical for early development in the field and preceded the use of cadaveric donors. Most donors are related genetically to the recipients, like a parent, a child, or a sibling of the recipient, but there are an increasing percentage of cases where donors are genetically unrelated like spouses, friends, or altruistic individuals. Donor shortages constitute the major barrier for kidney transplantation, and much effort has been made to increase the supply of living donors. The impact of donor source on the outcome of renal transplantation is not adequately studied in our country. Objectives The aim of the study was to evaluate the impact of donor source on the outcome of live donor kidney transplantation. Patients and Methods From March 1976 to December 2013, the number of patients that underwent living renal transplantation sharing at least one HLA haplotype with their donors was 2,485. We divided these patients into two groups: (1) 2,075 kidney transplant recipients (1,554 or 74.9% male and 521 or 25.1% female) for whom the donors were living related, (2) 410 kidney transplant recipients (297 or 72.4% male and 113 or 27.6% female) for whom the donors were living unrelated. All patients received immunosuppressive therapy, consisting of a calcineurin inhibitor, mycophenolate mofetil, or azathioprine and prednisolone. We compared acute rejection and complication rates, as well as long-term graft and patient survival of both groups. Demographic characteristics were compared using the chi-square test. Graft survival and patient survival were calculated using the Kaplan-Meier method. Results The percentages of patients with acute vascular rejection were significantly higher in the unrelated group, while percentages of patients with no rejection were significantly higher in the related group, but there were no significant

  16. Compact high-power red-green-blue laser light source generation from a single lithium tantalate with cascaded domain modulation.

    PubMed

    Xu, P; Zhao, L N; Lv, X J; Lu, J; Yuan, Y; Zhao, G; Zhu, S N

    2009-06-08

    1W quasi-white-light source has been generated from a single lithium tantalate with cascaded domain modulation. The quasi-white-light is combined by proper proportion of the red, green and blue laser light. The red and the blue result from a compact self-sum frequency optical parametric oscillation when pumped by a single green laser. The efficiency of quasi-white-light from the green pump reaches 27%. This compact design can be employed not only as a stable and powerful RGB light source but also an effective blue laser generator.

  17. 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

  18. Growth and development of Arabidopsis thaliana under single-wavelength red and blue laser light

    PubMed Central

    Ooi, Amanda; Wong, Aloysius; Ng, Tien Khee; Marondedze, Claudius; Gehring, Christoph; Ooi, Boon S.

    2016-01-01

    Indoor horticulture offers a sensible solution for sustainable food production and is becoming increasingly widespread. However, it incurs high energy and cost due to the use of artificial lighting such as high-pressure sodium lamps, fluorescent light or increasingly, the light-emitting diodes (LEDs). The energy efficiency and light quality of currently available horticultural lighting is suboptimal, and therefore less than ideal for sustainable and cost-effective large-scale plant production. Here, we demonstrate the use of high-powered single-wavelength lasers for indoor horticulture. They are highly energy-efficient and can be remotely guided to the site of plant growth, thus reducing on-site heat accumulation. Furthermore, laser beams can be tailored to match the absorption profiles of different plant species. We have developed a prototype laser growth chamber and demonstrate that plants grown under laser illumination can complete a full growth cycle from seed to seed with phenotypes resembling those of plants grown under LEDs reported previously. Importantly, the plants have lower expression of proteins diagnostic for light and radiation stress. The phenotypical, biochemical and proteome data show that the single-wavelength laser light is suitable for plant growth and therefore, potentially able to unlock the advantages of this next generation lighting technology for highly energy-efficient horticulture. PMID:27659906

  19. Growth and development of Arabidopsis thaliana under single-wavelength red and blue laser light.

    PubMed

    Ooi, Amanda; Wong, Aloysius; Ng, Tien Khee; Marondedze, Claudius; Gehring, Christoph; Ooi, Boon S

    2016-09-23

    Indoor horticulture offers a sensible solution for sustainable food production and is becoming increasingly widespread. However, it incurs high energy and cost due to the use of artificial lighting such as high-pressure sodium lamps, fluorescent light or increasingly, the light-emitting diodes (LEDs). The energy efficiency and light quality of currently available horticultural lighting is suboptimal, and therefore less than ideal for sustainable and cost-effective large-scale plant production. Here, we demonstrate the use of high-powered single-wavelength lasers for indoor horticulture. They are highly energy-efficient and can be remotely guided to the site of plant growth, thus reducing on-site heat accumulation. Furthermore, laser beams can be tailored to match the absorption profiles of different plant species. We have developed a prototype laser growth chamber and demonstrate that plants grown under laser illumination can complete a full growth cycle from seed to seed with phenotypes resembling those of plants grown under LEDs reported previously. Importantly, the plants have lower expression of proteins diagnostic for light and radiation stress. The phenotypical, biochemical and proteome data show that the single-wavelength laser light is suitable for plant growth and therefore, potentially able to unlock the advantages of this next generation lighting technology for highly energy-efficient horticulture.

  20. Surface Shear Stress Around a Single Flexible Live Plant and a Rigid Cylinder

    NASA Astrophysics Data System (ADS)

    Walter, B. A.; Gromke, C.; Leonard, K. C.; Clifton, A.; Lehning, M.

    2010-12-01

    The sheltering effect of vegetation against soil erosion and snow transport has direct implications on land degradation and local water storage as snow in many arid and semi arid regions. Plants influence the erosion, transport and redeposition of soil and snow by the wind through momentum absorption, local stress concentration, trapping particles in motion and reducing the area of sediment exposed to the wind. The shear stress distributions on the ground beneath plant canopies determine the onset and magnitude of differential soil and snow erosion on rough or vegetated surfaces, but this has been studied exclusively with artificial and rigid vegetation elements thus far. Real plants have highly irregular structures that can be extremely flexible and porous. They align with the flow at higher wind speeds, resulting in considerable changes to the drag and flow regimes relative to rigid imitations of comparable size. We present measurements in the SLF atmospheric boundary layer wind tunnel of the surface shear stress distribution around a live grass plant (Lolium Perenne) and a solid cylinder of comparable size. Irwin sensors are used to measure pressure differences close to the surface which can be calibrated with surface shear stress velocities. The basal to frontal area index of the plant and the cylinder as well as the Reynolds number of the two experimental setups have been checked for similarity and show good agreement. Distinctive differences between the shear stress pattern around the plant and the cylinder can be attributed to the influence of the plant’s porosity and flexibility. The sheltered zone behind the plant is narrower in cross-stream and longer in streamwise direction than that of the cylinder. For the plant, the lowest shear stresses in the sheltered zone are 50% lower than the mean surface shear stress (τ = 0.15 N/m2) in the undisturbed flow. The sheltering was higher behind the cylinder with values reduced by 70% relative to background.

  1. Is life span extension in single gene long-lived Caenorhabditis elegans mutants due to hypometabolism?

    PubMed

    Van Voorhies, Wayne A

    2003-06-01

    The nematode C. elegans is widely used in aging research largely because of the identification of numerous gene mutations that significantly increase worm longevity. While model organisms such as C. elegans can provide important insights into aging it is also important to consider the limitations of these systems. For example, ectothermic (poikilothermic) organisms are able to tolerate a much larger metabolic depression than humans and considering only chronological longevity when assaying for long-lived mutants provides a limited perspective on the mechanisms by which longevity is increased. In order to provide true insight into the aging process additional physiological processes, such as metabolic rate, must also be assayed. Currently it is controversial when long-lived C. elegans mutants retain normal metabolic function. Resolving this issue requires accurately measuring the metabolic rate of C. elegans under conditions that minimize environmental stress. Comparisons of metabolic rate between long-lived and wild-type C. elegans under more optimized conditions indicate that the extended longevity of at least some long-lived C. elegans mutants may be due to a reduction in metabolic rate, rather than an alteration of a metabolically-independent genetic mechanism specific to aging. Consistent with this assertion are studies showing that the disruption of mitochondrial function in C. elegans can extend worm's longevity, but typically causes worms to grow and develop more slowly than wild-type animals.

  2. Al3+-induced far-red fluorescence enhancement of conjugated polymer nanoparticles and its application in live cell imaging

    NASA Astrophysics Data System (ADS)

    LiuH. Liu, X. Hao, C. H. Duan,; H. Yang Contributed Equally To This Work., Heng; Hao, Xian; Duan, Chunhui; Yang, Hui; Lv, Yi; Xu, Haijiao; Wang, Hongda; Huang, Fei; Xiao, Debao; Tian, Zhiyuan

    2013-09-01

    Fluorescent nanoparticles (NPs) for Al3+ sensing with high selectivity were developed from a type of carbazole-based conjugated polymer with a two-dimensional donor-π bridge-acceptor (D-π-A) structure. These NPs are characterized by their small particle diameter (~18 nm), far-red fluorescence emission (centered ~710 nm), and Al3+-induced fluorescence enhancement with high selectivity owing to an Al3+-triggered inhibition on the intramolecular charge transfer (ICT) processes between the conjugated backbone and the pendant acceptors. This type of nanoparticle is easily suspended in aqueous solutions, indicating their practical applicability in physiological media, and their ability for intracellular Al3+ sensing was confirmed. As compared to other types of conjugated polymer based probes showing metal ion mediated fluorescence quenching, these as-prepared NPs possess analyte-enhanced fluorescence emission, which is analytically favored in terms of sensitivity and selectivity. Fluorescence emission with wavelengths in the biological window of maximum optical transparency (~700 to 1000 nm) is expected to impart a salient advantage for biological detection applications to these as-prepared probes. The superior features of merit of this new type of fluorescent probe, together with the validation of practicability for intracellular Al3+ ion sensing, are indicative of their potential for application in fluorescence-based imaging and sensing, such as investigations on Al3+-related physiological and pathological processes.Fluorescent nanoparticles (NPs) for Al3+ sensing with high selectivity were developed from a type of carbazole-based conjugated polymer with a two-dimensional donor-π bridge-acceptor (D-π-A) structure. These NPs are characterized by their small particle diameter (~18 nm), far-red fluorescence emission (centered ~710 nm), and Al3+-induced fluorescence enhancement with high selectivity owing to an Al3+-triggered inhibition on the intramolecular charge

  3. 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.

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

    PubMed

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

    2016-05-05

    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.

  5. 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.

  6. 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

    2015-02-11

    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.

  7. 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

  8. Real-time Imaging and Tuning Subcellular Structures and Membrane Transport Kinetics of Single Live Cells at Nanosecond Regime

    PubMed Central

    Xu, Hongwu; Nallathamby, Prakash D.; Xu, Xiao-Hong Nancy

    2009-01-01

    We developed an electric-field exposure microchannel system with 230-nanometer thin-layer gold electrodes, and interfaced it with a single living cell imaging station and a 10-nanosecond-electric-pulse (10nsEP) generator. This design allows us to image intracellular molecules and structures, membrane transport and viability of single leukemic cells (HL60) while the cells are exposed to 10nsEPs of 0–179 kV/cm, permitting the study of subcellular responses at nanosecond regime. The electrodes confine a thin-layer section of the cells exposed to 10nsEPs, offering unprecedented high spatial resolution (230-nm at z-direction of E and imaging plane) for imaging intracellular molecules of single cells affected by 10nsEPs. We found that nucleic acids, membrane transport rates and viability of single cells depend on the number and electric-field-strength (E) of 10nsEPs, showing the cumulative effect of 10nsEPs on intracellular molecules and structures and suggesting the possibility of tuning them one-pulse-at-a-time. Using lower E (51 kV/cm) of 10nsEPs, we could manipulate nucleic acids of single living cells without disrupting their cellular membrane and viability. As E increases to 80, 124 and 179 kV/cm, membrane integrity and viability of cells exhibit higher dependence on the number of 10nsEPs in a non-linear fashion, showing that critical E and pulse number are needed to surmount cellular transport barriers and membrane integrity. PMID:19795898

  9. Direct observation of a long-lived single-atom catalyst chiseling atomic structures in graphene.

    PubMed

    Wang, Wei Li; Santos, Elton J G; Jiang, Bin; Cubuk, Ekin Dogus; Ophus, Colin; Centeno, Alba; Pesquera, Amaia; Zurutuza, Amaia; Ciston, Jim; Westervelt, Robert; Kaxiras, Efthimios

    2014-02-12

    Fabricating stable functional devices at the atomic scale is an ultimate goal of nanotechnology. In biological processes, such high-precision operations are accomplished by enzymes. A counterpart molecular catalyst that binds to a solid-state substrate would be highly desirable. Here, we report the direct observation of single Si adatoms catalyzing the dissociation of carbon atoms from graphene in an aberration-corrected high-resolution transmission electron microscope (HRTEM). The single Si atom provides a catalytic wedge for energetic electrons to chisel off the graphene lattice, atom by atom, while the Si atom itself is not consumed. The products of the chiseling process are atomic-scale features including graphene pores and clean edges. Our experimental observations and first-principles calculations demonstrated the dynamics, stability, and selectivity of such a single-atom chisel, which opens up the possibility of fabricating certain stable molecular devices by precise modification of materials at the atomic scale.

  10. 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.

  11. 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.

  12. Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains.

    PubMed

    Mücksch, Jonas; Spielmann, Thiemo; Sisamakis, Evangelos; Widengren, Jerker

    2015-05-01

    We demonstrate the applicability of Single Plane Illumination Microscopy to Transient State Imaging (TRAST), offering sensitive microenvironmental information together with optical sectioning and reduced overall excitation light exposure of the specimen. The concept is verified by showing that transition rates can be determined accurately for free dye in solution and that fluorophore transition rates can be resolved pixel-wise in live cells. Furthermore, we derive a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging.

  13. Adult Living Donor Liver Transplantation with ABO-Incompatible Grafts: A German Single Center Experience

    PubMed Central

    Goralczyk, Armin D.; Obed, Aiman; Schnitzbauer, Andreas; Doenecke, Axel; Tsui, Tung Yu; Scherer, Marcus N.; Ramadori, Giuliano; Lorf, Thomas

    2009-01-01

    Adult living donor liver transplantations (ALDLTs) across the ABO blood group barrier have been reported in Asia, North Americas, and Europe, but not yet in Germany. Several strategies have been established to overcome the detrimental effects that are attached with such a disparity between donor and host, but no gold standard has yet emerged. Here, we present the first experiences with three ABO-incompatible adult living donor liver transplantations in Germany applying different immunosuppressive strategies. Four patient-donor couples were considered for ABO-incompatible ALDLT. In these patients, resident ABO blood group antibodies (isoagglutinins) were depleted by plasmapheresis or immunoadsorption and replenishment was inhibited by splenectomy and/or B-cell-targeted immunosuppression. Despite different treatments ALDLT could safely be performed in three patients and all patients had good initial graft function without signs for antibody-mediated rejection (AMR). Two patients had long-term graft survival with stable graft function. We thus propose the feasibility of ABO-incompatible ALDLT with these protocols and advocate further expansion of ABO incompatible ALDLT in multicenter trials to improve efficacy and safety. PMID:20148072

  14. Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.

    PubMed

    Hayashi-Takanaka, Yoko; Yamagata, Kazuo; Wakayama, Teruhiko; Stasevich, Timothy J; Kainuma, Takashi; Tsurimoto, Toshiki; Tachibana, Makoto; Shinkai, Yoichi; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2011-08-01

    Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

  15. 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.

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

    PubMed

    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 (k{b}), the maximum velocity of fluid flow (u{max}), 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 Y{d} 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.

  17. 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.

  18. Characteristics of Living and Deceased Suicidal Military Personnel Based on Single Versus Multiple Suicide Attempt Status

    DTIC Science & Technology

    2012-03-27

    Suicide Prevention and Significance of Tertiary Prevention Efforts There are three stages of prevention in regard to illness and injury (Green...Finally, tertiary prevention is treatment to prevent the recurrence of illness or injury (Green & MILITARY SINGLE VERSUS MULTIPLE SUICIDE ATTEMPTS 3...Kreuter, 2005). Tertiary prevention is the aim of the present study as its purpose is to investigate the factors that are

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Energy, water and space use by free-living red kangaroos Macropus rufus and domestic sheep Ovis aries in an Australian rangeland.

    PubMed

    Munn, A J; Dawson, T J; McLeod, S R; Dennis, T; Maloney, S K

    2013-08-01

    We used doubly labelled water to measure field metabolic rates (FMR) and water turnover rates (WTR) in one of Australia's largest native herbivores, the red kangaroo (Macropus rufus) and one of Australia's dominant livestock species, the wool-breed Merino sheep, under free-living conditions in a typical Australian rangeland. Also, we used GPS technology to examine animal space use, along with the comparisons of urine concentration, diet, diet digestibility, and subsequent grazing pressures. We found smaller space-use patterns than previously reported for kangaroos, which were between 14 and 25 % those of sheep. The FMR of a 25-kg kangaroo was 30 % that of a 45-kg sheep, while WTR was 15 % and both were associated with smaller travel distances, lower salt intakes, and higher urine concentration in kangaroos than sheep. After accounting for differences in dry matter digestibility of food eaten by kangaroos (51 %) and sheep (58 %), the relative grazing pressure of a standard (mature, non-reproductive) 25-kg kangaroo was 35 % that of a 45-kg sheep. Even for animals of the same body mass (35 kg), the relative grazing pressure of the kangaroo was estimated to be only 44 % that of the sheep. After accounting for the energetic costs of wool growth by sheep, the FMRs of our sheep and kangaroos were 2-3 times their expected BMRs, which is typical for mammalian FMR:BMRs generally. Notably, data collected from our free-living animals were practically identical to those from animals confined to a semi-natural enclosure (collected in an earlier study under comparable environmental conditions), supporting the idea that FMRs are relatively constrained within species.

  5. 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.

  6. 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.

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

    PubMed

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

    2014-07-29

    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.

  8. 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

  9. Single-unit analysis of the human posterior hypothalamus and red nucleus during deep brain stimulation for aggressivity.

    PubMed

    Micieli, Robert; Rios, Adriana Lucia Lopez; Aguilar, Ricardo Plata; Posada, Luis Fernando Botero; Hutchison, William D

    2017-04-01

    OBJECTIVE Deep brain stimulation (DBS) of the posterior hypothalamus (PH) has been reported to be effective for aggressive behavior in a number of isolated cases. Few of these case studies have analyzed single-unit recordings in the human PH and none have quantitatively analyzed single units in the red nucleus (RN). The authors report on the properties of ongoing neuronal discharges in bilateral trajectories targeting the PH and the effectiveness of DBS of the PH as a treatment for aggressive behavior. METHODS DBS electrodes were surgically implanted in the PH of 1 awake patient with Sotos syndrome and 3 other anesthetized patients with treatment-resistant aggressivity. Intraoperative extracellular recordings were obtained from the ventral thalamus, PH, and RN and analyzed offline to discriminate single units and measure firing rates and firing patterns. Target location was based on the stereotactic coordinates used by Sano et al. in their 1970 study and the location of the dorsal border of the RN. RESULTS A total of 138 units were analyzed from the 4 patients. Most of the PH units had a slow, irregular discharge (mean [± SD] 4.5 ± 2.7 Hz, n = 68) but some units also had a higher discharge rate (16.7 ± 4.7 Hz, n = 15). Two populations of neurons were observed in the ventral thalamic region as well, one with a high firing rate (mean 16.5 ± 6.5 Hz, n = 5) and one with a low firing rate (mean 4.6 ± 2.8 Hz, n = 6). RN units had a regular firing rate with a mean of 20.4 ± 9.9 Hz and displayed periods of oscillatory activity in the beta range. PH units displayed a prolonged period of inhibition following microstimulation compared with RN units that were not inhibited. Patients under anesthesia showed a trend for lower firing rates in the PH but not in the RN. All 4 patients displayed a reduction in their aggressive behavior after surgery. CONCLUSIONS During PH DBS, microelectrode recordings can provide an additional mechanism to help identify the PH target and

  10. 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.

  11. Direct observation of single amyloid-β(1-40) oligomers on live cells: binding and growth at physiological concentrations.

    PubMed

    Johnson, Robin D; Schauerte, Joseph A; Wisser, Kathleen C; Gafni, Ari; Steel, Duncan G

    2011-01-01

    Understanding how amyloid-β peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aβ interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aβ concentrations required for most biochemical analyses, the metastable nature of Aβ aggregates, and the complex variety of Aβ species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aβ(1-40) oligomers bind to living neuroblastoma cells at physiological Aβ concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aβ species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aβ oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aβ species that interact with neurons at physiological concentrations.

  12. Direct single molecule measurement of TCR triggering by agonist pMHC in living primary T cells.

    PubMed

    O'Donoghue, Geoff P; Pielak, Rafal M; Smoligovets, Alexander A; Lin, Jenny J; Groves, Jay T

    2013-07-03

    T cells discriminate between self and foreign antigenic peptides, displayed on antigen presenting cell surfaces, via the TCR. While the molecular interactions between TCR and its ligands are well characterized in vitro, quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling. We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes. Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70, as a readout of TCR triggering. Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems. Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio. Thus any signal amplification must occur downstream of TCR triggering. DOI:http://dx.doi.org/10.7554/eLife.00778.001.

  13. Fluorescence molecule counting for single-molecule studies in crowded environment of living cells without and with broken ergodicity.

    PubMed

    Földes-Papp, Zeno; Baumann, Gerd

    2011-05-01

    We present a new approach to distinguish between non-ergodic and ergodic behavior. Performing ensemble averaging in a subpopulation of individual molecules leads to a mean value that can be similar to the mean value obtained in an ergodic system. The averaging is carried out by minimizing the variation between the sum of the temporal averaged mean square deviation of the simulated data with respect to the logarithmic scaling behavior of the subpopulation. For this reason, we first introduce a kind of Continuous Time Random Walks (CTRW), which we call Limited Continuous Time Random Walks (LCTRW) on fractal support. The random waiting time distributions are sampled at points which fulfill the condition N <1, where N is the Poisson probability of finding a single molecule in the femtoliter-sized observation volume ΔV at the single-molecule level. Given a subpopulation of different single molecules of the same kind, the ratio T/ T(m) between the measurement time T and the meaningful time T(m), which is the time for observing just one and the same single molecule, is the experimentally accessible quantity that allows to compare different molecule numbers in the subpopulation. In addition, the mean square displacement traveled by the molecule during the time t is determined by an upper limit of the geometric dimension of the living cell or its nucleus.

  14. 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.

  15. Living Donor Liver Transplantation for Combined Hepatocellular Carcinoma and Cholangiocarcinoma: Experience of a Single Center.

