Sample records for label reveals stim1

  1. A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure

    PubMed Central

    Hauser, Christina T.; Tsien, Roger Y.

    2007-01-01

    Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (>220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (<15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn2+ ions binds tightly but reversibly to hexahistidine (His6) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca2+ from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His6 tags are accessible to the dye or antibodies, demonstrating externalization. PMID:17360414

  2. Human muscle economy myoblast differentiation and excitation-contraction coupling use the same molecular partners, STIM1 and STIM2.

    PubMed

    Darbellay, Basile; Arnaudeau, Serge; Ceroni, Dimitri; Bader, Charles R; Konig, Stephane; Bernheim, Laurent

    2010-07-16

    Our recent work identified store-operated Ca(2+) entry (SOCE) as the critical Ca(2+) source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370-5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca(2+) mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca(2+) content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca(2+)-content during repeated KCl-induced Ca(2+) releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling.

  3. Human Muscle Economy Myoblast Differentiation and Excitation-Contraction Coupling Use the Same Molecular Partners, STIM1 and STIM2*

    PubMed Central

    Darbellay, Basile; Arnaudeau, Serge; Ceroni, Dimitri; Bader, Charles R.; Konig, Stephane; Bernheim, Laurent

    2010-01-01

    Our recent work identified store-operated Ca2+ entry (SOCE) as the critical Ca2+ source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370–5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca2+ mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca2+ content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca2+-content during repeated KCl-induced Ca2+ releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling. PMID:20436167

  4. Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site

    NASA Astrophysics Data System (ADS)

    Wang, Xizhuo; Wang, Youjun; Zhou, Yandong; Hendron, Eunan; Mancarella, Salvatore; Andrake, Mark D.; Rothberg, Brad S.; Soboloff, Jonathan; Gill, Donald L.

    2014-02-01

    STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca2+ sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca2+ channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

  5. Molecular Dynamics Simulations of Membrane-Bound STIM1 to Investigate Conformational Changes during STIM1 Activation upon Calcium Release.

    PubMed

    Mukherjee, Sreya; Karolak, Aleksandra; Debant, Marjolaine; Buscaglia, Paul; Renaudineau, Yves; Mignen, Olivier; Guida, Wayne C; Brooks, Wesley H

    2017-02-27

    Calcium is involved in important intracellular processes, such as intracellular signaling from cell membrane receptors to the nucleus. Typically, calcium levels are kept at less than 100 nM in the nucleus and cytosol, but some calcium is stored in the endoplasmic reticulum (ER) lumen for rapid release to activate intracellular calcium-dependent functions. Stromal interacting molecule 1 (STIM1) plays a critical role in early sensing of changes in the ER's calcium level, especially when there is a sudden release of stored calcium from the ER. Inactive STIM1, which has a bound calcium ion, is activated upon ion release. Following activation of STIM1, there is STIM1-assisted initiation of extracellular calcium entry through channels in the cell membrane. This extracellular calcium entering the cell then amplifies intracellular calcium-dependent actions. At the end of the process, ER levels of stored calcium are reestablished. The main focus of this work was to study the conformational changes accompanying homo- or heterodimerization of STIM1. For this purpose, the ER luminal portion of STIM1 (residues 58-236), which includes the sterile alpha motif (SAM) domain plus the calcium-binding EF-hand domains 1 and 2 attached to the STIM1 transmembrane region (TM), was modeled and embedded in a virtual membrane. Next, molecular dynamics simulations were performed to study the conformational changes that take place during STIM1 activation and subsequent protein-protein interactions. Indeed, the simulations revealed exposure of residues in the EF-hand domains, which may be important for dimerization steps. Altogether, understanding conformational changes in STIM1 can help in drug discovery when targeting this key protein in intracellular calcium functions.

  6. STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

    NASA Astrophysics Data System (ADS)

    Zhou, Yandong; Wang, Xizhuo; Wang, Xianming; Loktionova, Natalia A.; Cai, Xiangyu; Nwokonko, Robert M.; Vrana, Erin; Wang, Youjun; Rothberg, Brad S.; Gill, Donald L.

    2015-09-01

    The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)--the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer-dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

  7. Regulation of STIM1 and SOCE by the ubiquitin-proteasome system (UPS).

    PubMed

    Keil, Jeffrey M; Shen, Zhouxin; Briggs, Steven P; Patrick, Gentry N

    2010-10-18

    The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca(2+) entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3's), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca(2+) homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function.