    PubMed

    Chang, Cheng-Chih; Chen, Ying-Ju; Huang, Tzu-Hao; Chen, Chun-Han; Kuo, Fang-Ying; Eng, Hock-Liew; Yong, Chee-Chien; Liu, Yueh-Wei; Lin, Ting-Lung; Li, Wei-Feng; Lin, Yu-Hung; Lin, Chih-Che; Wang, Chih-Chi; Chen, Chao-Long

    2017-02-28

    BACKGROUND Because the outcome of liver transplantation for cholangiocarcinoma is often poor, cholangiocarcinoma is a contraindication for liver transplantation in most centers. Combined hepatocellular carcinoma and cholangiocarcinoma is a rare type of primary hepatic malignancy containing features of hepatocellular carcinoma and cholangiocarcinoma. Diagnosing combined hepatocellular carcinoma and cholangiocarcinoma pre-operatively is difficult. Because of sparse research presentations worldwide, we report our experience with living donor liver transplantation for combined hepatocellular carcinoma and cholangiocarcinoma. MATERIAL AND METHODS A total of 710 patients underwent living donor liver transplantation at our institution from April 2006 to June 2014; 377 of them received transplantation because of hepatocellular carcinoma with University of California San Francisco (UCSF) staging criteria fulfilled pre-operatively. Eleven patients (2.92%) were diagnosed with combined hepatocellular carcinoma and cholangiocarcinoma confirmed pathologically from explant livers; we reviewed these cases retrospectively. Long-term survival was compared between patients diagnosed with combined hepatocellular carcinoma and cholangiocarcinoma and patients diagnosed with hepatocellular carcinoma. RESULTS The mean age of the patients in our series was 60.2 years, and the median follow-up period was 23.9 months. Four patients were diagnosed with a recurrence during the follow-up period, including one intra-hepatic and three extra-hepatic recurrences. Four patients died due to tumor recurrence. Except for patients with advanced-stage cancer, disease-free survival of patients with combined hepatocellular carcinoma and cholangiocarcinoma compared with that of patients with hepatocellular carcinoma was 80% versus 97.2% in 1 year, and 46.7% versus 92.5% in 3 years (p<0.001), and overall survival was 90% versus 97.2% in 1 year, and 61.7% versus 95.1% in 3 years (p<0.001). CONCLUSIONS

  16. Tuberculosis screening among homeless persons with AIDS living in single-room-occupancy hotels.

    PubMed

    Layton, M C; Cantwell, M F; Dorsinville, G J; Valway, S E; Onorato, I M; Frieden, T R

    1995-11-01

    Congregate facilities for homeless persons with the acquired immunodeficiency syndrome (AIDS) are often endemic for tuberculosis. We evaluated tuberculosis screening methods at single-room-occupancy hotels housing persons with AIDS. Residents were screened by cross matching the New York City Tuberculosis Registry, interviewing for tuberculosis history, skin testing, and chest radiography. Cases were classified as either previously or newly diagnosed. Among the 106 participants, 16 (15%) previously diagnosed tuberculosis cases were identified. Participants' tuberculosis histories were identified by the questionnaire (100%) or by registry match (69%). Eight participants (50%) were noncompliant with therapy. These findings prompted the establishment of a directly observed therapy program on site.

  17. Real-Time Visualization and Quantification of Contractile Ring Proteins in Single Living Cells

    PubMed Central

    Davidson, Reshma; Liu, Yajun; Gerien, Kenneth S.; Wu, Jian-Qiu

    2017-01-01

    Single-cell microscopy provides a powerful tool to visualize cellular and subcellular processes in wild-type and mutant cells by observing fluorescently tagged proteins. Here, we describe three simple methods to visualize fission yeast cells: gelatin slides, coverslip-bottom dishes, and tetrad fluorescence microscopy. These imaging methods and data analysis using free software make it possible to quantify protein localization, dynamics, and concentration with high spatial and temporal resolution. In fission yeast, the actomyosin contractile ring is essential for cytokinesis. We use the visualization and quantification of contractile ring proteins as an example to demonstrate how to use these methods. PMID:26519302

  18. Extracting the stepping dynamics of molecular motors in living cells from trajectories of single particles.

    PubMed

    Bruno, Augusto; Bruno, Luciana; Levi, Valeria

    2013-01-01

    Molecular motors are responsible of transporting a wide variety of cargos in the cytoplasm. Current efforts are oriented to characterize the biophysical properties of motors in cells with the aim of elucidating the mechanisms of these nanomachines in the complex cellular environment. In this study, we present an algorithm designed to extract motor step sizes and dwell times between steps from trajectories of motors or cargoes driven by motors in cells. The algorithm is based on finding patterns in the trajectory compatible with the behavior expected for a motor step, i.e., a region of confined motion followed by a jump in the position to another region of confined motion with similar characteristics to the previous one. We show that this algorithm allows the analysis of 2D trajectories even if they present complex motion patterns such as active transport interspersed with diffusion and does not require the assumption of a given step size or dwell period. The confidence on the step detection can be easily obtained and allows the evaluation of the confidence of the dwell and step size distributions. To illustrate the possible applications of this algorithm, we analyzed trajectories of myosin-V driven organelles in living cells.

  19. Source of Scatter in the Creep Lives of NiAl(Hf) Single Crystals Revealed

    NASA Technical Reports Server (NTRS)

    1996-01-01

    In recent years, there has been an increased emphasis in developing NiAl-based alloys for high- temperature applications in aircraft engines. In comparison to commercial superalloys, binary NiAl has a higher melting temperature, lower density, larger thermal conductivity, and better oxidation resistance. These properties make it a desirable material to replace superalloys as blades and vanes in aircraft engines. Despite this attractive combination of properties, binary NiAl cannot be used as a reliable structural material because of its low-temperature brittleness and poor high-temperature creep strength. GE Aircraft Engines in Cincinnati, Ohio, has recently developed NiAl(Hf) alloys that have creep strengths comparable to commercial superalloys while maintaining the other desirable properties of binary NiAl. The microstructures of these alloys consist of finely distributed G-phase (Ni_(16)Hf_(6)Si_(7)) precipitates, which strengthen the NiAl matrix. However, while the creep properties of these alloys were being evaluated, considerable scatter was observed in the creep lives of specimens tested under identical stress and temperature conditions. Although these alloys had nominally the same composition, the test specimens were obtained from four different ingots (A, B, C, and D) that had been heat treated under similar conditions. The NASA Lewis Research Center began the present study at the request of GE Aircraft Engines under a Space Act Agreement to identify the source of this scatter.

  20. 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

  1. Patient-Reported Outcomes Following Living Kidney Donation: A Single Center Experience.

    PubMed

    Rodrigue, James R; Vishnevsky, Tanya; Fleishman, Aaron; Brann, Tracy; Evenson, Amy R; Pavlakis, Martha; Mandelbrot, Didier A

    2015-09-01

    This article describes the development and implementation of an initiative at one transplant center to annually assess psychosocial outcomes of living kidney donors. The current analysis focuses on a cohort of adults (n = 208) who donated a kidney at BIDMC between September 2005 and August 2012, in which two post-donation annual assessments could be examined. One and two year post-donation surveys were returned by 59 % (n = 123) and 47 % (n = 98) of LKDs, respectively. Those who did not complete any survey were more likely to be younger (p = 0.001), minority race/ethnicity (p < 0.001), and uninsured at the time of donation (p = 0.01) compared to those who returned at least one of the two annual surveys. The majority of donors reported no adverse physical or psychosocial consequences of donation, high satisfaction with the donation experience, and no donation decision regret. However, a sizable minority of donors felt more pain intensity than expected and recovery time was much slower than expected, and experienced a clinically significant decline in vitality. We describe how these outcomes are used to inform clinical practice at our transplant center as well as highlight challenges in donor surveillance over time.

  2. 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.

  3. 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

  4. Live-cell single-molecule labeling and analysis of myosin motors with quantum dots

    PubMed Central

    Hatakeyama, Hiroyasu; Nakahata, Yoshihito; Yarimizu, Hirokazu; Kanzaki, Makoto

    2017-01-01

    Quantum dots (QDs) are a powerful tool for quantitatively analyzing dynamic cellular processes by single-particle tracking. However, tracking of intracellular molecules with QDs is limited by their inability to penetrate the plasma membrane and bind to specific molecules of interest. Although several techniques for overcoming these problems have been proposed, they are either complicated or inconvenient. To address this issue, in this study, we developed a simple, convenient, and nontoxic method for labeling intracellular molecules in cells using HaloTag technology and electroporation. We labeled intracellular myosin motors with this approach and tracked their movement within cells. By simultaneously imaging myosin movement and F-actin architecture, we observed that F-actin serves not only as a rail but also as a barrier for myosin movement. We analyzed the effect of insulin on the movement of several myosin motors, which have been suggested to regulate intracellular trafficking of the insulin-responsive glucose transporter GLUT4, but found no significant enhancement in myosin motor motility as a result of insulin treatment. Our approach expands the repertoire of proteins for which intracellular dynamics can be analyzed at the single-molecule level. PMID:28035048

  5. High-sensitivity measurements of multiple kinase activities in live single cells.

    PubMed

    Regot, Sergi; Hughey, Jacob J; Bajar, Bryce T; Carrasco, Silvia; Covert, Markus W

    2014-06-19

    Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.

  6. Morphometric and biochemical characterization of red beet (Beta vulgaris L.) hairy roots obtained after single and double transformations.

    PubMed

    Thimmaraju, R; Venkatachalam, L; Bhagyalakshmi, N

    2008-06-01

    It is known that T-DNA of Agrobacterium rhizogenes affects processes of plant development and activates the synthesis of secondary metabolites in transformed plant cells. In the present investigation, we provide evidence that different strains of A. rhizogenes significantly affect morphometric, morphological and functional characteristics of hairy roots of red beet (Beta vulgaris L.). Infection with four strains of A. rhizogenes (A4, A 2/83, A 20/83 and LMG-150) resulted in ten clones of hairy roots, which were named accordingly as A4(1), A4(2), A4(3), A 2/83(1), A 2/83(2), A 2/83(3), A 20/83(1), A 20/83(2), A 20/83(3) and LMG-150. Their growth characteristics, pigment content, levels of endogenous auxin and T-DNA copy number showed significant differences probably due to the physiological status of the host cell rather than the T-DNA copy number. Although A 2/83 showed highest hairy root induction capacity, the best hairy root clone was obtained with strain LMG-150 that produced highest biomass and pigments. In this root clone, the enzyme peroxidase was found involved in altering the endogenous auxin pool. When root clone LMG-150 was re-transformed to insert additional individual rol genes, two double transformed clones were obtained, one for rolABC and the other for rolC gene where the former produced higher biomass and betalaine than the latter. Despite the established fact that rol genes of T-DNA influence endogenous phytohormones, no direct correlation among the single transformants and the double transformants was found. This is the first report, in our knowledge, where a hairy root clone has been used to obtain double transformants.

  7. 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

    2013-02-02

    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.

  8. 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.

  9. 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.

  10. 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.

  11. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging.

    PubMed

    Ritsma, Laila; Ellenbroek, Saskia I J; Zomer, Anoek; Snippert, Hugo J; de Sauvage, Frederic J; Simons, Benjamin D; Clevers, Hans; van Rheenen, Jacco

    2014-03-20

    The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5(+) cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5- Confetti mice. We find that Lgr5(+) cells in the upper part of the niche (termed 'border cells') can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed 'central cells', experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5(+) cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.

  12. 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.

  13. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging

    NASA Astrophysics Data System (ADS)

    Ritsma, Laila; Ellenbroek, Saskia I. J.; Zomer, Anoek; Snippert, Hugo J.; de Sauvage, Frederic J.; Simons, Benjamin D.; Clevers, Hans; van Rheenen, Jacco

    2014-03-01

    The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5+ cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5-Confetti mice. We find that Lgr5+ cells in the upper part of the niche (termed `border cells') can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed `central cells', experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5+ cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.

  14. Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

    PubMed Central

    Nadler, André; Haberkant, Per; Kirkpatrick, Joanna; Schifferer, Martina; Stein, Frank; Hauke, Sebastian; Porter, Forbes D.; Schultz, Carsten

    2017-01-01

    Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner. PMID:28154130

  15. 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...

  16. Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

    NASA Astrophysics Data System (ADS)

    Lagerholm, B. Christoffer; Andrade, Débora M.; Clausen, Mathias P.; Eggeling, Christian

    2017-02-01

    Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below the diffraction limit of conventional microscopy. However, a major disparity in interpretation of data from SPT and STED-FCS remains, namely the proposed existence of a very fast (unhindered) lateral diffusion coefficient, ⩾5 µm2 s-1, in the plasma membrane of live cells at very short length scales, ≈⩽ 100 nm, and time scales, ≈1-10 ms. This fast diffusion coefficient has been advocated in several high-speed SPT studies, for lipids and membrane proteins alike, but the equivalent has not been detected in STED-FCS measurements. Resolving this ambiguity is important because the assessment of membrane dynamics currently relies heavily on SPT for the determination of heterogeneous diffusion. A possible systematic error in this approach would thus have vast implications in this field. To address this, we have re-visited the analysis procedure for SPT data with an emphasis on the measurement errors and the effect that these errors have on the measurement outputs. We subsequently demonstrate that STED-FCS and SPT data, following careful consideration of the experimental errors of the SPT data, converge to a common interpretation which for the case of a diffusing phospholipid analogue in the plasma membrane of live mouse embryo fibroblasts results in an unhindered, intra-compartment, diffusion coefficient of  ≈0.7-1.0 µm2 s-1, and a compartment size of about 100-150 nm.

  17. 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.

  18. Single-Molecule Tracking Photoactivated Localization Microscopy to Map Nano-Scale Structure and Dynamics in Living Spines

    PubMed Central

    MacGillavry, Harold D.; Blanpied, Thomas A.

    2013-01-01

    Super-resolution microscopy has rapidly become an indispensable tool in cell biology and neuroscience by enabling measurement in live cells of structures smaller than the classical limit imposed by diffraction. The most widely applied super-resolution method currently is localization microscopy, which takes advantage of the ability to determine the position of individual fluorescent molecules with nanometer accuracy even in cells. By iteratively measuring sparse subsets of photoactivatable fluorescent proteins, protein distribution in macromolecular structures can be accurately reconstructed. Moreover, the motion trajectories of individual molecules within cells can be measured, providing unique ability to measure transport kinetics, exchange rates, and binding affinities of even small subsets of molecules with high temporal resolution and great spatial specificity. This unit describes protocols to measure and quantify the distribution of scaffold proteins within single synapses of cultured hippocampal neurons, and to track and measure the diffusion of intracellular constituents of the neuronal plasma membrane. PMID:25429311

  19. Fluorescent Saxitoxins for Live Cell Imaging of Single Voltage-Gated Sodium Ion Channels beyond the Optical Diffraction Limit

    PubMed Central

    Ondrus, Alison E.; Lee, Hsiao-lu D.; Iwanaga, Shigeki; Parsons, William H.; Andresen, Brian M.; Moerner, W.E.; Bois, J. Du

    2013-01-01

    SUMMARY A desire to better understand the role of voltagegated sodium channels (NaVs) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select NaV isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to NaVs in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective NaV labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal NaV distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia. PMID:22840778

  20. Optical nanosensors for chemical analysis inside single living cells. 1. Fabrication, characterization, and methods for intracellular delivery of PEBBLE sensors.

    PubMed

    Clark, H A; Hoyer, M; Philbert, M A; Kopelman, R

    1999-11-01

    Spherical optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced in sizes including 20 and 200 nm in diameter. These sensors are fabricated in a microemulsion and consist of fluorescent indicators entrapped in a polyacrylamide matrix. A generalized polymerization method has been developed that permits production of sensors containing any hydrophilic dye or combination of dyes in the matrix. The PEBBLE matrix protects the fluorescent dye from interference by proteins, allowing reliable in vivo calibrations of dyes. Sensor response times are less than 1 ms. Cell viability assays indicate that the PEBBLEs are biocompatible, with negligible biological effects compared to control conditions. Several sensor delivery methods have been studied, including liposomal delivery, gene gun bombardment, and picoinjection into single living cells.

  1. Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes.

    PubMed

    Ewers, Helge; Smith, Alicia E; Sbalzarini, Ivo F; Lilie, Hauke; Koumoutsakos, Petros; Helenius, Ari

    2005-10-18

    The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, analysis of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30-60 nm in diameter), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5-10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside molecules by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus/receptor complex by cortical actin filaments.

  2. Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes

    PubMed Central

    Ewers, Helge; Smith, Alicia E.; Sbalzarini, Ivo F.; Lilie, Hauke; Koumoutsakos, Petros; Helenius, Ari

    2005-01-01

    The lateral mobility of individual murine polyoma virus–like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, analysis of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30–60 nm in diameter), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5–10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside molecules by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus/receptor complex by cortical actin filaments. PMID:16219700

  3. 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.

  4. Precision Optogenetic Tool for Selective Single- and Multiple-Cell Ablation in a Live Animal Model System.

    PubMed

    Makhijani, Kalpana; To, Tsz-Leung; Ruiz-González, Rubén; Lafaye, Céline; Royant, Antoine; Shu, Xiaokun

    2017-01-19

    Cell ablation is a strategy to study cell lineage and function during development. Optogenetic methods are an important cell-ablation approach, and we have previously developed a mini singlet oxygen generator (miniSOG) tool that works in the living Caenorhabditis elegans. Here, we use directed evolution to generate miniSOG2, an improved tool for cell ablation via photogenerated reactive oxygen species. We apply miniSOG2 to a far more complex model animal system, Drosophila melanogaster, and demonstrate that it can be used to kill a single neuron in a Drosophila larva. In addition, miniSOG2 is able to photoablate a small group of cells in one of the larval wing imaginal discs, resulting in an adult with one incomplete and one normal wing. We expect miniSOG2 to be a useful optogenetic tool for precision cell ablation at a desired developmental time point in live animals, thus opening a new window into cell origin, fate and function, tissue regeneration, and developmental biology.

  5. 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

  6. 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

  7. Long-lived charge-separated configuration of a push-pull archetype of Disperse Red 1 end-capped poly[9,9-bis(4-diphenylaminophenyl)fluorene].

    PubMed

    El-Khouly, Mohamed E; Chen, Yu; Zhuang, Xiaodong; Fukuzumi, Shunichi

    2009-05-13

    The photoinduced electron-transfer process in Disperse Red 1 end-capped poly[9,9-bis(4-diphenylaminophenyl)-2,7-fluorene], a promising material for electronic and optoelectronic devices, is reported here. The charge-separated configuration was found to be long-lived, with a lifetime of up to 2.2 ms in the polar benzonitrile, as inferred from time-resolved absorption measurements.

  8. Fluorescence resonance energy transfer on single living cells. Application to binding of monovalent haptens to cell-bound immunoglobulin E.

    PubMed Central

    Kubitscheck, U; Kircheis, M; Schweitzer-Stenner, R; Dreybrodt, W; Jovin, T M; Pecht, I

    1991-01-01

    We have determined the specific binding of 2,4-dinitrophenyl (DNP)-haptens to two different monoclonal immunoglobulin (IgE) molecules bound to Fc epsilon-receptors on the cell surface of single, living rat basophilic leukemia cells subclone 2H3 cells. The measurements were performed at 4 degrees, 15 degrees, and 25 degrees C using a recently developed technique that permits the quantitative determination of fluorescence resonance energy transfer between two fluorophores on single cells in a microscope from the photobleaching kinetics of the donor fluorophore. We introduce here a method for performing binding studies on individual attached cells. At 25 degrees C, the titration studies yielded equilibrium binding constants Kint of 9 x 10(8), 8 x 10(8), and 8 x 10(7) M-1 for the monovalent haptens N-2,4-DNP-epsilon-amino-n-caproic acid, N epsilon-2,4-DNP-L-lysine, and N-2,4-DNP-gamma-amino-n-butyric acid, respectively. Our data indicate that the affinity constants for the first two haptens binding to IgE on adherent cells are 4 to 11 times larger than that of the corresponding values obtained by fluorescence quenching experiments with the same haptens and IgE molecules either in solution or bound to cells in suspension. PMID:1832974

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

    PubMed

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

    2014-11-05

    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.

  10. 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

    2015-05-07

    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.

  11. Explore various co-substrates for simultaneous electricity generation and Congo red degradation in air-cathode single-chamber microbial fuel cell.

    PubMed

    Cao, Yunqing; Hu, Yongyou; Sun, Jian; Hou, Bin

    2010-08-01

    Microbial fuel cell (MFC) holds a great promise to harvest electricity directly from a wide range of ready degradable organic matters and enhance degradation of some recalcitrant contaminants. Glucose, acetate sodium and ethanol were separately examined as co-substrates for simultaneous bioelectricity generation and Congo red degradation in a proton exchange membrane (PEM) air-cathode single-chamber MFC. The batch test results showed that more than 98% decolorization at the dye concentration of 300 mg/L were achieved within 36 h for all tested co-substrates during electricity generation. The decolorization rate was different with the co-substrates used. The fastest decolorization rate was achieved with glucose followed by ethanol and sodium acetate. Accumulated intermediates were observed during Congo red degradation which was demonstrated by UV-Visible spectra and high performance liquid chromatography (HPLC). Electricity generation was sustained and not significantly affected by the Congo red degradation. Glucose, acetate sodium and ethanol produced maximum power densities of 103 mW/m(2), 85.9 mW/m(2) and 63.2 mW/m(2), respectively, and the maximum voltage output decreased by only 7% to 15%. Our results demonstrated the feasibility of using various co-substrates for simultaneous decolorization of Congo red and bioelectricity generation in the MFC and showed that glucose was the preferred co-substrate.

  12. Single-dose live-attenuated Nipah virus vaccines confer complete protection by eliciting antibodies directed against surface glycoproteins

    PubMed Central

    DeBuysscher, Blair L.; Scott, Dana; Marzi, Andrea; Prescott, Joseph; Feldmann, Heinz

    2016-01-01

    Background Nipah virus (NiV), a zoonotic pathogen causing severe respiratory illness and encephalitis in humans, emerged in Malaysia in 1998 with subsequent outbreaks on an almost annual basis since 2001 in parts of the Indian subcontinent. The high case fatality rate, human-to-human transmission, wide-ranging reservoir distribution and lack of licensed intervention options are making NiV a serious regional and potential global public health problem. The objective of this study was to develop a fast-acting, single-dose NiV vaccine that could be implemented in a ring vaccination approach during outbreaks. Methods In this study we have designed new live-attenuated vaccine vectors based on recombinant vesicular stomatitis viruses (rVSV) expressing NiV glycoproteins (G or F) or nucleoprotein (N) and evaluated their protective efficacy in Syrian hamsters, an established NiV animal disease model. We further characterized the humoral immune response to vaccination in hamsters using ELISA and neutralization assays and performed serum transfer studies. Results Vaccination of Syrian hamsters with a single dose of the rVSV vaccine vectors resulted in strong humoral immune responses with neutralizing activities found only in those animals vaccinated with rVSV expressing NiV G or F proteins. Vaccinated animals with neutralizing antibody responses were completely protected from lethal NiV disease, whereas animals vaccinated with rVSV expressing NiV N showed only partial protection. Protection of NiV G or F vaccinated animals was conferred by antibodies, most likely the neutralizing fraction, as demonstrated by serum transfer studies. Protection of N-vaccinated hamsters was not antibody-dependent indicating a role of adaptive cellular responses for protection. Conclusions The rVSV vectors expressing Nipah virus G or F are prime candidates for new ‘emergency vaccines’ to be utilized for NiV outbreak management. PMID:24631094

  13. Rapid isolation of nuclei from living immune cells by a single centrifugation through a multifunctional lysis gradient.

    PubMed

    Poglitsch, Marko; Katholnig, Karl; Säemann, Marcus D; Weichhart, Thomas

    2011-10-28

    Due to their low protein content and limited nuclear detergent stability, primary human immune cells such as monocytes or T lymphocytes represent a great challenge for standard nuclear isolation protocols. Nuclei clumping during the multiple centrifugation steps or contamination of isolated nuclei with cytoplasmic proteins due to membrane lysis is a frequently observed problem. Here we describe a versatile and novel method for the isolation of clean and intact nuclei from primary human monocytes, which can be applied for virtually any cell type. Living cells were applied on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. Mild cell lysis as well as efficient washing of the nuclei was performed during the course of one single low g-force centrifugation step. The isolation procedure, which we call lysis gradient centrifugation (LGC), results in the recovery of 90-95% of highly pure nuclei. This easy and highly reproducible procedure allows an effective preparation of nuclei and the cytoplasm in only 15 min with the ability to handle as little as one million cells per sample and easy parallel processing of multiple samples.

  14. 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

  15. 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

  16. Temperature-Dependent Model of Multi-step Transcription Initiation in Escherichia coli Based on Live Single-Cell Measurements

    PubMed Central

    Lloyd-Price, Jason; Tran, Huy; Ribeiro, Andre S.

    2016-01-01

    Transcription kinetics is limited by its initiation steps, which differ between promoters and with intra- and extracellular conditions. Regulation of these steps allows tuning both the rate and stochasticity of RNA production. We used time-lapse, single-RNA microscopy measurements in live Escherichia coli to study how the rate-limiting steps in initiation of the Plac/ara-1 promoter change with temperature and induction scheme. For this, we compared detailed stochastic models fit to the empirical data in maximum likelihood sense using statistical methods. Using this analysis, we found that temperature affects the rate limiting steps unequally, as nonlinear changes in the closed complex formation suffice to explain the differences in transcription dynamics between conditions. Meanwhile, a similar analysis of the PtetA promoter revealed that it has a different rate limiting step configuration, with temperature regulating different steps. Finally, we used the derived models to explore a possible cause for why the identified steps are preferred as the main cause for behavior modifications with temperature: we find that transcription dynamics is either insensitive or responds reciprocally to changes in the other steps. Our results suggests that different promoters employ different rate limiting step patterns that control not only their rate and variability, but also their sensitivity to environmental changes. PMID:27792724

  17. Ultralow frequency Stokes and anti-Stokes Raman spectroscopy of single living cells and microparticles using a hot rubidium vapor filter.

    PubMed

    Lin, Jinda; Li, Yong-qing

    2014-01-01

    We report on ultralow frequency Stokes and anti-Stokes Raman spectroscopy of single living cells and microsized particles in an aqueous medium with a frequency shift down to 10 cm(-1) by the combination of a hot rubidium (Rb) vapor filter, a confocal pinhole, and optical trapping. A single frequency-stabilized diode laser beam at 780.2 nm is used to optically trap and excite a single living cell or microparticle, and the Rayleigh scattering light from the particle is effectively blocked with a Rb vapor cell and a confocal pinhole. Ultralow frequency Raman spectra of the trapped cells or microparticles in both Stokes and anti-Stokes regions are then measured with a single-stage CCD spectrograph.

  18. 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.

  19. Laparoendoscopic single-site (LESS) vs laparoscopic living-donor nephrectomy: a systematic review and meta-analysis.

    PubMed

    Autorino, Riccardo; Brandao, Luis Felipe; Sankari, Bashir; Zargar, Homayoun; Laydner, Humberto; Akça, Oktay; De Sio, Marco; Mirone, Vincenzo; Chueh, Shih-Chieh J; Kaouk, Jihad H

    2015-02-01

    The aim of this study was to provide a systematic review and meta-analysis of reports comparing laparoendoscopic single-site (LESS) living-donor nephrectomy (LDN) vs standard laparoscopic LDN (LLDN). A systematic review of the literature was performed in September 2013 using PubMed, Scopus, Ovid and The Cochrane library databases. Article selection proceeded according to the search strategy based on Preferred Reporting Items for Systematic Reviews and Meta-analyses criteria. Weighted mean differences (WMDs) were used to measure continuous variables and odds ratios (ORs) to measure categorical ones. Nine publications meeting eligibility criteria were identified, including 461 LESS LDN and 1006 LLDN cases. There were more left-side cases in the LESS LDN group (96.5% vs 88.6%, P < 0.001). Meta-analysis of extractable data showed that LLDN had a shorter operative time (WMD 15.06 min, 95% confidence interval [CI] 4.9-25.1; P = 0.003), without a significant difference in warm ischaemia time (WMD 0.41 min, 95% CI -0.02 to 0.84; P = 0.06). Estimated blood loss was lower for LESS LDN (WMD -22.09 mL, 95% CI -29.5 to -14.6; P < 0.001); however, this difference was not clinically significant. There was a greater likelihood of conversion for LESS LDN (OR 13.21, 95% CI 4.65-37.53; P < 0.001). Hospital stay was similar (WMD -0.11 days, 95% CI -0.33 to 0.12; P = 0.35), as well as the visual analogue pain score at discharge (WMD -0.31, 95% CI -0.96 to 0.35; P = 0.36), but the analgesic requirement was lower for LESS LDN (WMD -2.58 mg, 95% CI -5.01 to -0.15; P = 0.04). Moreover, there was no difference in the postoperative complication rate (OR 1.00, 95% CI 0.65-1.54; P = 0.99). Renal function of the recipient, as based on creatinine levels at 1 month, showed similar outcomes between groups (WMD 0.10 mg/dL, -0.09 to 0.29; P = 0.29). In conclusion, LESS LDN represents an emerging option for living kidney donation. This procedure offers comparable surgical and early

  20. 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

    2015-11-03

    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.

  1. Polymerization Induced Self-Assembly of Alginate Based Amphiphilic Graft Copolymers Synthesized by Single Electron Transfer Living Radical Polymerization.

    PubMed

    Kapishon, Vitaliy; Whitney, Ralph A; Champagne, Pascale; Cunningham, Michael F; Neufeld, Ronald J

    2015-07-13

    Alginate-based amphiphilic graft copolymers were synthesized by single electron transfer living radical polymerization (SET-LRP), forming stable micelles during polymerization induced self-assembly (PISA). First, alginate macroinitiator was prepared by partial depolymerization of native alginate, solubility modification and attachment of initiator. Depolymerized low molecular weight alginate (∼12 000 g/mol) was modified with tetrabutylammonium, enabling miscibility in anhydrous organic solvents, followed by initiator attachment via esterification yielding a macroinitiator with a degree of substitution of 0.02, or 1-2 initiator groups per alginate chain. Then, methyl methacrylate was polymerized from the alginate macroinitiator in mixtures of water and methanol, forming poly(methyl methacrylate) grafts, prior to self-assembly, of ∼75 000 g/mol and polydispersity of 1.2. PISA of the amphiphilic graft-copolymer resulted in the formation of micelles with diameters of 50-300 nm characterized by light scattering and electron microscopy. As the first reported case of LRP from alginate, this work introduces a synthetic route to a preparation of alginate-based hybrid polymers with a precise macromolecular architecture and desired functionalities. The intended application is the preparation of micelles for drug delivery; however, LRP from alginate can also be applied in the field of biomaterials to the improvement of alginate-based hydrogel systems such as nano- and microhydrogel particles, islet encapsulation materials, hydrogel implants, and topical applications. Such modified alginates can also improve the function and application of native alginates in food and agricultural applications.

  2. Detection of sodium and potassium in single human red blood cells by 193-nm laser ablative sampling: a feasibility demonstration.

    PubMed

    Ng, C W; Cheung, N H

    2000-01-01

    The feasibility of quantifying sodium and potassium in single human erythrocytes was demonstrated by spectrochemical analysis of emissions from plasmas produced by 193-nm laser ablation of blood cells confined in a sheath flow. In one scheme, single blood cells that happened to be in the ablation volume were sampled. In another scheme, individual blood cells were first sighted and then synchronously ablated downstream. Plasma emission spectra of single ablated cells were captured, and the ratios of the analyte line intensity to the root-mean-square fluctuation of the continuum background were measured to be about 18 for sodium and 30 for potassium.

  3. 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

  4. 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.

  5. 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

  6. [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

  7. Stress system dynamics during "life as it is lived": an integrative single-case study on a healthy woman.

    PubMed

    Schubert, Christian; Geser, Willi; Noisternig, Bianca; Fuchs, Dietmar; Welzenbach, Natalie; König, Paul; Schüßler, Gerhard; Ocaña-Peinado, Francisco M; Lampe, Astrid

    2012-01-01

    Little is known about the dynamic characteristics of stress system activity during "life as it is lived". Using as representative a study design as possible, this investigation sought to gain insights into this area. A healthy 25-year-old woman collected her entire urine over a period of 63 days in 12-h intervals (126 measurements) to determine cortisol and neopterin (immune activation marker) levels. In addition, she filled out questionnaires on emotional state and daily routine in 12-h intervals, and was interviewed weekly to identify emotionally negative and positive everyday incidents. Adjusted cross-correlational analyses revealed that stressful incidents were associated with cyclic response patterns in both urinary cortisol and urinary neopterin concentrations. Urinary cortisol levels first decreased 12-24 h after stressful incidents occurred (lag 1: -.178; p = 0.048) and then increased a total of 72-84 h later (lag 6: +.224; p = 0.013). Urinary neopterin levels first increased 0-12 h before the occurrence of stressful incidents (-lag 1: +.185; p = 0.040) and then decreased a total of 48-60 h following such stressors (lag 4: -.181; p = 0.044). Decreases in urinary neopterin levels were also found 24-36 and 48-60 h after increases in pensiveness (lag 2: -.215; p = 0.017) and depressiveness (lag 4: -.221; p = 0.014), respectively. Findings on emotionally positive incidents sharply contrasted with those dealing with negative experiences. Positive incidents were followed first by urinary cortisol concentration increases within 12 h (lag 0: +.290; p = 0.001) and then by decreases after a total of 60-72 h (lag 5: -.186; p = 0.039). Urinary neopterin levels first decreased 12-24 h before positive incidents occurred (-lag 2: -.233; p = 0.010) and then increased a total of 12-24 h following these incidents (lag 1: +.222; p = 0.014). As with previous investigations on patients with systemic lupus erythematosus (SLE), this study showed that stress system response can be

  8. Comparison of the urinary excretion of quercetin glycosides from red onion and aglycone from dietary supplements in healthy subjects: a randomized, single-blinded, cross-over study.

    PubMed

    Shi, Yuanlu; Williamson, Gary

    2015-05-01

    Some intervention studies have shown that quercetin supplementation can regulate certain biomarkers, but it is not clear how the doses given relate to dietary quercetin (e.g. from onion). We conducted a two-period, two-sequence crossover study to compare the bioavailability of quercetin when administered in the form of a fresh red onion meal (naturally glycosylated quercetin) or dietary supplement (aglycone quercetin) under fasting conditions. Six healthy, non-smoking, adult males with BMI 22.7 ± 4.0 kg m(-2) and age 35.3 ± 12.3 y were grouped to take the two study meals in random order. In each of the 2 study periods, one serving of onion soup (made from 100 g fresh red onion, providing 156.3 ± 3.4 μmol (47 mg) quercetin) or a single dose of a quercetin dihydrate tablet (1800 ± 150 μmol (544 mg) of quercetin) were administered following 3 d washout. Urine samples were collected up to 24 h, and after enzyme deconjugation, quercetin was quantified by LC-MS. The 24 h urinary excretion of quercetin (1.69 ± 0.79 μmol) from red onion in soup was not significantly different to that (1.17 ± 0.44 μmol) for the quercetin supplement tablet (P = 0.065, paired t-test). This means that, in practice, 166 mg of quercetin supplement would be comparable to about 10 mg of quercetin aglycone equivalents from onion. These data allow intervention studies on quercetin giving either food or supplements to be more effectively compared.

  9. 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

    2015-04-06

    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.

  10. An AFLP-based linkage map of Japanese red pine (Pinus densiflora) using haploid DNA samples of megagametophytes from a single maternal tree.

    PubMed

    Kim, Yong-Yul; Choi, Hyung-Soon; Kang, Bum-Yong

    2005-10-31

    We have constructed an AFLP-based linkage map of Japanese red pine (Pinus densiflora Siebold et Zucc.) using haploid DNA samples of 96 megagametophytes from a single maternal tree, selection clone Kyungbuk 4. Twenty-eight primer pairs generated a total of 5,780 AFLP fragments. Five hundreds and thirteen fragments were verified as genetic markers with two alleles by their Mendelian segregation. At the linkage criteria LOD 4.0 and maximum recombination fraction 0.25(theta), a total of 152 markers constituted 25 framework maps for 19 major linkage groups. The maps spanned a total length of 2,341 cM with an average framework marker spacing of 18.4 cM. The estimated genome size was 2,662 cM. With an assumption of equal marker density, 82.2% of the estimated genome would be within 10 cM of one of the 230 linked markers, and 68.1% would be within 10 cM of one of the 152 framework markers. We evaluated map completeness in terms of LOD value, marker density, genome length, and map coverage. The resulting map will provide crucial information for future genomic studies of the Japanese red pine, in particular for QTL mapping of economically important breeding target traits.

  11. Dynamical color-controllable lasing with extremely wide tuning range from red to green in a single alloy nanowire using nanoscale manipulation.

    PubMed

    Liu, Zhicheng; Yin, Leijun; Ning, Hao; Yang, Zongyin; Tong, Limin; Ning, Cun-Zheng

    2013-10-09

    Multicolor lasing and dynamic color-tuning in a wide spectrum range are challenging to realize but critically important in many areas of technology and daily life, such as general lighting, display, multicolor detection, and multiband communication. By exploring nanoscale growth and manipulation, we have demonstrated the first active dynamical color control of multicolor lasing, continuously tunable between red and green colors separated by 107 nm in wavelength. This is achieved in a purposely engineered single CdSSe alloy nanowire with composition varied along the wire axis. By looping the wide-gap end of the alloy nanowire through nanoscale manipulation, two largely independent (only weakly coupled) laser cavities are formed respectively for the green and red color modes. Our approach simultaneously overcomes the two fundamental challenges for multicolor lasing in material growth and cavity design. Such multicolor lasing and continuous color tuning in a wide spectral range represents a new paradigm shift and would eventually enable color-by-design and white-color lasers for lighting, illumination, and many other applications.

  12. Degradation behavior and thermal properties of red (650 nm) high-power diode single emitters and laser bars

    NASA Astrophysics Data System (ADS)

    Tomm, Jens W.; Tien, Tran Q.; Ziegler, Mathias; Weik, Fritz; Sumpf, Bernd; Zorn, Martin; Zeimer, Ute; Erbert, Götz

    2007-02-01

    The degradation behavior of broad-area laser diodes and bars emitting at 650 nm under constant power operation is investigated. In addition to the increase in operation current the temperature of the laser facets was monitored using Raman spectroscopy. The formation of defects was studied using photocurrent spectroscopy while cathodoluminescence provided insight into the position of extended non-radiative defects at different stages of degradation. Although the facet does not show any visible alteration even for failed devices, its immediate vicinity appears to be the starting point of the observed gradual degradation effects. At the same time the local facet temperature is increased. The observed aging behavior is compared to the known degradation scenarios for devices emitting at 808 nm. In both cases there is a clear correlation between packaging-induced strain and observed degradation effects as demonstrated by the results obtained for bars. For the red devices a correlation between optical load and facet temperature exists which proves that here facet heating is indeed caused by re-absorption processes. Furthermore, the gradual degradation process is not accompanied by the creation of dark bands along 100 directions as observed earlier for 808 nm devices. The observed gradual degradation of the 650 nm devices is primarily accompanied by the formation of deep-level point defects, followed by the creation of macroscopic areas of reduced luminescence intensity. Packaging induced strains become important when gradual bar degradation is monitored at early stages.

  13. 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.

  14. Vibrationally resonant imaging of a single living cell by supercontinuum-based multiplex coherent anti-Stokes Raman scattering microspectroscopy

    NASA Astrophysics Data System (ADS)

    Kano, Hideaki; Hamaguchi, Hiro-O.

    2005-02-01

    Supercontinuum-based multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy has been applied to vibrational imaging of a living fission yeast cell. We have successfully extracted only a vibrationally resonant CARS image from a characteristic spectral profile in the C-H stretching vibrational region. Using our simple but sensitive analysis, the vibrational contrast is significantly improved in comparison with a CARS imaging at a fixed Raman shift. The CARS image of a living yeast cell indicates several areas at which the signal is remarkably strong. They are considered to arise from mitochondria.

  15. Localization, Dynamics, and Function of Survivin Revealed by Expression of Functional SurvivinDsRed Fusion Proteins in the Living Cell

    PubMed Central

    Temme, Achim; Rieger, Michael; Reber, Friedemann; Lindemann, Dirk; Weigle, Bernd; Diestelkoetter-Bachert, Petra; Ehninger, Gerhard; Tatsuka, Masaaki; Terada, Yasuhiko; Rieber, Ernst Peter

    2003-01-01

    Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein. PMID:12529428

  16. Long-term outcomes of living-related small intestinal transplantation in children: A single-center experience.

    PubMed

    Garcia Aroz, Sandra; Tzvetanov, Ivo; Hetterman, Elizabeth Anne; Jeon, Hoonbae; Oberholzer, Jose; Testa, Giuliano; John, Eunice; Benedetti, Enrico

    2017-03-12

    Pediatric patients with irreversible intestinal failure present a significant challenge to meet the nutritional needs that promote growth. From 2002 to 2013, 13 living-related small intestinal transplantations were performed in 10 children, with a median age of 18 months. Grafts included isolated living-related intestinal transplantation (n=7), and living-related liver and small intestine (n=6). The immunosuppression protocol consisted of induction with thymoglobulin and maintenance therapy with tacrolimus and steroids. Seven of 10 children are currently alive with a functioning graft and good quality of life. Six of the seven children who are alive have a follow-up longer than 10 years. The average time to initiation of oral diet was 32 days (range, 13-202 days). The median day for ileostomy takedown was 77 (range, 18-224 days). Seven children are on an oral diet, and one of them is on supplements at night through a g-tube. We observed an improvement in growth during the first 3 years post-transplant and progressive weight gain throughout the first year post-transplantation. Growth catch-up and weight gain plateaued after these time periods. We concluded that living donor intestinal transplantation potentially offers a feasible, alternative strategy for long-term treatment of irreversible intestinal failure in children.

  17. Imaging single cells in a beam of live cyanobacteria with an X-ray laser (CXIDB ID 27)

    SciTech Connect

    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.

  18. Red Emission B, N, S-co-Doped Carbon Dots for Colorimetric and Fluorescent Dual Mode Detection of Fe(3+) Ions in Complex Biological Fluids and Living Cells.

    PubMed

    Liu, Yinghua; Duan, Wenxiu; Song, Wei; Liu, Juanjuan; Ren, Cuiling; Wu, Jiang; Liu, Dan; Chen, Hongli

    2017-04-03

    Colorimetric and fluorescent dual mode detection methods have gained much attention in recent years; however, it is still desirable to develop new colorimetric and fluorescent dual mode nanosensors with more simple preparation procedures, low cost, and excellent biocompatibility. Herein, a colorimetric and fluorescent nanosensor based on B, N, S-co-doped carbon dots (BNS-CDs) was synthesized by one-step hydrothermal treatment of 2,5-diaminobenzenesulfonic acid and 4-aminophenylboronic acid hydrochloride. Using this nanosensor, a highly sensitive assay of Fe(3+) in the range of 0.3-546 μM with a detection limit of 90 nM was provided by quenching the red emission fluorescence. It is more attractive that Fe(3+) can also be visualized by this nanosensor via evident color changes of the solution (from red to blue) under sunlight without the aid of an ultraviolet (UV) lamp. Furthermore, the designed nanosensor can be applied for efficient detection of intracellular Fe(3+) with excellent biocompatibility and cellular imaging capability, and it holds great promise in biomedical applications.

  19. High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system

    PubMed Central

    Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

    2016-01-01

    Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases. PMID:27841364

  20. 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…

  1. Exercise-induced oxidative stress in elderly subjects: the effect of red orange supplementation on the biochemical and cellular response to a single bout of intense physical activity.

    PubMed

    Pittaluga, M; Sgadari, A; Tavazzi, B; Fantini, C; Sabatini, S; Ceci, R; Amorini, A M; Parisi, P; Caporossi, D

    2013-03-01

    Aging is characterized by an impaired capacity to maintain the redox balance both in physiological and pathological situations associated with an increased production of reactive oxygen species. Since the extent of this phenomenon may be influenced by an antioxidants-rich diet, we investigated the effect of supplementation with fresh red orange juice (ROJ) on biochemical and cellular biomarkers of oxidative stress in healthy, trained elderly women after a single bout of exhaustive exercise (EE). To this purpose, a sample of 22 females, 15 (69.0 ± 5.1 years) taking the ROJ supplementation and 7 (68.1 ± 2.7 years) as Control group, was constituted. Blood samples were collected immediately before, 30 minutes, and 24 hr after a single bout of EE, at baseline and after 4 weeks. Our results demonstrate that markers of DNA damage or apoptosis were not affected by EE both in Control and ROJ group, and by ROJ, whereas, exercise temporarily affected the redox balance in both groups. Controls didn't change their response to EE after the experimental period, but experimental group after ROJ supplementation had lower EE-induced MDA, consumed less ascorbic acid, and had less activation of the hypoxanthine/xanthine system, i.e., they seemed to be protected from hypoxia/reoxygenation mechanisms.

  2. 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

  3. Imaging single cells in a beam of live cyanobacteria with an X-ray laser (CXIDB ID 26)

    SciTech Connect

    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.

  4. 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

  5. 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.

  6. FOXO3 variants are beneficial for longevity in Southern Chinese living in the Red River Basin: A case-control study and meta-analysis.