  8. Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines.

    PubMed

    Zheng, Sisi; Zhou, Lijuan; Ma, Guolin; Zhang, Tian; Liu, Jindou; Li, Jia; Nguyen, Nhung T; Zhang, Xiaoyan; Li, Wanjie; Nwokonko, Robert; Zhou, Yandong; Zhao, Fukuan; Liu, Jingguo; Huang, Yun; Gill, Donald L; Wang, Youjun

    2018-06-22

    Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca 2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca 2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca 2+ and Ca 2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca 2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca 2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca 2+ but is dispensable for the maintenance of long-term ER Ca 2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM1 1-491 and STIM1 1-666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

  9. STIM1L traps and gates Orai1 channels without remodeling the cortical ER

    PubMed Central

    Saüc, Sophie; Bulla, Monica; Nunes, Paula; Orci, Lelio; Marchetti, Anna; Antigny, Fabrice; Bernheim, Laurent; Cosson, Pierre; Frieden, Maud; Demaurex, Nicolas

    2015-01-01

    STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca2+ entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca2+ influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca2+ imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1−/−/Stim2−/− fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca2+ elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides. PMID:25736291

  10. Inside-out Ca2+ signalling prompted by STIM1 conformational switch

    NASA Astrophysics Data System (ADS)

    Ma, Guolin; Wei, Ming; He, Lian; Liu, Chongxu; Wu, Bo; Zhang, Shenyuan L.; Jing, Ji; Liang, Xiaowen; Senes, Alessandro; Tan, Peng; Li, Siwei; Sun, Aomin; Bi, Yunchen; Zhong, Ling; Si, Hongjiang; Shen, Yuequan; Li, Minyong; Lee, Mi-Sun; Zhou, Weibin; Wang, Junfeng; Wang, Youjun; Zhou, Yubin

    2015-07-01

    Store-operated Ca2+ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca2+ entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca2+ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca2+ signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

  11. Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum–plasma membrane junctions

    PubMed Central

    Wu, Minnie M.; Covington, Elizabeth D.; Lewis, Richard S.

    2014-01-01

    Following endoplasmic reticulum (ER) Ca2+ depletion, STIM1 and Orai1 complexes assemble autonomously at ER–plasma membrane (PM) junctions to trigger store-operated Ca2+ influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps Orai1 in the PM through binding of its calcium release-activated calcium activation domain. We tested this model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian, while Orai1 is slightly subdiffusive. After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER–PM junctions. While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and Orai1, which engage in constant diffusional exchange with extrajunctional pools. PMID:25057023

  12. RNA interference against stromal interacting molecule-1 (STIM1) ameliorates ethanol-induced hepatotoxicity.

    PubMed

    Cui, Ruibing; Li, Rong; Guo, Xiaolan; Jia, Xiaoqing; Yan, Ming

    2018-06-01

    Previously we have demonstrated that stromal interacting molecule-1 (STIM1) was involved in ethanol induced liver injury. However, the exact pathogenic mechanism of STIM1 in alcoholic liver disease (ALD) is still unknown. We constructed plasmid vectors encoding short-hairpin RNA against STIM1 to investigate its role in ALD in the rat liver cell line BRL and in Sprague-Dawley rats. The results showed that STIM1 targeted sh-RNA (Sh-STIM1) significantly ameliorated ethanol-induced BRL cells injury and liver injury in rats with 20 weeks-induced alcoholic liver disease. Inhibition of STIM1 also reduced intracellular calcium ion concentration, reactive oxygen species (ROS) production, lipid peroxidation, NF-kappa B activation and TNF-α production under ethanol exposure. STIM1 may play an important role in the pathogenesis of alcoholic liver disease. Silencing STIM1 may be effective in preventing alcoholic liver disease. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Essential role of STIM1 in the development of cardiomyocyte hypertrophy

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

    Ohba, Takayoshi; Watanabe, Hiroyuki; Murakami, Manabu

    2009-11-06

    Store-operated Ca{sup 2+} entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48 h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential rolemore » in the development of cardiomyocyte hypertrophy.« less

  14. Role of STIM1 (Stromal Interaction Molecule 1) in Hypertrophy-Related Contractile Dysfunction.

    PubMed

    Troupes, Constantine D; Wallner, Markus; Borghetti, Giulia; Zhang, Chen; Mohsin, Sadia; von Lewinski, Dirk; Berretta, Remus M; Kubo, Hajime; Chen, Xiongwen; Soboloff, Jonathan; Houser, Steven