    PubMed

    Sun, Liang; Hu, Caiyou; Zheng, Chenguang; Qian, Yu; Liang, Qinghua; Lv, Zeping; Huang, Zezhi; Qi, KeYan; Gong, Huan; Zhang, Zheng; Huang, Jin; Zhou, Qin; Yang, Ze

    2015-04-27

    Forkhead box class O (FOXO) transcription factors play a crucial role in longevity across species. Several polymorphisms in FOXO3 were previously reported to be associated with human longevity. However, only one Chinese replication study has been performed so far. To verify the role of FOXO3 in southern Chinese in the Red River Basin, a community-based case-control study was conducted, and seven polymorphisms were genotyped in 1336 participants, followed by a meta-analysis of eight case-control studies that included 5327 longevity cases and 4608 controls. In our case-control study, we found rs2802288*A and rs2802292*G were beneficial to longevity after Bonferroni correction (pallele = 0.005, OR = 1.266; pallele = 0.026, OR = 1.207). In addition, in the longevity group, carriers with rs2802288*A and rs2802292*G presented reduced HbA1c (p = 0.001), and homozygotes of rs2802292*GG presented improved HOMA-IR (p = 0.014). The meta-analysis further revealed the overall contribution of rs2802288*A and rs2802292*G to longevity. However, our stratified analysis revealed that rs2802292*G might act more strongly in Asians than Europeans, for enhancement of longevity. In conclusion, our study provides convincing evidence for a significant association between the rs2802288*A and rs2802292*G gene variants in FOXO3 and human longevity, and adds the Southern Chinese in the Red River Basin to the growing number of human replication populations.

  7. 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

    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

  8. 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.

  9. 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

  10. 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.

  11. Inventing a co-axial atomic resolution patch clamp to study a single resonating protein complex and ultra-low power communication deep inside a living neuron cell.

    PubMed

    Ghosh, Subrata; Sahu, Satyajit; Agrawal, Lokesh; Shiga, Takashi; Bandyopadhyay, Anirban

    2016-12-01

    To read the signals of single molecules in vitro on a surface, or inside a living cell or organ, we introduce a coaxial atom tip (coat) and a coaxial atomic patch clamp (COAPAP). The metal-insulator-metal cavity of these probes extends to the atomic scale (0.1[Formula: see text]nm), it eliminates the cellular or environmental noise with a S/N ratio 10(5). Five ac signals are simultaneously applied during a measurement by COAT and COAPAP to shield a true signal under environmental noise in five unique ways. The electromagnetic drive in the triaxial atomic tips is specifically designed to sense anharmonic vibrational and transmission signals for any system between 0.1[Formula: see text]nm and 50[Formula: see text]nm where the smallest nanopatch clamp cannot reach. COAT and COAPAP reliably pick up the atomic scale vibrations under the extreme noise of a living cell. Each protein's distinct electromagnetic, mechanical, electrical and ionic vibrational signature studied in vitro in a protected environment is found to match with the ones studied inside a live neuron. Thus, we could confirm that by using our probe blindly we could hold on to a single molecule or its complex in the invisible domain of a living cell. Our decade long investigations on perfecting the tools to measure bio-resonance of all forms and simultaneously in all frequency domains are summarized. It shows that the ratio of emission to absorption resonance frequencies of a biomaterial is around [Formula: see text], only a few in the entire em spectrum are active that regulates all other resonances, like mechanical, ionic, etc.

  12. 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).

  13. 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…

  14. Markers of Decompression Stress of Mass Stranded/Live Caught and Released vs. Single Stranded Marine Mammals

    DTIC Science & Technology

    2012-09-30

    University of Pennsylvania Philadelphia PA 19104 Kathleen Moore International Fund for Animal Welfare Yarmouth Port MA 02675 Randall Wells...stress in cetaceans . After undertaking a baseline experiment in Steller sea lions, this project will compare and contrast health, body condition, and...presence of bubbles with markers for decompression stress in mass stranded cetaceans , and compare these indices with single stranded individuals

  15. Single versus Multiple Suicide Attempts: A Prospective Examination of Psychiatric Factors and Wish to Die/Wish to Live Index

    DTIC Science & Technology

    2013-04-18

    to another base, his post-traumatic stress and depressive symptoms worsened. Prior to his suicide , he made two unsuccessful attempts. His mother...one suicide attempt in women. Trauma history. Individuals with Posttraumatic Stress Disorder may display self-destructive and impulsive behaviors...Afghanistan war veterans reporting SINGLE VERSUS MULTIPLE SUICIDE ATTEMPTS 103       subthreshold and threshold Posttraumatic Stress

  16. Children in Single-Parent Families Living in Poverty Have Fewer Supports after Welfare Reform. IWPR Research in Brief.

    ERIC Educational Resources Information Center

    Lyter, Deanna M.; Sills, Melissa; Oh, Gi-Taik

    Since the 1996 passage of the Personal Responsibility and Work Opportunity Act (welfare reform), impoverished children in single-parent families receive less aid than under the previous system, and the most disadvantaged of these children have slipped deeper into poverty. This research brief summarizes a study that explored the economic well-being…

  17. Live Healthy, Live Longer

    MedlinePlus

    ... Human Services. More Health News on: Exercise and Physical Fitness Health Screening Healthy Living Recent Health News Related MedlinePlus Health Topics Exercise and Physical Fitness Health Screening Healthy Living About MedlinePlus Site Map ...

  18. The effect of live feeds bathed with the red seaweed Asparagopsis armata on the survival, growth and physiology status of Sparus aurata larvae.

    PubMed

    Castanho, S; Califano, G; Soares, F; Costa, R; Mata, L; Pousão-Ferreira, P; Ribeiro, L

    2017-03-17

    Larval rearing is affected by a wide range of microorganisms that thrive in larviculture systems. Some seaweed species have metabolites capable of reducing the bacterial load. However, no studies have yet tested whether including seaweed metabolites on larval rearing systems has any effects on the larvae development. This work assessed the development of Sparus aurata larvae fed preys treated with an Asparagopsis armata product. Live prey, Brachionus spp. and Artemia sp., were immersed in a solution containing 0.5% of a commercial extract of A. armata (Ysaline 100, YSA) for 30 min, before being fed to seabream larvae (n = 4 each). In the control, the live feed was immersed in clear water. Larval parameters such as growth, survival, digestive capacity (structural-histology and functional-enzymatic activity), stress level (cortisol content), non-specific immune response (lysozyme activity), anti-bacterial activity (disc-diffusion assay) and microbiota quantification (fish larvae gut and rearing water) were monitored. Fish larvae digestive capacity, stress level and non-specific immune response were not affected by the use of YSA. The number of Vibrionaceae was significantly reduced both in water and larval gut when using YSA. Growth was enhanced for YSA treatment, but higher mortality was also observed, especially until 10 days after hatching (DAH). The mortality peak observed at 8 DAH for both treatments, but higher for YSA, indicates larval higher susceptibility at this development stage, suggesting that lower concentrations of YSA should be used until 10 DAH. The application of YSA after 10 DAH onwards promotes a safer rearing environment.

  19. 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).

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

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

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

  1. 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

  2. Quantum dots as a sensor for quantitative visualization of surface charges on single living cells with nano-scale resolution.

    PubMed

    Huang, Yao-Xiong; Zheng, Xin-Jing; Kang, Li-Li; Chen, Xing-Yao; Liu, Wen-Jing; Huang, Bao-Tian; Wu, Zheng-Jie

    2011-01-15

    We developed a technique using quantum dot (QD) as a sensor for quantitative visualization of the surface charge on biological cells with nano-scale resolution. The QD system was designed and synthesized using amino modified CdSe/ZnS nanoparticles. In a specially designed buffer solution, they are positively charged and can homogeneously disperse in the aqueous environment to label all the negative charges on the surfaces of living cells. Using a wide-field optical sectioning microscopy to achieve 2D/3D imaging of the QD-labeled cells, we determined the charge densities of different kinds of cells from normal to mutant ones. The information about the surface charge distribution is significant in evaluating the structure, function, biological behavior and even malignant transformation of cells.

  3. Eye redness

    MedlinePlus

    Bloodshot eyes; Red eyes; Scleral injection; Conjunctival injection ... There are many causes of a red eye or eyes. Some are medical emergencies. Others are a cause for concern, but not an emergency. Many are nothing to worry about. Eye ...

  4. 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

  5. Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

    PubMed Central

    Hiroshima, Michio; Saeki, Yuko; Okada-Hatakeyama, Mariko; Sako, Yasushi

    2012-01-01

    Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces of MCF-7 human breast cancer cells. The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al. (2006) EMBO J 25:4215–4222], suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow. The dissociation rate constant from the liganded monomer was intermediate. The dynamic changes in the association and dissociation kinetics in relation to the dimerization of ErbB displayed negative cooperativity, which resulted in apparent low- and high-affinity sites of HRG association on the cell surface. PMID:22891299

  6. 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

  7. Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes.

    PubMed

    Katz, Zachary B; English, Brian P; Lionnet, Timothée; Yoon, Young J; Monnier, Nilah; Ovryn, Ben; Bathe, Mark; Singer, Robert H

    2016-01-13

    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.

  8. Red Sea

    Atmospheric Science Data Center

    2013-04-16

    article title:  The Red Sea     View Larger Image ... 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 ... 2000 - The Red Sea between the East Africa coast and Saudi Arabian peninsula. project:  MISR category:  ...

  9. 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.

  10. 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

  11. Health-seeking behavior and social networks of the aged living in single-room occupancy hotels.

    PubMed

    Cohen, C I; Sokolovsky, J

    1979-06-01

    The elderly who reside in single-room occupancy (SRO) hotels often have been depicted as "isolates," lacking the interest or ability to engage primary or secondary support systems. This characterization has not enhanced understanding of how the SRO aged are able to survive in the community. With the use of network analysis techniques, this study demonstrates the inaccuracy of the assertion that these old persons lack significant social support. The data pointed to differences in network size, complexity, intensity, connectedness, and directionality in relation to varying degrees of physical and psychiatric health.

  12. Does situs inversus totalis preclude liver donation in living donor liver transplantation? A series of 3 cases from single institution

    PubMed Central

    N., Selvakumar; Goyal, Neerav; Nayeem, Mohammed; Vohra, Sandeep; Gupta, Subash

    2016-01-01

    Introduction Liver transplantation (LT) is the gold standard for decompensated Chronic Liver Disease (CLD) in individuals satisfying the selection criteria. Organ scarcity is the rate limiting step in liver transplantation across the globe. Expanding the donor pool is practiced by transplant surgeons across the globe in view of perennial donor organ scarcity and ever increasing organ demand. Presentation of case We have presented series of 3 cases of liver transplantation (LT) with modified left lobe (conventional right) graft from a situs inversus donor and implanting it as a conventional right lobe with a modified technique. The grafts had Type 1, Type 2 and Type 3 biliary anatomies. One graft had inferior hepatic veins also. All three patients had uneventful recoveries. The follow up period range is 4 years to 8 months. Discussion There are multiple case reports in the literature involving situs inversus donors in liver transplantation. Various techniques have also been described. We describe simple and effective technique which has proved successful to our patients. Conclusion SIT donors can be safely accepted for living donor liver transplantation. It is a technically challenging procedure both for donor liver harvesting and implantation in recipient. This is the first case series of LT using modified left lobe graft (conventional right) from a SIT donor with 2 different techniques. Biliary anastomosis is the tricky part of the operation. PMID:26895114

  13. The effect of the serum corona on interactions between a single nano-object and a living cell

    PubMed Central

    Dror, Yael; Sorkin, Raya; Brand, Guy; Boubriak, Olga; Urban, Jill; Klein, Jacob

    2017-01-01

    Nanoparticles (NPs) which enter physiological fluids are rapidly coated by proteins, forming a so-called corona which may strongly modify their interaction with tissues and cells relative to the bare NPs. In this work the interactions between a living cell and a nano-object, and in particular the effect on this of the adsorption of serum proteins, are directly examined by measuring the forces arising as an Atomic Force Microscope tip (diameter 20 nm) - simulating a nano-object - approaches and contacts a cell. We find that the presence of a serum protein corona on the tip strongly modifies the interaction as indicated by pronounced increase in the indentation, hysteresis and work of adhesion compared to a bare tip. Classically one expects an AFM tip interacting with a cell surface to be repelled due to cell elastic distortion, offset by tip-cell adhesion, and indeed such a model fits the bare-tip/cell interaction, in agreement with earlier work. However, the force plots obtained with serum-modified tips are very different, indicating that the cell is much more compliant to the approaching tip. The insights obtained in this work may promote better design of NPs for drug delivery and other nano-medical applications. PMID:28383528

  14. Neutral red staining for plant vacuoles.

    PubMed

    Schwab, Birgit; Hülskamp, Martin

    2010-06-01

    For almost 100 years, neutral red has been used to stain living cells and fixed tissue. It can be used as a general-purpose stain, a pH indicator (turning from red to yellow, as the medium becomes alkaline), or a nuclear stain. In this protocol, neutral red is used to stain plant vacuoles.

  15. Effect of size and temperature at vaccination on immunization and protection conferred by a live attenuated Francisella noatunensis immersion vaccine in red hybrid tilapia.

    PubMed

    Soto, Esteban; Brown, Nicholas; Gardenfors, Zackarias O; Yount, Shaun; Revan, Floyd; Francis, Stewart; Kearney, Michael T; Camus, Alvin

    2014-12-01

    Francisella noatunensis subsp. orientalis (Fno) is a pleomorphic, facultative intracellular, Gram-negative, emerging bacterial pathogen of marine and fresh water fish with worldwide distribution. In this study, the efficacy of an attenuated Fno intracellular growth locus C (iglC) mutant was evaluated for use as a live immersion vaccine, when administered to hybrid tilapia at two different stages of growth (5 g fry and 10 g fingerlings) and at two temperatures (25 °C and 30 °C). To determine vaccine efficacy, mortality, days to first death, and Fno genome equivalents (GE) in the spleens of survivors, as well as serum and mucus antibody levels, were evaluated after 30 d in fish challenged with a wild type virulent strain. Both size and temperature at vaccination played an important role in immunization and protection. Fry vaccinated at 25 °C were not protected when compared to non-vaccinated fry at 25 °C (p = 0.870). In contrast, 5 g fry vaccinated at 30 °C were significantly protected compared to non-vaccinated fry at 30 °C (p = 0.038). Although lower mortalities occurred, 10 g fingerlings vaccinated at 25 °C were not protected, compared to non-vaccinated fingerlings at 25 °C (p = 0.328), while, 10 g fingerlings vaccinated at 30 °C were significantly protected, compared to non-vaccinated fingerlings at 30 °C (p = 0.038). Additionally, overall mortality of 5 g fish was significantly higher than in 10 g fish. Mortality was also significantly higher in fish subjected to a 30 to 25 °C temperature change one week prior to challenge, than in fish maintained at the same temperature during vaccination and challenge. This information demonstrates that both temperature and size at vaccination are important factors when implementing immunization prophylaxis in cultured tilapia.

  16. 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%).

  17. 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

  18. Quantifying Transient 3D Dynamical Phenomena of Single mRNA Particles in Live Yeast Cell Measurements

    PubMed Central

    Calderon, Christopher P.; Thompson, Michael A.; Casolari, Jason M.; Paffenroth, Randy C.; Moerner, W. E.

    2013-01-01

    Single-particle tracking (SPT) has been extensively used to obtain information about diffusion and directed motion in a wide range of biological applications. Recently, new methods have appeared for obtaining precise (10s of nm) spatial information in three dimensions (3D) with high temporal resolution (measurements obtained every 4ms), which promise to more accurately sense the true dynamical behavior in the natural 3D cellular environment. Despite the quantitative 3D tracking information, the range of mathematical methods for extracting information about the underlying system has been limited mostly to mean-squared displacement analysis and other techniques not accounting for complex 3D kinetic interactions. There is a great need for new analysis tools aiming to more fully extract the biological information content from in vivo SPT measurements. High-resolution SPT experimental data has enormous potential to objectively scrutinize various proposed mechanistic schemes arising from theoretical biophysics and cell biology. At the same time, methods for rigorously checking the statistical consistency of both model assumptions and estimated parameters against observed experimental data (i.e. goodness-of-fit tests) have not received great attention. We demonstrate methods enabling (1) estimation of the parameters of 3D stochastic differential equation (SDE) models of the underlying dynamics given only one trajectory; and (2) construction of hypothesis tests checking the consistency of the fitted model with the observed trajectory so that extracted parameters are not over-interpreted (the tools are applicable to linear or nonlinear SDEs calibrated from non-stationary time series data). The approach is demonstrated on high-resolution 3D trajectories of single ARG3 mRNA particles in yeast cells in order to show the power of the methods in detecting signatures of transient directed transport. The methods presented are generally relevant to a wide variety of 2D and 3D SPT

  19. The health status and well-being of low-resource, housing-unstable, single-parent families living in violent neighbourhoods in Philadelphia, Pennsylvania

    PubMed Central

    Jacoby, Sara F.; Tach, Laura; Guerra, Terry; Wiebe, Douglas J.; Richmond, Therese S.

    2016-01-01

    The health and well-being of single-parent families living in violent neighbourhoods in US cities who participate in housing programmes is not well described. This two-phase, mixed-methods study explores the health status of families who were participants in a housing-plus programme in Philadelphia, Pennsylvania between 2011 and 2013 and the relationship between the characteristics of the neighbourhoods in which they lived and their perceptions of well-being and safety. In phase 1, data collected with standardised health status instruments were analysed using descriptive statistics and independent sample t-tests to describe the health of single parents and one randomly selected child from each parent’s household in comparison to population norms. In a subset of survey respondents, focus groups were conducted to generate an in-depth understanding of the daily lives and stressors of these families. Focus group data were analysed using content analysis to identify key descriptive themes. In phase 2, daily activity path mapping, surveys and interviews of parent–child dyads were collected to assess how these families perceive their health, neighbourhood and the influence of neighbourhood characteristics on the families’ day-to-day experience. Narratives and activity maps were combined with crime data from the Philadelphia Police Department to analyse the relationship between crime and perceptions of fear and safety. Phase 1 data demonstrated that parent participants met or exceeded the national average for self-reported physical health but fell below the national average across all mental health domains. Over 40% reported moderate to severe symptoms of depression. Parents described high levels of stress resulting from competing priorities, financial instability, and concern for their children’s well-being and safety. Analysis of phase 2 data demonstrated that neighbourhood characteristics exert influence over parents’ perceptions of their environment and how

  20. The health status and well-being of low-resource, housing-unstable, single-parent families living in violent neighbourhoods in Philadelphia, Pennsylvania.

    PubMed

    Jacoby, Sara F; Tach, Laura; Guerra, Terry; Wiebe, Douglas J; Richmond, Therese S

    2017-03-01

    The health and well-being of single-parent families living in violent neighbourhoods in US cities who participate in housing programmes is not well described. This two-phase, mixed-methods study explores the health status of families who were participants in a housing-plus programme in Philadelphia, Pennsylvania between 2011 and 2013 and the relationship between the characteristics of the neighbourhoods in which they lived and their perceptions of well-being and safety. In phase 1, data collected with standardised health status instruments were analysed using descriptive statistics and independent sample t-tests to describe the health of single parents and one randomly selected child from each parent's household in comparison to population norms. In a subset of survey respondents, focus groups were conducted to generate an in-depth understanding of the daily lives and stressors of these families. Focus group data were analysed using content analysis to identify key descriptive themes. In phase 2, daily activity path mapping, surveys and interviews of parent-child dyads were collected to assess how these families perceive their health, neighbourhood and the influence of neighbourhood characteristics on the families' day-to-day experience. Narratives and activity maps were combined with crime data from the Philadelphia Police Department to analyse the relationship between crime and perceptions of fear and safety. Phase 1 data demonstrated that parent participants met or exceeded the national average for self-reported physical health but fell below the national average across all mental health domains. Over 40% reported moderate to severe symptoms of depression. Parents described high levels of stress resulting from competing priorities, financial instability, and concern for their children's well-being and safety. Analysis of phase 2 data demonstrated that neighbourhood characteristics exert influence over parents' perceptions of their environment and how they permit

  1. 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

  2. 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

  3. 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.

  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.

  6. The molecular yo-yo method: Live jump detection improves throughput of single-molecule force spectroscopy for out-of-equilibrium transitions

    NASA Astrophysics Data System (ADS)

    Mack, A. H.; Schlingman, D. J.; Kamenetska, M.; Collins, R.; Regan, L.; Mochrie, S. G. J.

    2013-08-01

    By monitoring multiple molecular transitions, force-clamp, and trap-position-clamp methods have led to precise determinations of the free energies and free energy landscapes for molecular states populated in equilibrium at the same or similar forces. Here, we present a powerful new elaboration of the force-clamp and force-jump methods, applicable to transitions far from equilibrium. Specifically, we have implemented a live jump detection and force-clamp algorithm that intelligently adjusts and maintains the force on a single molecule in response to the measured state of that molecule. We are able to collect hundreds of individual molecular transitions at different forces, many times faster than previously, permitting us to accurately determine force-dependent lifetime distributions and reaction rates. Application of our method to unwinding and rewinding the nucleosome inner turn, using optical tweezers reveals experimental lifetime distributions that comprise a statistically meaningful number of transitions, and that are accurately single exponential. These measurements significantly reduce the error in the previously measured rates, and demonstrate the existence of a single, dominant free energy barrier at each force studied. A key benefit of the molecular yo-yo method for nucleosomes is that it reduces as far as possible the time spent in the tangentially bound state, which minimizes the loss of nucleosomes by dissociation.

  7. The molecular yo-yo method: live jump detection improves throughput of single-molecule force spectroscopy for out-of-equilibrium transitions.

    PubMed

    Mack, A H; Schlingman, D J; Kamenetska, M; Collins, R; Regan, L; Mochrie, S G J

    2013-08-01

    By monitoring multiple molecular transitions, force-clamp, and trap-position-clamp methods have led to precise determinations of the free energies and free energy landscapes for molecular states populated in equilibrium at the same or similar forces. Here, we present a powerful new elaboration of the force-clamp and force-jump methods, applicable to transitions far from equilibrium. Specifically, we have implemented a live jump detection and force-clamp algorithm that intelligently adjusts and maintains the force on a single molecule in response to the measured state of that molecule. We are able to collect hundreds of individual molecular transitions at different forces, many times faster than previously, permitting us to accurately determine force-dependent lifetime distributions and reaction rates. Application of our method to unwinding and rewinding the nucleosome inner turn, using optical tweezers reveals experimental lifetime distributions that comprise a statistically meaningful number of transitions, and that are accurately single exponential. These measurements significantly reduce the error in the previously measured rates, and demonstrate the existence of a single, dominant free energy barrier at each force studied. A key benefit of the molecular yo-yo method for nucleosomes is that it reduces as far as possible the time spent in the tangentially bound state, which minimizes the loss of nucleosomes by dissociation.

  8. Single-molecule imaging of beta-actin mRNAs in the cytoplasm of a living cell.

    PubMed

    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(MACRO)) at the leading edge was 0.3 microm(2)/s. On the other hand, D(MACRO) in the perinuclear region was 0.02 microm(2)/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize beta-actin mRNAs, led to an increase in D(MACRO) to 0.2 microm(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. Single-particle tracking and modulation of cell entry pathways of a tetrahedral DNA nanostructure in live cells.

    PubMed

    Liang, Le; Li, Jiang; Li, Qian; Huang, Qing; Shi, Jiye; Yan, Hao; Fan, Chunhai

    2014-07-21

    DNA is typically impermeable to the plasma membrane due to its polyanionic nature. Interestingly, several different DNA nanostructures can be readily taken up by cells in the absence of transfection agents, which suggests new opportunities for constructing intelligent cargo delivery systems from these biocompatible, nonviral DNA nanocarriers. However, the underlying mechanism of entry of the DNA nanostructures into the cells remains unknown. Herein, we investigated the endocytotic internalization and subsequent transport of tetrahedral DNA nanostructures (TDNs) by mammalian cells through single-particle tracking. We found that the TDNs were rapidly internalized by a caveolin-dependent pathway. After endocytosis, the TDNs were transported to the lysosomes in a highly ordered, microtubule-dependent manner. Although the TDNs retained their structural integrity within cells over long time periods, their localization in the lysosomes precludes their use as effective delivery agents. To modulate the cellular fate of the TDNs, we functionalized them with nuclear localization signals that directed their escape from the lysosomes and entry into the cellular nuclei. This study improves our understanding of the entry into cells and transport pathways of DNA nanostructures, and the results can be used as a basis for designing DNA-nanostructure-based drug delivery nanocarriers for targeted therapy.

  10. 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.

  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. Deep Sequencing of Distinct Preparations of the Live Attenuated Varicella-Zoster Virus Vaccine Reveals a Conserved Core of Attenuating Single-Nucleotide Polymorphisms

    PubMed Central

    Yamanishi, Koichi; Gomi, Yasuyuki; Gershon, Anne A.; Breuer, Judith

    2016-01-01

    ABSTRACT The continued success of the live attenuated varicella-zoster virus vaccine in preventing varicella-zoster and herpes zoster is well documented, as are many of the mutations that contribute to the attenuation of the vOka virus for replication in skin. At least three different preparations of vOka are marketed. Here, we show using deep sequencing of seven batches of vOka vaccine (including ZostaVax, VariVax, VarilRix, and the Oka/Biken working seed) from three different manufacturers (VariVax, GSK, and Biken) that 137 single-nucleotide polymorphism (SNP) mutations are present in all vaccine batches. This includes six sites at which the vaccine allele is fixed or near fixation, which we speculate are likely to be important for attenuation. We also show that despite differences in the vaccine populations between preparations, batch-to-batch variation is minimal, as is the number and frequency of mutations unique to individual batches. This suggests that the vaccine manufacturing processes are not introducing new mutations and that, notwithstanding the mixture of variants present, VZV live vaccines are extremely stable. IMPORTANCE The continued success of vaccinations to prevent chickenpox and shingles, combined with the extremely low incidence of adverse reactions, indicates the quality of these vaccines. The vaccine itself is comprised of a heterogeneous live attenuated virus population and thus requires deep-sequencing technologies to explore the differences and similarities in the virus populations between different preparations and batches of the vaccines. Our data demonstrate minimal variation between batches, an important safety feature, and provide new insights into the extent of the mutations present in this attenuated virus. PMID:27440875

  13. Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

    PubMed Central

    Zhen, Chao Yu; Tatavosian, Roubina; Huynh, Thao Ngoc; Duc, Huy Nguyen; Das, Raibatak; Kokotovic, Marko; Grimm, Jonathan B; Lavis, Luke D; Lee, Jun; Mejia, Frances J; Li, Yang; Yao, Tingting; Ren, Xiaojun

    2016-01-01

    The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes. DOI: http://dx.doi.org/10.7554/eLife.17667.001 PMID:27723458

  14. 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.

  15. Phylogeny of Parasitic Parabasalia and Free-Living Relatives Inferred from Conventional Markers vs. Rpb1, a Single-Copy Gene

    PubMed Central

    Malik, Shehre-Banoo; Brochu, Cynthia D.; Bilic, Ivana; Yuan, Jing; Hess, Michael; Logsdon, John M.; Carlton, Jane M.

    2011-01-01

    Background Parabasalia are single-celled eukaryotes (protists) that are mainly comprised of endosymbionts of termites and wood roaches, intestinal commensals, human or veterinary parasites, and free-living species. Phylogenetic comparisons of parabasalids are typically based upon morphological characters and 18S ribosomal RNA gene sequence data (rDNA), while biochemical or molecular studies of parabasalids are limited to a few axenically cultivable parasites. These previous analyses and other studies based on PCR amplification of duplicated protein-coding genes are unable to fully resolve the evolutionary relationships of parabasalids. As a result, genetic studies of Parabasalia lag behind other organisms. Principal Findings Comparing parabasalid EF1α, α-tubulin, enolase and MDH protein-coding genes with information from the Trichomonas vaginalis genome reveals difficulty in resolving the history of species or isolates apart from duplicated genes. A conserved single-copy gene encodes the largest subunit of RNA polymerase II (Rpb1) in T. vaginalis and other eukaryotes. Here we directly sequenced Rpb1 degenerate PCR products from 10 parabasalid genera, including several T. vaginalis isolates and avian isolates, and compared these data by phylogenetic analyses. Rpb1 genes from parabasalids, diplomonads, Parabodo, Diplonema and Percolomonas were all intronless, unlike intron-rich homologs in Naegleria, Jakoba and Malawimonas. Conclusions/Significance The phylogeny of Rpb1 from parasitic and free-living parabasalids, and conserved Rpb1 insertions, support Trichomonadea, Tritrichomonadea, and Hypotrichomonadea as monophyletic groups. These results are consistent with prior analyses of rDNA and GAPDH sequences and ultrastructural data. The Rpb1 phylogenetic tree also resolves species- and isolate-level relationships. These findings, together with the relative ease of Rpb1 isolation, make it an attractive tool for evaluating more extensive relationships within

  16. Binding Strength and Dynamics of Invariant Natural Killer Cell T Cell Receptor/CD1d-Glycosphingolipid Interaction on Living Cells by Single Molecule Force Spectroscopy*

    PubMed Central

    Bozna, Bianca L.; Polzella, Paolo; Rankl, Christian; Zhu, Rong; Salio, Mariolina; Shepherd, Dawn; Duman, Memed; Cerundolo, Vincenzo; Hinterdorfer, Peter

    2011-01-01

    Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells. PMID:21454514

  17. Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy.

    PubMed

    Bozna, Bianca L; Polzella, Paolo; Rankl, Christian; Zhu, Rong; Salio, Mariolina; Shepherd, Dawn; Duman, Memed; Cerundolo, Vincenzo; Hinterdorfer, Peter

    2011-05-06

    Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

  18. Are long-lived trees poised for evolutionary change? Single locus effects in the evolution of gene expression networks in spruce.

    PubMed

    Verta, Jukka-Pekka; Landry, Christian R; Mackay, John J

    2013-05-01

    Genetic variation in gene expression traits contributes to phenotypic diversity and may facilitate adaptation following environmental change. This is especially important in long-lived organisms where adaptation to rapid changes in the environment must rely on standing variation within populations. However, the extent of expression variation in most wild species remains to be investigated. We address this question by measuring the segregation of expression levels in white spruce [Picea glauca (Moench), Voss] in a transcriptome-wide manner and examining the underlying evolutionary and biological processes. We applied a novel approach for the genetic analysis of expression variation by measuring its segregation in haploid meiotic seed tissue. We identified over 800 transcripts whose abundances are most likely controlled by variants in single loci. Cosegregation analysis of allelic expression levels was used to construct regulatory associations between genes and define regulatory networks. The majority (67%) of segregating transcripts were under linkage. Regulatory associations were typically among small groups of genes (2-3 transcripts), indicating that most segregating expression levels can evolve independently from one another. One notable exception was a large putative trans effect that altered the expression of 180 genes that includes key regulators of protein metabolism, highlighting a regulatory cascade affected by variation in a single locus in this conserved metabolic pathway. Overall, segregating expression variation was associated with stress response- and duplicated genes, whose evolution may be linked to functional innovations. These observations indicate that expression variation might be important in facilitating diversity of molecular responses to environmental stresses in wild trees.

  19. 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

  20. Red Sky with Red Mesa

    ScienceCinema

    None

    2016-07-12

    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.

  1. Development of UV-excitable red and near-infrared fluorescent labels and their application for simultaneous multicolor bioimaging by single-wavelength excitation.

    PubMed

    Mizuno, Tetsuya; Umezawa, Keitaro; Shindo, Yutaka; Citterio, Daniel; Oka, Kotaro; Suzuki, Koji

    2013-09-01

    We report a new type of UV-excitable red/NIR-emissive fluorescent dyads (PKF series). Conjugation of a pyrene and a novel bright red/near-infrared (NIR) fluorophore resulted in large quasi-Stokes shift while retaining intense fluorescence emission and sharp spectral bands. Labeling of PKF dyads to biomolecules was performed by means of introduction of a succinimidyl ester. Simultaneous Ca(2+)/albumin dual-color intracellular imaging by PKF in combination with fura-2 (UV-excitable/VIS-emissive Ca(2+) indicator) reveals its usefulness as a new bioimaging tool.

  2. Red Hill

    EPA Pesticide Factsheets

    Information about the Red Hill Bulk Fuel Storage Facility in Hawaii Administrative Order on Consent (AOC), an enforceable agreement of the Hawaii Department of Health, the Environmental Protection Agency, and the U.S. Navy -- Defense Logistics Agency.

  3. A review of the Callogobius (Teleostei: Gobiidae) from the Red Sea with the description of a new species.

    PubMed

    Delventhal, Naomi R; Mooi, Randall D; Bogorodsky, Sergey V; Mal, Ahmad O

    2016-10-31

    Five species of Callogobius Bleeker have been previously reported from the Red Sea: C. amikami Goren, Miroz & Baranes, C. clarki (Goren), C. dori Goren, C. flavobrunneus (Smith), and C. maculipinnis (Fowler). Records of C. bifasciatus (Smith) in the Red Sea are referable to C. clarki. Callogobius amikami has been previously known only from a single specimen, the holotype from the Red Sea, and two photographs, a live juvenile from Oman and a live specimen at an aquarium at Coral World, Eilat. We obtained a possible additional juvenile from the Red Sea, although we are unable to definitively determine its identity. Red Sea specimens previously identified as C. maculipinnis [or C. irrasus (Smith)] represent a new species, distinguished from the latter by normally having four sets of transverse mandibular rows on each side (rather than three); this species is described here as Callogobius pilosimentum sp. nov. Four specimens of an additional, undescribed species of Callogobius, C. sp. A, have been collected from the Red Sea, but we withhold a formal description because this species is currently under study by colleagues. Callogobius sclateri (Steindachner), previously known from the Indo-West Pacific, is reported from the Red Sea for the first time. A key to all seven species is provided. Each species is photographed, habitat is described and a brief description with detailed comparisons is provided. The new species and C. clarki are endemic to the Red Sea.

  4. Monomeric red fluorescent proteins with a large Stokes shift.

    PubMed

    Piatkevich, Kiryl D; Hulit, James; Subach, Oksana M; Wu, Bin; Abdulla, Arian; Segall, Jeffrey E; Verkhusha, Vladislav V

    2010-03-23

    Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 mum the breast cancer cells show significant polarization towards vessels in living mice.

  5. Transgenic mouse model expressing P53R172H, luciferase, EGFP, and KRASG12D in a single open reading frame for live imaging of tumor

    PubMed Central

    Ju, Hye-Lim; Calvisi, Diego F.; Moon, Hyuk; Baek, Sinhwa; Ribback, Silvia; Dombrowski, Frank; Cho, Kyung Joo; Chung, Sook In; Han, Kwang-Hyub; Ro, Simon Weonsang

    2015-01-01

    Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53R172H and KRASG12D, are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53R172H and KRASG12D in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53R172H and KRASG12D. PMID:25623590

  6. Early systemic inflammatory response in mice after a single oral gavage with live Escherichia coli is evidenced by increased TNF-alpha and nitric oxide production.

    PubMed

    Nemec, Ana; Jerin, Aleš; Zdovc, Irena; Budefeld, Tomaž; Verstraete, Frank J M; Eržen, Damijan; Sentjurc, Marjeta; Petelin, Milan; Hitti, Tina; Pavlica, Zlatko

    2012-06-01

    Twenty-four female BALB/c mice were orally inoculated with 10(8) CFU Escherichia coli ATCC 25922 and euthanized 2.5, 7, 13 and 25 h post-inoculation. The levels of organ nitric oxide (NO) and plasma endotoxin, TNF-alpha and nitrite/nitrate (NO(x)) were compared to those found in sham-inoculated mice, to evaluate systemic host-response to a low-level oral exposure to Gram-negative bacteria. Organ bacterial culture and immunohistochemistry for iNOS were performed on lungs, liver, kidneys and brain from all mice. Organ NO and plasma TNF-alpha levels were higher in E. coli-inoculated animals, but no differences were detected in plasma endotoxin levels, NO(x) or iNOS immunostaining for any of the animal groups. Single oral gavage with live E. coli stimulates an early systemic immune response in clinically healthy mice as evidenced by increased plasma TNF-alpha and organ NO levels, but bacteremia and endotoxemia are not related to this inflammatory response.

  7. Development of TV003/TV005, a single dose, highly immunogenic live attenuated dengue vaccine; what makes this vaccine different from the Sanofi-Pasteur CYD™ vaccine?

    PubMed

    Whitehead, Stephen S

    2016-01-01

    Dengue is caused by four serotype-distinct dengue viruses (DENVs), and developing a multivalent vaccine against dengue has not been straightforward since partial immunity to DENV may predispose to more severe disease upon subsequent DENV infection. The vaccine that is furthest along in development is CYD™, a live attenuated tetravalent vaccine (LATV) produced by Sanofi Pasteur. Although the multi-dose vaccine demonstrated protection against severe dengue, its overall efficacy was limited by DENV serotype, serostatus at vaccination, region and age. The National Institute of Allergy and Infectious Diseases has developed the LATV dengue vaccines TV003/TV005. A single dose of either TV003 or TV005 induced seroconversion to four DENV serotypes in 74-92% (TV003) and 90% (TV005) of flavivirus seronegative adults and elicited near-sterilizing immunity to a second dose of vaccine administered 6-12 months later. The important differences in the structure, infectivity and immune responses to TV003/TV005 are compared with CYD™.

  8. 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.

  9. 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.

  10. Sambar deer (Cervus unicolor) x red deer (C. elaphus) interspecies hybrids.

    PubMed

    Muir, P D; Semiadi, G; Asher, G W; Broad, T E; Tate, M L; Barry, T N

    1997-01-01

    The artificial insemination of 400 red deer hinds with sambar deer semen resulted in 31 pregnancies at day 40 (24 at day 100) and the birth of four calves. Only one female calf was born alive. The artificial insemination of 10 sambar deer hinds with red deer semen resulted in five pregnancies at day 40, of which none went to term. Gel electrophoresis of three blood proteins confirmed the live calf as the first documented sambar deer x red deer hydrid. G-banded karyotypes were consistent with the calf (2n = 62; six unpaired and one paired metacentric autosomes) being the offspring of a red deer dam (2n = 68; single pair of metacentric autosomes) and a sambar deer sire (2n = 56; seven pairs of metacentric autosomes).

  11. The house mouse (Mus musculus L.) exerts strong differential grain consumption preferences among hard red and white spring wheat (Triticum aestivum L.) varieties in a single-elimination tournament design.

    PubMed

    Morris, Craig F; Fuerst, E Patrick; McLean, Derek J; Momont, Kathleen; James, Caleb P

    2014-11-01

    Wheat (Triticum aestivum L.) plays a central role in the health and nutrition of humans. Yet, little is known about possible flavor differences among different varieties. We have developed a model system using the house mouse (Mus musculus L.) to determine feeding preferences as a prelude to extending results to human sensory analysis. Here, we examine the application of a single-elimination tournament design to the analysis of consumption preferences of a set of hard red and hard white spring wheat varieties. A single-elimination tournament design in this case pairs 2 wheat varieties and only 1 of the 2 is advanced to further tests. Preferred varieties were advanced until an overall "winner" was identified; conversely, less desirable varieties were advanced such that an overall "loser" was identified. Hollis and IDO702 were the winner and loser, respectively, for the hard red varieties, and Clear White 515 and WA8123 were the winner and loser, respectively, for the hard white varieties. When using the more powerful protocol of 14 mice and a 4-d trial, differences in mean daily consumption preferences of 2 varieties were separated at P-values as small as 2 × 10(-8) . The single-elimination tournament design is an efficient means of identifying the most and least desirable varieties among a larger set of samples. One application for identifying the 2 extremes in preference within a group of varieties would be to use them as parents of a population to identify quantitative trait loci for preference.

  12. Fast quantitative ROS detection based on dual-color single rare-earth nanoparticle imaging reveals signaling pathway kinetics in living cells.

    PubMed

    Abdesselem, M; Ramodiharilafy, R; Devys, L; Gacoin, T; Alexandrou, A; Bouzigues, C I

    2017-01-05

    Reactive oxygen species (ROS), and notably hydrogen peroxide H2O2, are cellular second messengers that are known to control a variety of signaling processes. They can finely regulate the dynamics of signal transduction, cell response and ultimately tissue function. However, there are very few local, quantitative and time-resolved descriptions of their cellular organization at the scale of molecular reactions, due to the lack of efficient sensors. We thus developed a novel nanoprobe-based ROS detection system using the simultaneous imaging of single lanthanide nanoparticles (YAG:Ce and chemically reduced Gd0.6Eu0.4VO4). We reveal that both particle luminescence signals are controlled by their H2O2 local environment. By simultaneously tracking their luminescence, we devised a new approach providing a quantitative (0.5 μM accuracy in the 1-10 μM range) H2O2 measurement with a 500 ms time resolution, surpassing all existing methods by two orders of magnitude, and revealing previously inaccessible molecular events controlling ROS concentration. We used this nanoprobe in living cells to track fast signaling pathways, by measuring the dynamics of H2O2 intracellular concentrations, induced by endothelin-1 (ET-1) stimulation. We thus revealed the mechanisms controlling ROS production, notably the activity modulation of the ROS-producing enzyme NADPH oxidase by fast (<10 s) EGFR transactivation, and measured quantitatively their kinetic parameters through a minimal analytical model. Altogether, these results illustrate how lanthanide nanoparticle-based sensors are a powerful tool to dynamically probe molecular mechanisms shaping the oxidative cell response.

  13. Single red upconversion and near-infrared downconversion luminescence properties of cubic ZrO2:Y3+-Yb3+-Er3+ nanophosphors via microwave hydrothermal synthesis

    NASA Astrophysics Data System (ADS)

    Liao, Jinsheng; Wang, Qi; Nie, Liling; You, Weixiong; Chen, Jinglin

    2016-12-01

    The Yb3+-Er3+-Y3+ tri-doped cubic ZrO2 (brief as YSZ:Yb, Er) nanophosphors with different Yb3+ (Er3+) doping concentrations have been prepared by microwave hydrothermal method followed by further calcining treatment. X-ray diffraction (XRD), transmission electron microscope (TEM), and photoluminescence spectra were used to characterize the properties of YSZ:Yb, Er nanophosphors. Under the 980 nm excitation, the sample gives a set of upconversion light: very strong red emissions near 655 and 679 nm (4F9/2 → 4I15/2), very weak green (525 nm 2H11/2 → 4I15/2, 544 nm and 557 nm 4S3/2 → 4I15/2). Moreover, the sample gives near-infrared downconversion emission (1530 nm 4I13/2 → 4I15/2). Concentration dependent studies revealed that the optimal composition was realized for the 15 at% Yb3+ and 1 at% Er3+-doping concentration with a strong pure red emission. All the results indicate that YSZ:Yb, Er nanophosphors could be a promising biological labeling materials.

  14. Direct Interconversion of BINOL and H8-BINOL-Based Chiral Brønsted Acids Using Single-Step Red/Ox Manipulations.

    PubMed

    Tay, Jia-Hui; Arguelles, Alonso J; Nagorny, Pavel

    2015-08-07

    A direct single-step hydrogenation of BINOL-based chiral phosphoric acids, N-triflyl phosphoramides, and disulfonimides to the corresponding H8-BINOL Brønsted acids in excellent yields and chemoselectivities is described. In addition, the conditions for the single-step oxidation of H8-BINOL-based Brønsted acids into the corresponding BINOL-based acids have been identified and employed to accomplish these interconversions in 41-81% yield.

  15. 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.

  16. American Red Cross

    MedlinePlus

    ... Espanol Local Red Cross ( ) Change Chapter Edit Zip Code Edit Zip Code Shop the Red Cross Store Toggle Navigation Menu ... Espanol Local Red Cross ( ) Change Chapter Edit Zip Code Edit Zip Code Shop the Red Cross Store ...

  17. 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 ...

  18. Pharmacokinetic Properties of Single and Repeated Injection of Liposomal Platelet Substitute in a Rat Model of Red Blood Cell Transfusion-Induced Dilutional Thrombocytopenia.

    PubMed

    Hashimoto, Mai; Taguchi, Kazuaki; Ogaki, Shigeru; Watanabe, Hiroshi; Kinoshita, Manabu; Nishikawa, Kahoko; Takeoka, Shinji; Ikeda, Yasuo; Handa, Makoto; Otagiri, Masaki; Maruyama, Toru

    2015-11-01

    A preclinical study of dodecapeptide ((400)HHLGGAKQAGDV(411)) (H12)-(adenosine diphosphate, ADP)-liposomes for use as a synthetic platelet (PLT) substitute under conditions of red blood cell (RBC) transfusion-induced dilutional thrombocytopenia is limited to pharmacological effect. In this study, the pharmacokinetics of H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were evaluated. As evidenced by the use of (14) C, (3) H double-radiolabeled H12-(ADP)-liposomes in which the encapsulated ADP and liposomal membrane were labeled with (14) C and (3) H, respectively, the H12-(ADP)-liposomes remained intact in the blood circulation for up to 3 h after injection, and were mainly distributed to the liver and spleen. The encapsulated ADP was mainly eliminated in the urine, whereas the outer membrane was mainly eliminated in the feces. These successive pharmacokinetic properties of the H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were similar to those in healthy rats, except for the shorter retention time in the circulation. When H12-(ADP)-liposomes were repeatedly injected into RBC transfusion-induced dilutional thrombocytopenic rats at intervals of 5 days at a dose of 10 mg lipids/kg, the second dose of injected H12-(ADP)-liposomes were rapidly cleared from the circulation, namely, via the accelerated blood clearance phenomenon. These novel pharmacokinetic findings provide useful information for the further development of H12-(ADP)-liposomes as a PLT substitute.

  19. Impact of red blood cells count on the relationship between high density lipoproteins and the prevalence and extent of coronary artery disease: a single centre study [corrected].

    PubMed

    Schaffer, Alon; Verdoia, Monica; Cassetti, Ettore; Barbieri, Lucia; Perrone-Filardi, Pasquale; Marino, Paolo; De Luca, Giuseppe

    2015-07-01

    We have hypothesized that high red blood cells (RBC) count can potentially play an atheroprotective role in patients with coronary atherosclerosis. We, therefore, have investigated the relationship between high density lipoproteins cholesterol (HDL-C) and RBC levels in patients undergoing coronary angiography. Coronary artery disease (CAD) is a major cause of mortality. Impaired lipid profile represents a major risk factor for atherosclerosis. High density lipoprotein (HDL) is a key factor in atherosclerosis disease development. RBC can mimic HDL's reverse cholesterol transportation with a potential atheroprotective role. Coronary angiography has been evaluated in 3,534 patients. Fasting samples were collected for haematology and lipids levels assessment. Coronary disease was defined for at least 1 vessel stenosis >50 %. Patients were divided according to HDL-C and RBC tertiles. Lower HDL-C was significantly associated to the prevalence of CAD (84.8 vs 78.5 vs 67.3 %, p ≤ 0.001; adjusted OR [95 % CI] = 1.55 [1.3-1.8], p < 0.001) and severe CAD (30 % vs 30 % vs 24.4 %, p = 0.002; adjusted OR [95 % CI] = 1.08 [1.01-1.16], p = 0.02), this relationship was maintained even dividing our population according to RBC tertiles (p < 0.001).In conclusion, HDL-C levels are directly related to RBC count and inversely to the prevalence and extent of coronary disease. Higher RBC levels can reduce the risk of CAD in patients with lower HDL-C levels, suggesting an important atheroprotective role.

  20. Controllable red, green, blue (RGB) and bright white upconversion luminescence of Lu2O3:Yb3+/Er3+/Tm3+ nanocrystals through single laser excitation at 980 nm.

    PubMed

    Yang, Jun; Zhang, Cuimiao; Peng, Chong; Li, Chunxia; Wang, Lili; Chai, Ruitao; Lin, Jun

    2009-01-01

    Light fantastic! Lu(2)O(3):Yb(3+)/Er(3+)/Tm(3+) nanocrystals with controllable red, green, blue (RGB) and bright white upconversion luminescence by a single laser excitation of 980 nm have been successfully synthesized (see picture). Due to abundant UC PL colors, it can potentially be used as fluorophores in the field of color displays, back light, UC lasers, photonics, and biomedicine.Lu(2)O(3):Yb(3+)/Er(3+)/Tm(3+) nanocrystals have been successfully synthesized by a solvothermal process followed by a subsequent heat treatment at 800 degrees C. Powder X-ray diffraction, transmission electron microscopy, upconversion photoluminescence spectra, and kinetic decay were used to characterize the samples. Under single-wavelength diode laser excitation of 980 nm, the bright blue emissions of Lu(2)O(3):Yb(3+), Tm(3+) nanocrystals near 477 and 490 nm were observed due to the (1)G(4)-->(3)H(6) transition of Tm(3+). The bright green UC emissions of Lu(2)O(3):Er(3+) nanocrystals appeared near 540 and 565 nm were observed and assigned to the (2)H(11/2)-->(4)I(15/2) and (4)S(3/2)-->(4)I(15/2) transitions, respectively, of Er(3+). The ratio of the intensity of green luminescence to that of red luminescence decreases with an increase of concentration of Yb(3+) in Lu(2)O(3):Er(3+) nanocrystals. In sufficient quantities of Yb(3+) with resprct to Er(3+), the bright red UC emission of Lu(2)O(3):Yb(3+)/Er(3+) centered at 662 nm was predominant, due to the (4)F(9/2)-->(4)I(15/2) transition of Er(3+). Based on the generation of red, green, and blue emissions in the different doped Lu(2)O(3):RE(3+) nanocrystals, it is possible to produce the luminescence with a wide spectrum of colors, including white, by the appropriate doping of Yb(3+), Tm(3+), and Er(3+) in the present Lu(2)O(3) nanocrystals. Namely, Lu(2)O(3):3 %Yb(3+)/0.2 %Tm(3+)/0.4 %Er(3+) nanocrystals show suitable intensities of blue, green, and red (RGB) emission, resulting in the production of perfect and bright white light

  1. 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.

  2. Detection and analysis of human serum albumin nanoparticles within phagocytic cells at the resolution of individual live cell or single 3D multicellular spheroid

    NASA Astrophysics Data System (ADS)

    Afrimzon, Elena; Zurgil, Naomi; Sobolev, Maria; Shafran, Yana; Langer, Klaus; Zlatev, Iavor; Wronski, Robert; Windisch, Manfred; von Briesen, Hagen; Schmidt, Reinhold; Pietrzik, Claus; Deutsch, Mordechai

    2015-12-01

    Since nanoparticles (NPs) have shown great potential in various biomedical applications, live cell response to NPs should be thoroughly explored prior to their in vivo use. In the current study, live cell array (LCA) methodology and unique cell-based assays were used to study the interaction of magnetite (HSA-Mag NP) loaded human serum albumin NPs with phagocytic cells. The LCA enabled cell culturing during HSA-Mag NP accumulation and monolayer or spheroid formation, concomitantly with on-line monitoring of NP internalization. These platforms were also utilized for imaging intercellular links between living cells preloaded with HSA-Mag NP in 2D and 3D resolution. HSA-Mag NP uptake by cells was quantified by imaging, and analyzed using time-resolved measurements. Image analysis of the individual cells in cell populations showed accumulation of HSA-Mag NP by promonocytes and glial cells in a dose- and time-dependent manner. High variability of NP accumulation in individual cells within cell populations, as well as cell subgroups, was evident in both cell types. Following 24 h interaction, uptake of HSA-Mag NP was about 10 times more efficient in glial cells than in activated promonocytes. The presented assays may facilitate detection and analysis of the amount of NPs within individual cells, as well as the rate of NP accumulation and processing in different subsets of living cells. Such data are crucial for estimating predicted drug dosage delivered by NPs, as well as to study possible mechanisms for NP interference with live cells.

  3. 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.

  4. Autogenic synthesis of green- and red-emitting single-phase Pr(2)O(2)CO(3) and PrO(1.833) luminescent nanopowders.

    PubMed

    Calderon Moreno, Jose M; Pol, Vilas G; Suh, Soong-Hyuck; Popa, Monica

    2010-11-01

    This Article reveals a rare synthesis of pure Pr(2)O(2)CO(3) (POC) nanopowder by thermolysis (700 °C) of a single chemical precursor in an autogenic reaction. The autogenic thermolysis of praseodymium acetate is a solvent-free, efficient, and straightforward approach yielding luminescent POC nanoparticles. The as-prepared POC nanopowder converted to PrO(1.833) (PO) powder via combustion. Methodical morphological, structural, and compositional characterizations of POC and PO powders are carried out, supported by mechanistic elucidation and the photoluminescent properties.

  5. Inventing atomic resolution scanning dielectric microscopy to see a single protein complex operation live at resonance in a neuron without touching or adulterating the cell.

    PubMed

    Agrawal, Lokesh; Sahu, Satyajit; Ghosh, Subrata; Shiga, Takashi; Fujita, Daisuke; Bandyopadhyay, Anirban

    2016-12-01

    A substantial ion flow in a normally wet protein masks any other forms of signal transmission. We use hysteresis and linear conduction (both are artifacts) as a marker to precisely wet a protein, which restricts the ionic conduction (hysteresis disappears), and at the same time, it is not denatured (quantized conductance and Raman spectra are intact). Pure electric visualization of proteins at work by eliminating the screening of ions, electrons, would change the way we study biology. Here we discuss the technical challenges resolved for imaging a protein or live cell using nonlinear dielectric response (spatial distribution of conductance, capacitance and phase, GCP trio). We electromagnetically triggered electrical, mechanical, thermal and ionic resonant vibrations in a protein. During resonant oscillations, we imaged the protein using resonant scanning tunneling microscopy of biomaterials (Brestum) and during ionic firing we imaged live what happens inside an axon core of a neuron by using our atomic scale scanning dielectric microscopy (Asadim). Both Asadim and Brestum are housed in a homebuilt scanning tunneling microscope (bio-STM) and a special micro-grid developed by us (patent JP-5187804) for fractal supercomputing. We found the trick to turn a membrane transparent and see inside without making any physical contact. We image live that a protein molecule adopts a unique configuration for each resonance frequency, - thus far unknown to biology. "Membrane alone fires" is found to be wrong after a century, micro-neuro-filaments communicate prior to firing to decide its necessity and then regulate it suitably. We introduce a series of technologies e.g., fractal grid, point contact, micro THz antenna, to discover that from atomic structure to a living cell, the biomaterials vibrate collectively.

  6. Long-term follow-up in adult living donors for combined liver/bowel transplant in pediatric recipients: a single center experience.

    PubMed

    Ghafari, Jamie L; Bhati, Chandra; John, Eunice; Tzvetanov, Ivo G; Testa, Giuliano; Jeon, Hoonbae; Oberholzer, Jose; Benedetti, Enrico

    2011-06-01

    Pediatric candidates for combined liver/bowel transplant (LBTx) experience a very high mortality on the cadaver waiting list. Our transplant center has successfully used adult living donors to treat pediatric candidates for LBTx. We report the long-term follow-up of this unique cohort of organ donors. The charts of six adult donors for LBTx performed between 2004 and 2007 were reviewed. All the pertinent clinical data were carefully reviewed and integrated with phone interviews of all donors. A total of six children (average age 13.5 months) received living donor LBTx. Average follow-up for the donors was 42 months (range 29-51). The donors' median age was 25 yr (19-32); five women and one man. The average median hospital stay was nine days. There were no peri-operative complications. At present all donors remain in good health. Three of the five mothers became pregnant after donation. Five of the six children are currently alive and well whereas one died with functioning grafts six months post-transplant due to plasmoblastic lymphoma. Living donor LBTx is an effective therapy for combined hepatic and intestinal failure in children less than five yr. The donor operation can be performed with minimal morbidity.

  7. Structural comparisons lead to the definition of a new superfamily of NAD(P)(H)-accepting oxidoreductases: the single-domain reductases/epimerases/dehydrogenases (the 'RED' family).

    PubMed Central

    Labesse, G; Vidal-Cros, A; Chomilier, J; Gaudry, M; Mornon, J P

    1994-01-01

    Using both primary- and tertiary-structure comparisons, we have established new structural similarities shared by reductases, epimerases and dehydrogenases not previously known to be related. Despite the low sequence identity (down to 10%), short consensus segments are identified. We show that the sequence, the active site and the supersecondary structure are well conserved in these proteins. New homologues (the protochlorophyllide reductases) are detected, and we define a new superfamily composed of single-domain dinucleotide-binding enzymes. Rules for the cofactor-binding specificity are deduced from our sequence alignment. The involvement of some amino acids in catalysis is discussed. Comparison with two-domain dehydrogenases allows us to distinguish two general mechanisms of divergent evolution. Images Figure 1 PMID:7998963

  8. High affinity scFv-hapten pair as a tool for quantum dot labeling and tracking of single proteins in live cells.

    PubMed

    Iyer, Gopal; Michalet, Xavier; Chang, Yun-Pei; Pinaud, Fabien F; Matyas, Stephanie E; Payne, Gregory; Weiss, Shimon

    2008-12-01

    We describe a general approach to label cell surface proteins using quantum dots (QD) for single-molecule tracking. QDs coated with small-hapten modified peptides are targeted to cell surface fusion proteins containing the corresponding single-chain fragment antibody (scFv). The approach is illustrated with the small hapten fluorescein (FL) and a high-affinity anti-FL scFv fused to two different proteins in yeast and murine neuronal cell line N2a.

  9. Pesa Large Red Dry Bean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Released in 2006, Pesa was derived from the single cross Rojo x Kablanketi made in Dec-Jan 1992-93. The parent ‘Rojo’ is a large red-seeded cultivar released by SUA in 1997. It has I bc-12 resistance to BCMV and BCMNV, resistance to the prevalent races of ALS, and moderate resistance to CBB, and H...

  10. Simultaneous Congo red decolorization and electricity generation in air-cathode single-chamber microbial fuel cell with different microfiltration, ultrafiltration and proton exchange membranes.

    PubMed

    Hou, Bin; Sun, Jian; Hu, Yong-you

    2011-03-01

    Different microfiltration membrane (MFM), proton exchange membrane (PEM) and ultrafiltration membranes (UFMs) with different molecular cutoff weights of 1K (UFM-1K), 5K (UFM-5K) and 10K (UFM-10K) were incorporated into air-cathode single-chamber microbial fuel cells (MFCs) which were explored for simultaneous azo dye decolorization and electricity generation to investigate the effect of membrane on the performance of the MFC. Batch test results showed that the MFC with an UFM-1K produced the highest power density of 324 mW/m(2) coupled with an enhanced coulombic efficiency compared to MFM. The MFC with UMF-10K achieved the fastest decolorization rate (4.77 mg/L h), followed by MFM (3.61 mg/L h), UFM-5K (2.38 mg/L h), UFM-1K (2.02 mg/Lh) and PEM (1.72 mg/Lh). These results demonstrated the possibility of using various membranes in the system described here, and showed that UFM-1K was the best one based on the consideration of both cost and performance.

  11. 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

  12. Red cell transfusion "trigger": a review.

    PubMed

    Petrides, Marian

    2003-07-01

    Despite the publication of several consensus guidelines that set forth recommendations for the transfusion of red cells, actual clinical practice continues to vary widely. Animal data and studies in human volunteers and patients support a red cell transfusion threshold of 7 to 8 g/dl in most patients. However, conflicting data, particularly in cardiac patients and in the elderly, suggest that it may be impossible to define a single red cell "trigger" for all patients. A well-designed, randomized, controlled trial is still needed to establish a safe threshold for red cell transfusion in adults with coronary artery disease.

  13. Unique and Protective Contributions of Parenting and Classroom Processes to the Adjustment of African American Children Living in Single-Parent Families.

    ERIC Educational Resources Information Center

    Brody, Gene H.; Dorsey, Shannon; Forehand, Rex; Armistead, Lisa

    2002-01-01

    Examined contributions parenting and classroom processes make to 7- to 15-year-olds' self-regulation and adjustment among single-parent Black families. Found that parenting and classroom processes contributed uniquely to children's adjustment through the development of self-regulation, and that each process could serve a protective or stabilizing…

  14. 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 review of…

  15. 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…

  16. Healthy Living

    MedlinePlus

    ... Environment & Health Healthy Living Pollution Reduce, Reuse, Recycle Science – How It Works The Natural World Games Brainteasers Puzzles Riddles Songs Activities Be a Scientist Coloring Science Experiments Stories Lessons Topics Games Activities Lessons MENU ...

  17. Bachelor Living

    ERIC Educational Resources Information Center

    Germer, Sondra

    1974-01-01

    Male high school students in a Bachelor Living Class observed methods of child care including bottle feeding, spoon feeding, changing diapers, and method of holding. The purpose was for the students to grasp a better understanding of child development. (EK)

  18. 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)

  19. Assisted Living

    MedlinePlus

    ... Transportation Back to top How to Choose a Facility? The following suggestions can help you get started ... for a safe, comfortable and appropriate assisted living facility: Think ahead. What will the resident’s future needs ...

  20. 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. ...

  1. Greener Living

    EPA Pesticide Factsheets

    Learn about how to live a more environmentally friendly life by reducing your environmental footprint, enhancing sustainability, using clean energy, water efficiency, composting, selecting a fuel efficient vehicle, and reducing waste.

  2. 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.

  3. 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

  4. 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.

  5. Mapping candidate QTLs related to plant persistency in red clover.

    PubMed

    Klimenko, Irina; Razgulayeva, Nadejda; Gau, Mitsuru; Okumura, Kenji; Nakaya, Akihiro; Tabata, Satoshi; Kozlov, Nicolay N; Isobe, Sachiko

    2010-04-01

    Red clover (Trifolium pratense L.) is a diploid (2n = 14), self-incompatible legume that is widely cultivated as a forage legume in cold geographical regions. Because it is a short-lived perennial species, improvement of plant persistency is the most important objective for red clover breeding. To develop a marker-assisted selection (MAS) approach for red clover, we identified candidate QTLs related to plant persistency. Two full-sib mapping populations, 272 x WF1680 and HR x R130, were used for QTL identification. Resistance to Sclerotinia trifoliorum and Fusarium species, as well as to winter hardiness, was investigated in the laboratory and in field experiments in Moscow region (Russia), and Sapporo (Japan). With the genotype data derived from microsatellite and other DNA markers, candidate QTLs were identified by simple interval mapping (SIM), Kruskal-Wallis analysis (KW analysis) and genotype matrix mapping (GMM). A total of 10 and 23 candidate QTL regions for plant persistency were identified in the 272 x WF1680 and the HR x R130 mapping populations, respectively. The QTLs identified by multiple mapping approaches were mapped on linkage group (LG) 3 and LG6. The significant QTL interactions identified by GMM explained the higher phenotypic variation than single effect QTLs. Identification of haplotypes having positive effect QTLs in each parent were first demonstrated in this study for pseudo-testcross mapping populations in plant species using experimental data.

  6. Single-molecule tracking in live Vibrio cholerae reveals that ToxR recruits the membrane-bound virulence regulator TcpP to the toxT promoter.

    PubMed

    Haas, Beth L; Matson, Jyl S; DiRita, Victor J; Biteen, Julie S

    2015-04-01

    Vibrio cholerae causes the human disease cholera by producing a potent toxin. The V. cholerae virulence pathway involves an unusual transcription step: the bitopic inner-membrane proteins TcpP and ToxR activate toxT transcription. As ToxT is the primary direct transcription activator in V. cholerae pathogenicity, its regulation by membrane-localized activators is key in the disease process. However, the molecular mechanisms by which membrane-localized activators engage the transcription process have yet to be uncovered in live cells. Here we report the use of super-resolution microscopy, single-molecule tracking, and gene knockouts to examine the dynamics of individual TcpP proteins in live V. cholerae cells with < 40 nm spatial resolution on a 50 ms timescale. Single-molecule trajectory analysis reveals that TcpP diffusion is heterogeneous and can be described by three populations of TcpP motion: one fast, one slow, and one immobile. By comparing TcpP diffusion in wild-type V. cholerae to that in mutant strains lacking either toxR or the toxT promoter, we determine that TcpP mobility is greater in the presence of its interaction partners than in their absence. Our findings support a mechanism in which ToxR recruits TcpP to the toxT promoter for transcription activation.

  7. Internet-based video-group delivery of Healthy Relationships--a "prevention with positives" intervention: report on a single group pilot test among women living with HIV.

    PubMed

    Marhefka, Stephanie L; Iziduh, Sharon; Fuhrmann, Hollie J; Lopez, Bernice; Glueckauf, Robert; Lynn, Vickie; Baldwin, Julie

    2013-01-01

    Women living with HIV (WLH) face challenges related to stigma, disclosure of HIV status, and negotiating safer sex. Several effective behavioral interventions, such as Healthy Relationships (HR), help WLH address these challenges and are disseminated by the USA Centers for Disease Control and Prevention's (CDC) Diffusion of Effective Behavioral Interventions project. However, many WLH living in poor urban or rural locations cannot access interventions such as HR because implementation is not feasible. Video-conferencing technology holds promise for expanding access to effective behavioral interventions for WLH. Following a systematic adaptation to the video-conferencing format, this pilot study tested the delivery of HR via video-group (VG) among WLH. The video-conferencing-based intervention, HR-VG, consisted of six, two-hour sessions led by two facilitators, and used structured activities and video-clips to build disclosure and safer sex skills. Four minority WLH received HR-VG at four different community-based intervention sites in a private room equipped with a video-phone for participating in HR-VG and a desktop computer for completing assessments via Audio Computer-Assisted Self Interview. Participants completed a baseline assessment prior to HR-VG and post-session assessment after each HR-VG session. The post-intervention assessment and video-focus group were completed following the last HR-VG session. Facilitators completed an assessment after each HR-VG session and an open-ended questionnaire following HR-VG. HR-VG was implemented in its entirety with minimal challenges. Both participants and facilitators reported feeling either "very comfortable" or "completely comfortable" with the technology and the overall intervention. Participants also reported high levels of unity and togetherness among the group. These preliminary findings suggest VG delivery of HR for WLH is both feasible and highly valued by participants. A follow-up randomized controlled trial

  8. 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.

  9. Live visualizations of single isolated tubulin protein self-assembly via tunneling current: effect of electromagnetic pumping during spontaneous growth of microtubule.

    PubMed

    Sahu, Satyajit; Ghosh, Subrata; Fujita, Daisuke; Bandyopadhyay, Anirban

    2014-12-03

    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.

  10. 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.

  11. Effect of donor age and parent-to-child transplant on living-related donor kidney transplantation: a single center's experience of 236 cases.

    PubMed

    Qiu, Jiang; Wang, Changxi; Liang, Xianwei; Chen, Guodong; Huang, Gang; Fu, Qian; Chen, Lizhong

    2015-07-01

    To study the impact of parent-to-child transplant and older donor age on recipients' post-transplant creatinine levels, a total of 236 patients who received living donor kidney transplantation were evaluated for kidney viability based on creatinine (Cr) level. Of the 236 pairings, 113 (48%) were parent-to-child followed by sibling transplants (66, 30%). Recipient Cr levels were significantly higher at 6 months and 3 years post-transplant in the parent-to-child transplants compared to other donor-recipient relationships. In addition, donor age (average age: 44.1 ± 11.5; range: 19-66) contributed to higher recipient post-transplant Cr levels (p < 0.01). Pre-transplant donor and recipient Cr levels tended to result in higher post-transplant Cr levels in recipients (p < 0.05). Multivariate logistic regression analysis revealed that the presence of both parent-to-child transplant and older donor significantly increased the risk of elevated post-transplant Cr levels in recipients with an estimated odds ratios ranging from 3.46 (95% CI: 1.71-6.98) at 6 months to 8.04 (3.14-20.56) at 3 years post-transplant. Donor age significantly affected transplant survival as measured by higher recipient post-transplant Cr levels. In addition, parent-to-child transplant pairings, along with older donor age, significantly increased the risk of elevated post-transplant Cr levels in recipients.

  12. Live visualizations of single isolated tubulin protein self-assembly via tunneling current: effect of electromagnetic pumping during spontaneous growth of microtubule

    PubMed Central

    Sahu, Satyajit; Ghosh, Subrata; Fujita, Daisuke; Bandyopadhyay, Anirban

    2014-01-01

    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. PMID:25466883

  13. Effect of small bowel bacterial overgrowth on the immunogenicity of single-dose live oral cholera vaccine CVD 103-HgR.

    PubMed

    Lagos, R; Fasano, A; Wasserman, S S; Prado, V; San Martin, O; Abrego, P; Losonsky, G A; Alegria, S; Levine, M M

    1999-11-01

    Several live oral vaccines (polio, bovine rotavirus, CVD 103-HgR cholera) are less immunogenic in developing than in industrialized countries. It was hypothesized that proximal small bowel bacterial overgrowth (common in children in less developed countries but rare in industrialized settings) diminishes the vibriocidal antibody response to CVD 103-HgR. In total, 202 fasting Santiago schoolchildren aged 5-9 years had lactulose breath H2 tests to detect proximal small bowel bacteria 1 day before ingesting CVD 103-HgR. Florid small bowel overgrowth was observed in 10 (5.6%) of 178 analyzable children. In children with florid overgrowth, vibriocidal seroconversion differed little from other children (60% vs. 67%), but the geometric mean titer was lower (160 vs. 368; P=.25). By logistic regression, increased peak breath H2 at small bowel time points was associated with diminished seroconversion (P=.04), as was the interaction of H2 value and weight (children >25 kg had lower seroconversion rates among subjects with heaviest overgrowth).

  14. Quantifying β-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells

    PubMed Central

    Kafri, Pinhas; Hasenson, Sarah E; Kanter, Itamar; Sheinberger, Jonathan; Kinor, Noa; Yunger, Sharon; Shav-Tal, Yaron

    2016-01-01

    Signal propagation from the cell membrane to a promoter can induce gene expression. To examine signal transmission through sub-cellular compartments and its effect on transcription levels in individual cells within a population, we used the Wnt/β-catenin signaling pathway as a model system. Wnt signaling orchestrates a response through nuclear accumulation of β-catenin in the cell population. However, quantitative live-cell measurements in individual cells showed variability in nuclear β-catenin accumulation, which could occur in two waves, followed by slow clearance. Nuclear accumulation dynamics were initially rapid, cell cycle independent and differed substantially from LiCl stimulation, presumed to mimic Wnt signaling. β-catenin levels increased simultaneously at adherens junctions and the centrosome, and a membrane-centrosome transport system was revealed. Correlating β-catenin nuclear dynamics to cyclin D1 transcriptional activation showed that the nuclear accumulation rate of change of the signaling factor, and not actual protein levels, correlated with the transcriptional output of the pathway. DOI: http://dx.doi.org/10.7554/eLife.16748.001 PMID:27879202

  15. Evaluation and improvement of a single nucleotide polymorphism-based PCR assay for rapid differentiation of live attenuated vaccine strains from field isolates of Erysipelothrix rhusiopathiae.

    PubMed

    Zhu, Weifeng; Li, Jingtao; Wang, Ya; Kang, Chao; Jin, Meilin; Chen, Huanchun

    2016-11-01

    A single nucleotide polymorphism-based PCR assay has been developed to differentiate the attenuated vaccine strain used in Japan from field isolates of Erysipelothrix rhusiopathiae found in pigs. However, this assay has been evaluated with only Japanese strains and isolates; therefore, it is unknown whether it could be used in other countries with E. rhusiopathiae strains and isolates of different genetic backgrounds. In our study, the PCR assay was evaluated using Chinese E. rhusiopathiae vaccine strains and field isolates. The PCR assay was able to differentiate the attenuated vaccine strains from the field isolates of E. rhusiopathiae in China but with a pattern different from that observed in Japan (only a single nucleotide polymorphism was detected in the Chinese vaccine strains compared with 5 in the Japanese vaccine strains). Importantly, either a DNA polymerase without 3' to 5' exonuclease activity or an exo(+) polymerase with an antibody inhibiting the proofreading activity was required. In conclusion, after evaluation and improvement, this fast differentiation assay can be extended from Japan to China.

  16. Nitric oxide scavenging by red cell microparticles.

    PubMed

    Liu, Chen; Zhao, Weixin; Christ, George J; Gladwin, Mark T; Kim-Shapiro, Daniel B

    2013-12-01

    Red cell microparticles form during the storage of red blood cells and in diseases associated with red cell breakdown and asplenia, including hemolytic anemias such as sickle cell disease. These small phospholipid vesicles that are derived from red blood cells have been implicated in the pathogenesis of transfusion of aged stored blood and hemolytic diseases, via activation of the hemostatic system and effects on nitric oxide (NO) bioavailability. Red cell microparticles react with the important signaling molecule NO almost as fast as cell-free hemoglobin, about 1000 times faster than red-cell-encapsulated hemoglobin. The degree to which this fast reaction with NO by red cell microparticles influences NO bioavailability depends on several factors that are explored here. In the context of stored blood preserved in ADSOL, we find that both cell-free hemoglobin and red cell microparticles increase as a function of duration of storage, and the proportion of extra erythrocytic hemoglobin in the red cell microparticle fraction is about 20% throughout storage. Normalized by hemoglobin concentration, the NO-scavenging ability of cell-free hemoglobin is slightly higher than that of red cell microparticles as determined by a chemiluminescence NO-scavenging assay. Computational simulations show that the degree to which red cell microparticles scavenge NO will depend substantially on whether they enter the cell-free zone next to the endothelial cells. Single-microvessel myography experiments performed under laminar flow conditions demonstrate that microparticles significantly enter the cell-free zone and inhibit acetylcholine, endothelial-dependent, and NO-dependent vasodilation. Taken together, these data suggest that as little as 5 μM hemoglobin in red cell microparticles, an amount formed after the infusion of one unit of aged stored packed red blood cells, has the potential to reduce NO bioavailability and impair endothelial-dependent vasodilation.

  17. Yellow and the Novel Aposematic Signal, Red, Protect Delias Butterflies from Predators.

    PubMed

    Wee, Jocelyn Liang Qi; Monteiro, Antónia

    2017-01-01

    Butterflies of the South Asian and Australian genus Delias possess striking colours on the ventral wings that are presumed to serve as warning signals to predators. However, this has not been shown empirically. Here we experimentally tested whether the colours of one member of this diverse genus, Delias hyparete, function as aposematic signals. We constructed artificial paper models with either a faithful colour representation of D. hyparete, or with all of its colours converted to grey scale. We also produced models where single colours were left intact, while others were converted to grey-scale or removed entirely. We placed all model types simultaneously in the field, attached to a live mealworm, and measured relative attack rates at three separate field sites. Faithful models of D. hyparete, suffered the least amount of attacks, followed by grey-scale models with unaltered red patches, and by grey-scale models with unaltered yellow patches. We conclude that red and yellow colours function as warning signals. By mapping dorsal and ventral colouration onto a phylogeny of Delias, we observed that yellow and red colours appear almost exclusively on the ventral wing surfaces, and that basal lineages have mostly yellow, white, and black wings, whereas derived lineages contain red colour in addition to the other colours. Red appears to be, thus, a novel adaptive trait in this lineage of butterflies.

  18. Yellow and the Novel Aposematic Signal, Red, Protect Delias Butterflies from Predators

    PubMed Central

    Wee, Jocelyn Liang Qi

    2017-01-01

    Butterflies of the South Asian and Australian genus Delias possess striking colours on the ventral wings that are presumed to serve as warning signals to predators. However, this has not been shown empirically. Here we experimentally tested whether the colours of one member of this diverse genus, Delias hyparete, function as aposematic signals. We constructed artificial paper models with either a faithful colour representation of D. hyparete, or with all of its colours converted to grey scale. We also produced models where single colours were left intact, while others were converted to grey-scale or removed entirely. We placed all model types simultaneously in the field, attached to a live mealworm, and measured relative attack rates at three separate field sites. Faithful models of D. hyparete, suffered the least amount of attacks, followed by grey-scale models with unaltered red patches, and by grey-scale models with unaltered yellow patches. We conclude that red and yellow colours function as warning signals. By mapping dorsal and ventral colouration onto a phylogeny of Delias, we observed that yellow and red colours appear almost exclusively on the ventral wing surfaces, and that basal lineages have mostly yellow, white, and black wings, whereas derived lineages contain red colour in addition to the other colours. Red appears to be, thus, a novel adaptive trait in this lineage of butterflies. PMID:28060944

  19. 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.

  20. Single-Center Experience of ABO-Incompatible Living-Donor Liver Transplantation With a New Simplified Intravenous Immunoglobulin Protocol: A Propensity Score-Matching Analysis.

    PubMed

    Kim, J D; Choi, D L; Kim, S-G; Lee, A-J

    2016-05-01

    The outcomes of patients who undergo ABO-incompatible (ABO-I) living-donor liver transplantation (LDLT) have markedly improved as strategies have become more innovative and advanced. Here, we describe 25 cases of ABO-I LDLT with a simplified protocol and compare the outcomes to those of ABO-compatible LDLT. We analyzed outcomes via a retrospective review of 182 adult LDLT cases including 25 ABO-I LDLTs from January 2011 to December 2014. Propensity scoring was used to compare the groups. The desensitization protocol included plasma exchange, rituximab, and intravenous immunoglobulin without local infusion therapy. The triple immunosuppression protocol consisted of tacrolimus and steroids with mycophenolate mofetil; a splenectomy was not routinely performed. The median age of recipients was 51 years (range, 35-66 years), and the median mean Model for End-Stage Liver Disease (MELD) score was 15 (range, 7-37). The initial ranges of isoagglutinin IgM and IgG titers were 1:1 to 1:256 and 1:4 to 1:2048, respectively. There were no significant differences in patient demographics or perioperative variables between the groups. Although significant rebound elevation in anti-ABO antibody during the postoperative period was observed in 3 cases, neither C4d staining nor clinical signs of antibody-mediated rejection was apparent in these cases. No diffuse intrahepatic biliary stricture was encountered in any ABO-I LDLT patient within a mean follow-up of 22.6 ± 17.2 months. Moreover, no significant difference in overall or graft survival was observed between the groups. ABO-I LDLT can be performed safely under this new simplified protocol and may be proposed when ABO-compatible donors are unavailable.

  1. 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.

  2. Independent Living.

    ERIC Educational Resources Information Center

    Nathanson, Jeanne H., Ed.

    1994-01-01

    This issue of "OSERS" addresses the subject of independent living of individuals with disabilities. The issue includes a message from Judith E. Heumann, the Assistant Secretary of the Office of Special Education and Rehabilitative Services (OSERS), and 10 papers. Papers have the following titles and authors: "Changes in the…

  3. 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…

  4. Living History

    ERIC Educational Resources Information Center

    Walsh, Mark

    2005-01-01

    John Tinker and Mary Beth Tinker are back in a classroom in their hometown, once again wearing black armbands and drawing attention to a war. Now in their 50s, the siblings are living symbols of constitutional rights for secondary school students. In 1965, they and a handful of others were suspended for wearing black armbands to their public…

  5. Robust and Balanced Immune Responses to All 4 Dengue Virus Serotypes Following Administration of a Single Dose of a Live Attenuated Tetravalent Dengue Vaccine to Healthy, Flavivirus-Naive Adults

    PubMed Central

    Kirkpatrick, Beth D.; Durbin, Anna P.; Pierce, Kristen K.; Carmolli, Marya P.; Tibery, Cecilia M.; Grier, Palmtama L.; Hynes, Noreen; Diehl, Sean A.; Elwood, Dan; Jarvis, Adrienne P.; Sabundayo, Beulah P.; Lyon, Caroline E.; Larsson, Catherine J.; Jo, Matthew; Lovchik, Janece M.; Luke, Catherine J.; Walsh, Mary C.; Fraser, Ellen A.; Subbarao, Kanta; Whitehead, Steven S.

    2015-01-01

    Background. The 4 serotypes of dengue virus, DENV-1–4, are the leading cause of arboviral disease globally. The ideal dengue vaccine would provide protection against all serotypes after a single dose. Methods. Two randomized, placebo-controlled trials were performed with 168 flavivirus-naive adults to demonstrate the safety and immunogenicity of a live attenuated tetravalent dengue vaccine (TV003), compared with those of a second tetravalent vaccine with an enhanced DENV-2 component (TV005), and to evaluate the benefit of a booster dose at 6 months. Safety data, viremia, and neutralizing antibody titers were evaluated. Results. A single dose of TV005 elicited a tetravalent response in 90% of vaccinees by 3 months after vaccination and a trivalent response in 98%. Compared with TV003, the higher-dose DENV-2 component increased the observed frequency of immunogenicity to DENV-2 in the TV005 trial. Both the first and second doses were well tolerated. Neither vaccine viremia, rash, nor a significant antibody boost were observed following a second dose. Conclusions. A single subcutaneous dose of TV005 dengue vaccine is safe and induces a tetravalent antibody response at an unprecedented frequency among vaccinees. A second dose has limited benefit and appears to be unnecessary. Studies to confirm these findings and assess vaccine efficacy will now move to populations in regions where DENV transmission is endemic. Clinical Trials Registration. NCT01072786 and NCT01436422. PMID:25801652

  6. 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.

  7. Utilisation patterns and cost of hospital care for people living with HIV in Ireland in 2012: a single-centre study.

    PubMed

    Brennan, Aline; Horgan, Mary; Jackson, Arthur; Browne, John P; Bergin, Colm J

    2017-03-01

    Data on the pattern and cost of health service use by HIV patients are required for evaluations of the cost-effectiveness of new drugs and technologies as well as being essential for service planning. The aim of this study was to identify the utilisation patterns and cost of hospital care for HIV patients in a single centre in Ireland in 2012. Data on the frequency and non-drug costs of all hospital resources used by HIV patients were extracted from a hospital activity-based costing system. Cost data were analysed using a generalised linear model. A total of 328 patients, 3672 patient months, were included in this study. Patients had a mean of 4.4 scheduled infectious disease outpatient appointments per patient year; 37% of patients also used another outpatient service, 15% in-patient services, 4% day-case service and 18% emergency department services in 2012. Patients with very advanced HIV disease continue to incur a disproportionate amount of the total cost of providing care. This study provides baseline utilisation and cost data for use of both infectious-disease and non-infectious disease hospital services and will be useful for service planning in light of the likely increases in resource demands.

  8. Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells.

    PubMed Central

    Tramier, Marc; Gautier, Isabelle; Piolot, Tristan; Ravalet, Sylvie; Kemnitz, Klaus; Coppey, Jacques; Durieux, Christiane; Mignotte, Vincent; Coppey-Moisan, Maïté

    2002-01-01

    By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG. PMID:12496124

  9. 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.

  10. Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process.

    PubMed Central

    Hofer, A M; Landolfi, B; Debellis, L; Pozzan, T; Curci, S

    1998-01-01

    Free [Ca2+] in agonist-sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK-21 fibroblasts were loaded with the low-affinity fluorescent calcium indicator mag-fura-2-AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist- and thapsigargin-sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA-AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre-loading the cells with BAPTA-AM permitted efficient refilling in Ca2+-free, EGTA-containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA-AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+]. PMID:9524121

  11. 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.

  12. Red and Green Fluorescence from Oral Biofilms

    PubMed Central

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

    2016-01-01

    Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries. PMID:27997567

  13. Red blood cell production

    MedlinePlus

    ... hemocytoblasts give rise to all of the formed elements in blood. If a hemocytoblast commits to becoming a cell called a proerythroblast, it will develop into a new red blood cell. The formation of a red ...

  14. Human semen as an early, sensitive biomarker of highly polluted living environment in healthy men: A pilot biomonitoring study on trace elements in blood and semen and their relationship with sperm quality and RedOx status.

    PubMed

    Bergamo, Paolo; Volpe, Maria Grazia; Lorenzetti, Stefano; Mantovani, Alberto; Notari, Tiziana; Cocca, Ennio; Cerullo, Stefano; Di Stasio, Michele; Cerino, Pellegrino; Montano, Luigi

    2016-12-01

    The Campania region in Italy is facing an environmental crisis due to the illegal disposal of toxic waste. Herein, a pilot study (EcoFoodFertility initiative) was conducted to investigate the use of human semen as an early biomarker of pollution on 110 healthy males living in various areas of Campania with either high or low environmental impact. The semen from the "high impact" group showed higher zinc, copper, chromium and reduced iron levels, as well as reduced sperm motility and higher sperm DNA Fragmentation Index (DFI). Redox biomarkers (total antioxidant capacity, TAC, and glutathione, GSH) and the activity of antioxidant enzymes in semen were lower in the "high impact" group. The percentage of immotile spermatozoa showed a significant inverse correlation with TAC and GSH. Overall, several semen parameters (reduced sperm quality and antioxidant defenses, altered chemical element pattern), which were associated with residence in a high polluted environment, could be used in a further larger scale study, as early biomarkers of environmental pollution.

  15. Redox alters yellow dragonflies into red.

    PubMed

    Futahashi, Ryo; Kurita, Ryoji; Mano, Hiroaki; Fukatsu, Takema

    2012-07-31

    Body color change associated with sexual maturation--so-called nuptial coloration--is commonly found in diverse vertebrates and invertebrates, and plays important roles for their reproductive success. In some dragonflies, whereas females and young males are yellowish in color, aged males turn vivid red upon sexual maturation. The male-specific coloration plays pivotal roles in, for example, mating and territoriality, but molecular basis of the sex-related transition in body coloration of the dragonflies has been poorly understood. Here we demonstrate that yellow/red color changes in the dragonflies are regulated by redox states of epidermal ommochrome pigments. Ratios of reduced-form pigments to oxidized-form pigments were significantly higher in red mature males than yellow females and immature males. The ommochrome pigments extracted from the dragonflies changed color according to redox conditions in vitro: from red to yellow in the presence of oxidant and from yellow to red in the presence of reductant. By injecting the reductant solution into live insects, the yellow-to-red color change was experimentally reproduced in vivo in immature males and mature females. Discontinuous yellow/red mosaicism was observed in body coloration of gynandromorphic dragonflies, suggesting a cell-autonomous regulation over the redox states of the ommochrome pigments. Our finding extends the mechanical repertoire of pigment-based body color change in animals, and highlights an impressively simple molecular mechanism that regulates an ecologically important color trait.

  16. Redox alters yellow dragonflies into red

    PubMed Central

    Futahashi, Ryo; Kurita, Ryoji; Mano, Hiroaki; Fukatsu, Takema

    2012-01-01

    Body color change associated with sexual maturation—so-called nuptial coloration—is commonly found in diverse vertebrates and invertebrates, and plays important roles for their reproductive success. In some dragonflies, whereas females and young males are yellowish in color, aged males turn vivid red upon sexual maturation. The male-specific coloration plays pivotal roles in, for example, mating and territoriality, but molecular basis of the sex-related transition in body coloration of the dragonflies has been poorly understood. Here we demonstrate that yellow/red color changes in the dragonflies are regulated by redox states of epidermal ommochrome pigments. Ratios of reduced-form pigments to oxidized-form pigments were significantly higher in red mature males than yellow females and immature males. The ommochrome pigments extracted from the dragonflies changed color according to redox conditions in vitro: from red to yellow in the presence of oxidant and from yellow to red in the presence of reductant. By injecting the reductant solution into live insects, the yellow-to-red color change was experimentally reproduced in vivo in immature males and mature females. Discontinuous yellow/red mosaicism was observed in body coloration of gynandromorphic dragonflies, suggesting a cell-autonomous regulation over the redox states of the ommochrome pigments. Our finding extends the mechanical repertoire of pigment-based body color change in animals, and highlights an impressively simple molecular mechanism that regulates an ecologically important color trait. PMID:22778425

  17. 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 ...

  18. Acaricidal activities of clove bud oil and red thyme oil using microencapsulation against HDMs.

    PubMed

    Kim, Joo Ran; Sharma, Suraj

    2011-01-01

    The purpose of this study was to produce a safer microcapsule loaded with clove bud oil and red thyme oil to reduce the population of house dust mites (HDMs). Gelatin-based microcapsules 4-85 µm in size were created, with agitation speed and type of oil playing a critical role in governing their size. Microcapsules made up of single spherical units less than 30 µm in diameter remained separate on the fibre, whereas larger microcapsules of over 30 µm ruptured or aggregated. Thermogravimetric analysis (TGA) demonstrated that microcapsules containing red thyme oil showed a more consistent range of oil loading, from 50 to 80%, than microcapsules containing clove bud oil, which ranged from 30 to 80% (more deviated). Mortality tests on Dermatophagoides farinae conducted on fabric with attached microcapsules showed that clove bud oil, containing a more phenolic monoterpenoid (eugenol), was more effective at reducing the live HDMs (94% mortality).

  19. Gendered Living Arrangements among Children with Disabilities

    ERIC Educational Resources Information Center

    Cohen, Philip N.; Petrescu-Prahova, Miruna

    2006-01-01

    Using data on disabilities from the 2000 Census, we found a consistent pattern of living arrangements that leaves children (aged 5-15 years) with disabilities living disproportionately with women. Children with disabilities are more likely to live with single parents, and especially their mothers, than are other children. Further, those who do not…

  20. The effect of red palm olein and refined palm olein on lipids and haemostatic factors in hyperfibrinogenaemic subjects.

    PubMed

    Scholtz, Susanna C; Pieters, Marlien; Oosthuizen, Welma; Jerling, Johann C; Bosman, Magdalena J C; Vorster, Hester H

    2004-01-01

    Little is known about the physiological effects of red palm olein (RPO). The effects of red palm olein and palm olein (POL) compared to sunflower oil (SFO), on lipids, haemostatic factors and fibrin network characteristics in hyperfibrinogenaemic volunteers were investigated. Fifty-nine free-living, hyperfibrinogenaemic volunteers participated in this randomized, controlled, single blind parallel study. After a 4-week run-in, during which subjects received sunflower oil products, they were paired and randomly assigned to one of three intervention groups receiving products containing 25 g/day ( approximately 12% of total energy intake) of either red palm olein, palm olein or sunflower oil for another 4 weeks. Anthropometric measurements, blood samples and dietary intakes were measured before run-in, and before and after intervention. The differences in changes in total serum cholesterol response between palm olein and red palm olein (+0.59 vs. +0.18 mmol/l; p=0.053), and between palm olein and sunflower oil (+0.59 vs. -0.003 mmol/l; p < or =0.01) were significant. The low-density lipoprotein cholesterol (LDLC) response in the palm olein-and sunflower oil-groups also differed significantly (+0.42 vs. -0.11 mmol/l; p < or =0.01). Tissue plasminogen activator antigen (tPA(ag)) decreased significantly in the red palm olein group compared to the palm olein-and sunflower oil-groups. No effects were found in other haemostatic variables. Palm olein and red palm olein had no independent effect on fibrin network characteristics. In conclusion, compared to palm olein, red palm olein had less detrimental effects on the lipid profile and decreased tissue plasminogen activator antigen. Studies in larger groups are advised for confirmation of results, elucidation of mechanisms and effects of nonglyceride constituents of red palm oil (PO).

  1. Establishment of adult Parelaphostrongylus tenuis, patent infections, and acquired immunity after experimental infection of white-tailed deer (Odocoileus virginianus) and red deer (Cervus elaphus elaphus).

    PubMed

    Duffy, Michael S; Greaves, Trent A; Burt, Michael D B

    2004-04-01

    Experimental Parelaphostrongylus tenuis infections were established in white-tailed deer (Odocoileus virginianus) and an atypical host, red deer (Cervus elaphus elaphus). Groups of deer were fed 10, 25, or 100 third-stage larvae (L3) of P. tenuis and received a single equivalent challenge exposure at varying intervals. Infections were monitored up to 6 yr in white-tailed deer and up to 2.8 yr in red deer. The prepatent period in white-tailed deer varied from 91 to 1,072 days (381 +/- 374) and in red deer from 105 to 358 days (167 +/- 77). Adult worms lived for up to 6 yr in white-tailed deer. Although most had patent infections until necropsy, latent periods were observed regardless of season. Adult worms lived for up to 2.8 yr in red deer, and patent infections persisted for 20-363 days (152 +/- 106). Patent infections were correlated with the presence of adult worms in blood vessels and sinuses of both deer species. Worms were restricted to the subdural space in all deer with latent and occult infections. Adult worm recovery in white-tailed deer fed 10 or 25 L3 corresponded to the mean intensities reported in natural infections of white-tailed deer Recovery from deer fed 100 L3 was not typical of natural infection intensities. Adult P. tenuis established in all groups of red deer, but neurologic disease was restricted to animals fed 100 L3. Acute neurologic disease was associated with subdural hemorrhage and occurred at 11 mo postinfection in 2 red deer. The absence of postchallenge patent periods and the persistence of occult infections indicated that challenge exposures did not establish. These data indicate that acquired immunity to P. tenuis was established by 6 mo postinfection in both white-tailed and red deer. Latent periods in white-tailed deer and latent infections in red deer reinforce the need for a reliable diagnostic assay.

  2. Single-Molecule Visualization of Living Polymerization

    DTIC Science & Technology

    2014-02-18

    siloxane bond†. Langmuir 24, 1343-1349 (2007). 4 M. Scholl, S. Ding, C. W. Lee & R. H. Grubbs. Synthesis and activity of a new generation of ruthenium ...Vougioukalakis & G. R. H. Ruthenium -based heterocyclic carbene-coordinated olefin metathesis catalysts. Chem Rev 110 (2010). 6 T. Lionnet, J. F. Allemand...2012). 10 X. Bantreil & S. P. Nolan. Synthesis of n-heterocyclic carbene ligands and derived ruthenium olefin metathesis catalysts. Nat Protoc 6, 69

  3. 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.

  4. 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.

  5. 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

  6. Intraocular live male filarial Loa loa worm.

    PubMed

    Eballe, André Omgbwa; Epée, Emillienne; Koki, Godefroy; Owono, Didier; Mvogo, Côme Ebana; Bella, Assumpta Lucienne

    2008-12-01

    We report a case of Loa loa filariasis in an 8-month-old child who presented with a 3-month history of irritated acute red eye and insomnia. Examination revealed a living and active adult Loa loa worm in the anterior chamber of the left eye. The worm was extracted under general anesthetic.

  7. 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

  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.

  9. 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.

  10. 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

  11. An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

    NASA Astrophysics Data System (ADS)

    Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

    2016-10-01

    Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

  12. 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.

  13. 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)

  14. 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.

  15. 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

  16. 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…

  17. 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.

  18. Stretching of red blood cells using an electro-optics trap

    PubMed Central

    Haque, Md. Mozzammel; Moisescu, Mihaela G.; Valkai, Sándor; Dér, András; Savopol, Tudor

    2014-01-01

    The stretching stiffness of Red Blood Cells (RBCs) was investigated using a combination of an AC dielectrophoretic apparatus and a single-beam optical tweezer. The experiments were performed at 10 MHz, a frequency high enough to avoid conductivity losses, but below the second turnover point between positive and negative dielectrophoresis. By measuring the geometrical parameters of single healthy human RBCs as a function of the applied voltage, the elastic modulus of RBCs was determined (µ = 1.80 ± 0.5 µN/m) and compared with similar values of the literature got by other techniques. The method is expected to be an easy-to-use, alternative tool to determine the mechano-elastic properties of living cells, and, on this basis, to distinguish healthy and diseased cells PMID:25657880

  19. High Red Blood Cell Count

    MedlinePlus

    Symptoms High red blood cell count By Mayo Clinic Staff A high red blood cell count is an increase in oxygen-carrying cells in your bloodstream. Red blood cells transport oxygen from your lungs to tissues throughout ...

  20. 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/ ...

  1. 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

  2. Characterization of MSB synapses in dissociated hippocampal culture with simultaneous pre- and postsynaptic live microscopy.

    PubMed

    Reilly, James E; Hanson, Hugo H; Fernández-Monreal, Mónica; Wearne, Susan L; 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.

  3. Videomicrofluorometry on living cells and discriminant factorial analysis to study cell cycle distributions.

    PubMed

    Savatier, J; Gbankoto, A; Vigo, J; Salmon, J M

    2004-01-01

    After a rapid overview of the approaches used to study cell cycle, a fluorescent digital imaging microscopy method is proposed. This method is improved by a factorial analysis relying on the evaluation of several parameters recorded on each living cell. Single lympho-blastoid living cells are labeled with three fluorescent markers: Hoechst 33342 for nuclear DNA, Rhodamine 123 for mitochondria and Nile Red for plasma membrane. For each cell, morphological and functional information parameters are obtained. A typological analysis is used to separate control cells into four groups: G0-G1, S, G2+M and polyploid cells Gn. These control cells define a learning population used to analyze untreated and adriamycine treated cells as supplementary individuals in a discriminant factorial analysis. Such an approach allows to accurately evidence the change of the values of some cellular parameters.

  4. 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.…

  5. Inhibition of Neutral red photolysis with different antioxidants.

    PubMed

    Rimpapa, Zlatan; Sofić, Emin; Sapcanin, Aida; Toromanović, Jasmin; Tahirović, Ismet

    2007-02-01

    Neutral red is a dye the azine structure which has been used as an acido-base indicator and a dye in histochemistry. In 1960 Goldhaber introduced Neutral red into the medium of resorbing bone cultures to localize the osteoclast in the living cultures. Using time-lapse microcinematography in order to follow the osteoclasts, he reported excellent contrast could be obtained with Neutral red due to the avidity of osteoclasts for this dye. Unfortunately, however, the photodynamic effect resulting from subsequent exposure of these cultures to light precluded this approach, and again in 1963. it was observed that the death of the osteoclasts was probably due to a photodynamic effect related to the dye in the cell, the presence of oxygen and the frequent exposure of light by our time-lapse photography. VIS and UV irradiation induced photolysis of Neutral red, and from Neutral red cation produced with photons a Neutral red radical. This Neutral red radical can be inhibited with action of an antioxidant, such as melatonin, glutathione, ascorbic acid, E vitamin, etc. We developed an assay with Neutral red photolysis which utilizes a VIS and UV irradiation technique for quantification the inhibition of photolysis with action of an antioxidant. In this method Neutral red acts double, as a free radical generator and as a photosensitizer.

  6. 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 ...

  7. Red Hill Updates

    EPA Pesticide Factsheets

    This and other periodic updates are intended to keep the public informed on major progress being made to protect public health and the environment at the Red Hill Underground Fuel Storage Facility in Hawaii.

  8. Aurora Australis, Red Crown

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This view of the Aurora Australis or Southern Lights (location unknown) shows a spiked band of red airglow called a 'Red Crown' above the Earth Limb. Calculated to be in the 80 - 120 km altitude region, auroral activity is due to exitation of atomic oxygen in the upper atmosphere by radiation from the van Allen Radiation Belts and is most common above the 65 degree north and south latitude range during the spring and fall of the year.

  9. 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

  10. Egypt and Red Sea

    NASA Technical Reports Server (NTRS)

    1982-01-01

    A panaramic view of eastern Egypt, The Red Sea and Saudi Arabia beyond (24.0N, 33.0E). In this desert country, where water is life, the high Aswan Dam and the impounded waters of the Nile River in the foreground assure water availability into the next century. The Red Sea beyond, part of the Suez Canal seaway, serves as a commercial link to the world and separates Egypt from Saudi Arabia.

  11. 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 ...

  12. Wavelength dependent specific plasmon resonance coupling of single silver nanoparticles with EGFP.

    PubMed

    Lee, Kerry J; Huang, Tao; Nallathamby, Prakash D; Xu, Xiao-Hong Nancy

    2015-11-14

    Noble metal nanoparticles (NPs) possess unique plasmonic properties, enabling them to serve as sub-diffraction light sources and nano- antennae for a wide range of applications. Here we report the specific interaction of single Ag NPs with single EGFP molecules and a high dependence of their interaction upon localized-surface-plasmon-resonance (LSPR) spectra of single Ag NPs and EGFP. The LSPR spectra of single red Ag NPs show a stunning 60 nm blue-shift during their incubation with EGFP, whereas they remain unchanged during their incubation with bovine serum albumin (BSA). Interestingly, the peak wavelengths of the LSPR spectra of green and blue Ag NPs remain essentially unchanged during their incubation with either EGFP or BSA. These interesting findings suggest that plasmon-resonance-energy-transfer (PRET) from single Ag NPs to EGFP might follow a two-photon excitation mechanism to excite EGFP and the fluorescence of the excited EGFP might couple with the plasmon of single NPs leading to a blue-shift of the red NPs. These distinctive phenomena are only observed by real-time single NP spectroscopic measurements. This study offers exciting new opportunities to design new sensing and imaging tools with high specificity and sensitivity to study long-range molecular interactions and dynamic events in single live cells, and to probe the underlying molecular mechanisms of PRET.

  13. Wavelength dependent specific plasmon resonance coupling of single silver nanoparticles with EGFP

    NASA Astrophysics Data System (ADS)

    Lee, Kerry J.; Huang, Tao; Nallathamby, Prakash D.; Xu, Xiao-Hong Nancy

    2015-10-01

    Noble metal nanoparticles (NPs) possess unique plasmonic properties, enabling them to serve as sub-diffraction light sources and nano- antennae for a wide range of applications. Here we report the specific interaction of single Ag NPs with single EGFP molecules and a high dependence of their interaction upon localized-surface-plasmon-resonance (LSPR) spectra of single Ag NPs and EGFP. The LSPR spectra of single red Ag NPs show a stunning 60 nm blue-shift during their incubation with EGFP, whereas they remain unchanged during their incubation with bovine serum albumin (BSA). Interestingly, the peak wavelengths of the LSPR spectra of green and blue Ag NPs remain essentially unchanged during their incubation with either EGFP or BSA. These interesting findings suggest that plasmon-resonance-energy-transfer (PRET) from single Ag NPs to EGFP might follow a two-photon excitation mechanism to excite EGFP and the fluorescence of the excited EGFP might couple with the plasmon of single NPs leading to a blue-shift of the red NPs. These distinctive phenomena are only observed by real-time single NP spectroscopic measurements. This study offers exciting new opportunities to design new sensing and imaging tools with high specificity and sensitivity to study long-range molecular interactions and dynamic events in single live cells, and to probe the underlying molecular mechanisms of PRET.

  14. 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…

  15. 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…

  16. [Health effects of living habits].

    PubMed

    Vuori, Ilkka

    2015-01-01

    Single healthy living habits such as non-smoking and regular physical activity decrease the risk of common non-communicable diseases, unsuccessful aging and premature death to a small to moderate degree. Their cumulative effects are, however, large. Only a small minority of people adhere well to all healthy living habits or even the healthiest ones. Consequently, the population attributable fractions of major public health problems due to unhealthy lifestyles are large. Substantial improvement of public health calls for policies and programs to influence the root causes of the lifestyles in the multiple environments and systems where they are developed, maintained, and changed.

  17. Heavy elements and mixing in red giants

    NASA Astrophysics Data System (ADS)

    Smith, Verne V.

    A brief overview of the s-process in red giants is presented, followed by discussions of three specific topics involving heavy-element s-process nucleosynthesis and mixing in red giants: (1) a comparison of neutron densities derived from observations and from the most recent stellar models, (2) how observations of technetium in S stars have led to a natural division of these stars into two separate groups, one of which is the result of single-star stellar evolution while the other is the result of mass transfer in a binary system, (3) a brief discussion of the recent speculative suggestion that gamma-ray induced photofission of heavy elements (Th and U) might be a source of the Tc observed in certain types of red giants.

  18. First Letters from Red Cross Nurses.

    PubMed

    2017-03-01

    Editor's note: From its first issue in 1900 to the present day, AJN has unparalleled archives detailing nurses' work and lives over more than a century. These articles not only chronicle nursing's growth as a profession within the context of the events of the day, but they also reveal prevailing societal attitudes about women, health care, and human rights. Today's nursing school curricula rarely include nursing's history, but it's a history worth knowing. To this end, From the AJN Archives highlights articles selected to fit today's topics and times.In this month's excerpt from a November 1914 article, Jane A. Delano, chair of the National Committee on Red Cross Nursing Service, describes the mobilization of 138 American Red Cross nurses for wartime service in England, France, Germany, Austria, and Russia. The nurses sailed together on a Red Cross steamer out of New York Harbor, and posted letters from the ship back to AJN. A nurse from Unit A (made up of Philadelphia nurses and assigned to France) wrote, "The days have been truly busy ones, more like the training school…" Their daily schedule included "lectures by doctors," quizzes, skills practice, and classes in French or German. To read the full article, go to http://bit.ly/2k7KR1U.For more than a century, American Red Cross nurses have provided care during wars and disasters and brought health care to underserved parts of the United States. The month of March is considered Red Cross Month, and we salute the staff and volunteers who continue this noble work. For more on the Red Cross in this issue, see On the Cover and Best of the Blog.

  19. 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.

  20. Optical modulation of single walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Strano, Michael S.

    2007-03-01

    Recent advances in the spectroscopy of single walled carbon nanotubes have significantly enhanced our ability to understand and control their surface chemistry, both covalently and non-covalently. Our work has focused on modulating the optical properties of semiconducting single walled carbon nanotubes as near infrared photoluminescent sensors for chemical analysis. Molecular detection using near-infrared light between 0.9 and 1.3 eV has important biomedical applications because of greater tissue penetration and reduced auto-fluorescent background in thick tissue or whole-blood media. In one system, the transition of DNA secondary structure modulates the dielectric environment of the single-walled carbon nanotube (SWNT) around which it is adsorbed. The SWNT band-gap fluorescence undergoes a red shift when an encapsulating 30-nucleotide oligomer is exposed to counter ions that screen the charged backbone. We demonstrate the detection of the mercuric ions in whole blood, tissue, and from within living mammalian cells using this technology. Similar results are obtained for DNA hybridization and the detection of single nucleotide polymorphism. We also report the synthesis and successful testing of near-infrared β-D-glucose sensors2 that utilize a different mechanism: a photoluminescence modulation via charge transfer. The results demonstrate new opportunities for nanoparticle optical sensors that operate in strongly absorbing media of relevance to medicine or biology.

  1. Green-to-red primed conversion of Dendra2 using blue and red lasers.

    PubMed

    Klementieva, N V; Lukyanov, K A; Markina, N M; Lukyanov, S A; Zagaynova, E V; Mishin, A S

    2016-11-18

    Recently, an unusual phenomenon of primed conversion of fluorescent protein Dendra2 by combined action of blue (488 nm) and near-infrared (700-780 nm) lasers was discovered. Here we demonstrate that primed conversion can be induced by red lasers (630-650 nm) common for most confocal and single molecule detection microscopes.

  2. 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.

  3. 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

  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. Demonstration project cuts red tape

    NASA Astrophysics Data System (ADS)

    Katzoff, Judith A.

    Results from a demonstration project aimed at cutting red tape in federal grants for basic research at universities are "very encouraging" so far, according to Don I. Phillips, executive director of the Government-University-Industry Research Roundtable. The roundtable is coordinating the experimental effort, with the participation of five major federal agencies as well as the Florida state university system and the University of Miami (a private institution in Coral Gables, Fla.).Under the project, special conditions apply to grants from the participating agencies to individual researchers at the participating universities: Investigators do not have to ask the agency's permission for changes in budget allocations, purchases of permanent equipment, or foreign travel. Grant money can be spent up to a year after the grant ends without a special request. Under this plan, researchers, with agency approval, can also ask to have their entire research program covered as a single administrative entity, rather than as several individual projects.

  6. 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.

  7. Red giants: then and now

    NASA Astrophysics Data System (ADS)

    Faulkner, John

    Fred Hoyle's work on the structure and evolution of red giants, particularly his pathbreaking contribution with Martin Schwarzschild (Hoyle and Schwarzschild 1955), is both lauded and critically assessed. In his later lectures and work with students in the early 1960s, Hoyle presented more physical ways of understanding some of the approximations used, and results obtained, in that seminal paper. Although later ideas by other investigators will be touched upon, Hoyle's viewpoint - that low-mass red giants are essentially white dwarfs with a serious mass-storage problem - is still extremely fruitful. Over the years, I have further developed his method of attack. Relatively recently, I have been able to deepen and broaden the approach, finally extending the theory to provide a unifying treatment of the structure of low-mass stars from the main sequence though both the red-giant and horizontal-branch phases of evolution. Many aspects of these stars that had remained puzzling, even mysterious, for decades have now fallen into place, and some questions have been answered that were not even posed before. With low-mass red giants as the simplest example, this recent work emphasizes that stars, in general, may have at least two distinct but very important centres: (I) a geometrical centre, and (II) a separate nuclear centre, residing in a shell outside a zero-luminosity dense core for example. This two-centre perspective leads to an explicit, analytical, asymptotic theory of low-mass red-giant structure. It enables one to appreciate that the problem of understanding why such stars become red giants is one of anticipating a remarkable yet natural structural bifurcation that occurs in them. This bifurcation occurs because of a combination of known and understandable facts just summarized namely that, following central hydrogen exhaustion, a thin nuclear-burning shell does develop outside a more-or-less dense core. In the resulting theory, both ρsh/ρolinec and

  8. 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.

  9. 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.

  10. '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...

  11. Red sea drillings.

    PubMed

    Ross, D A; Whitmarsh, R B; Ali, S A; Boudreaux, J E; Coleman, R; Fleisher, R L; Girdler, R; Manheim, F; Matter, A; Nigrini, C; Stoffers, P; Supko, P R

    1973-01-26

    Recent drilling in the Red Sea has shown that much of the basin is underlain by evaporites of a similar age to that of evaporites found in the Mediterranean Sea. These evaporites and their structural positions indicate that other brine areas are present-and, indeed, several others have been discovered.

  12. 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)

  13. RED2TEX: A TRIX RED to LATEX converter

    SciTech Connect

    Chase, L.; Langdon, A.B.

    1987-05-20

    A summary of RED2TEX is presented. RED2TEX converts standard TRIX RED format commands to TEX or LATEX commands for subsequent LATEX formatting. LATEX is a special version of the TEX document preparation system. LATEX adds to TEX a collection of commands that simplifies formatting. LATEX runs on the J-Vax and the LLL-LCC Pyramid machines. RED2TEX resides in Unix directory CHASE/TEX.

  14. 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.

  15. 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.

  16. Synthetic biology as red herring.

    PubMed

    Preston, Beth

    2013-12-01

    It has become commonplace to say that with the advent of technologies like synthetic biology the line between artifacts and living organisms, policed by metaphysicians since antiquity, is beginning to blur. But that line began to blur 10,000 years ago when plants and animals were first domesticated; and has been thoroughly blurred at least since agriculture became the dominant human subsistence pattern many millennia ago. Synthetic biology is ultimately only a late and unexceptional offshoot of this prehistoric development. From this perspective, then, synthetic biology is a red herring, distracting us from more thorough philosophical consideration of the most truly revolutionary human practice-agriculture. In the first section of this paper I will make this case with regard to ontology, arguing that synthetic biology crosses no ontological lines that were not crossed already in the Neolithic. In the second section I will construct a parallel case with regard to cognition, arguing that synthetic biology as biological engineering represents no cognitive advance over what was required for domestication and the new agricultural subsistence pattern it grounds. In the final section I will make the case with regard to human existence, arguing that synthetic biology, even if wildly successful, is not in a position to cause significant existential change in what it is to be human over and above the massive existential change caused by the transition to agriculture. I conclude that a longer historical perspective casts new light on some important issues in philosophy of technology and environmental philosophy.

  17. Quantitative phase imaging of live cells with near on-axis digital holographic microscopy using constrained optimization approach

    NASA Astrophysics Data System (ADS)

    Pandiyan, Vimal Prabhu; Khare, Kedar; John, Renu

    2016-10-01

    We demonstrate a single-shot near on-axis digital holographic microscope that uses a constrained optimization approach for retrieval of the complex object function in the hologram plane. The recovered complex object is back-propagated from the hologram plane to image plane using the Fresnel back-propagation algorithm. A numerical aberration compensation algorithm is employed for correcting the aberrations in the object beam. The reference beam angle is calculated automatically using the modulation property of Fourier transform without any additional recording. We demonstrate this approach using a United States Air Force (USAF) resolution target as an object on our digital holographic microscope. We also demonstrate this approach by recovering the quantitative phase images of live yeast cells, red blood cells and dynamics of live dividing yeast cells.

  18. Lipopolysaccharide induced acute red eye and corneal ulcers.

    PubMed

    Schultz, C L; Morck, D W; McKay, S G; Olson, M E; Buret, A

    1997-01-01

    Using a new animal model, the aims of this study were to assess the role played by purified lipopolysaccharide (LPS) and neutrophils in the pathogenesis of acute red-eye reactions (ARE) and corneal ulcers. In addition, IL-1 alpha was assessed for its implications in the formation of corneal ulcers. Following corneal abrasion, eyes of rabbits underwent single or double exposures to various doses of LPS from Pseudomonas aeruginosa or Serratia marcescens. This protocol induced ARE symptoms, and their severity depended on the dosage, number of LPS exposures, and type of LPS used (LPS from S. marcescens showing highest virulence). Corneal ulcers were induced by delivering a high dose of Serratia LPS (100 micrograms) followed by a low dose (10 micrograms). Histopathological examination revealed that both ARE and corneal ulceration were associated with prominent neutrophil infiltration. In addition, many lymphocytes and other monocytic cells infiltrated ulcerated ocular tissue. Tear fluids obtained from ulcerated eyes contained high concentrations of a protein recognized by anti-rabbit IL-1 alpha antibodies as demonstrated by immunoblotting studies. The results indicate that LPS can induce ARE and corneal ulceration in the absence of any live bacteria. Moreover, the findings implicate the accumulation of neutrophils and IL-1 alpha-related proteins in the pathogenesis of ARE and corneal ulcers.

  19. 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.

  20. 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.