    2017-07-07

    Pathological increases in cardiac afterload result in myocyte hypertrophy with changes in myocyte electrical and mechanical phenotype. Remodeling of contractile and signaling Ca 2+ occurs in pathological hypertrophy and is central to myocyte remodeling. STIM1 (stromal interaction molecule 1) regulates Ca 2+ signaling in many cell types by sensing low endoplasmic reticular Ca 2+ levels and then coupling to plasma membrane Orai channels to induce a Ca 2+ influx pathway. Previous reports suggest that STIM1 may play a role in cardiac hypertrophy, but its role in electrical and mechanical phenotypic alterations is not well understood. To define the contributions of STIM1-mediated Ca 2+ influx on electrical and mechanical properties of normal and diseased myocytes, and to determine whether Orai channels are obligatory partners for STIM1 in these processes using a clinically relevant large animal model of hypertrophy. Cardiac hypertrophy was induced by slow progressive pressure overload in adult cats. Hypertrophied myocytes had increased STIM1 expression and activity, which correlated with altered Ca 2 + -handling and action potential (AP) prolongation. Exposure of hypertrophied myocytes to the Orai channel blocker BTP2 caused a reduction of AP duration and reduced diastolic Ca 2+ spark rate. BTP2 had no effect on normal myocytes. Forced expression of STIM1 in cultured adult feline ventricular myocytes increased diastolic spark rate and prolonged AP duration. STIM1 expression produced an increase in the amount of Ca 2+ stored within the sarcoplasmic reticulum and activated Ca 2+ /calmodulin-dependent protein kinase II. STIM1 expression also increased spark rates and induced spontaneous APs. STIM1 effects were eliminated by either BTP2 or by coexpression of a dominant negative Orai construct. STIM1 can associate with Orai in cardiac myocytes to produce a Ca 2+ influx pathway that can prolong the AP duration and load the sarcoplasmic reticulum and likely contributes to the

  15. Initial activation of STIM1, the regulator of store-operated calcium entry

    PubMed Central

    Zhou, Yubin; Srinivasan, Prasanna; Razavi, Shiva; Seymour, Sam; Meraner, Paul; Gudlur, Aparna; Stathopulos, Peter B; Ikura, Mitsuhiko; Rao, Anjana; Hogan, Patrick G

    2013-01-01

    Physiological Ca2+ signalling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca2+ entry. STIM1 and STIM2 are Ca2+ sensors located in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca2+ stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca2+, but the mechanism transmitting activation to the STIM cytoplasmic domain has not been defined. Here we demonstrate, using Tb3+–acceptor energy transfer, that dimerization of STIM1 ER-luminal domains can initiate an extensive conformational change in murine STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane to communicate with ORAI channels. PMID:23851458

  16. Complex role of STIM1 in the activation of store-independent Orai1/3 channels

    PubMed Central

    Zhang, Wei; González-Cobos, José C.; Jardin, Isaac; Romanin, Christoph; Matrougui, Khalid

    2014-01-01

    Orai proteins contribute to Ca2+ entry into cells through both store-dependent, Ca2+ release–activated Ca2+ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) and leukotriene C4 (LTC4)-regulated Ca2+ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels. PMID:24567509

  17. Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry.

    PubMed

    Perni, Stefano; Dynes, Joseph L; Yeromin, Andriy V; Cahalan, Michael D; Franzini-Armstrong, Clara

    2015-10-06

    Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.

  18. Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry

    PubMed Central

    Perni, Stefano; Dynes, Joseph L.; Yeromin, Andriy V.; Cahalan, Michael D.; Franzini-Armstrong, Clara

    2015-01-01

    Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze–fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)–plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze–fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic “particles”; TG treatment led to aggregation of these particles into sharply delimited “puncta” positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules. PMID:26351694

  19. STIM1 Overexpression Promotes Colorectal Cancer Progression, Cell Motility and COX-2 Expression

    PubMed Central

    Wang, Jaw-Yuan; Sun, Jianwei; Huang, Ming-Yii; Wang, Yu-Shiuan; Hou, Ming-Feng; Sun, Yan; He, Huifang; Krishna, Niveditha; Chiu, Siou-Jin; Lin, Shengchen; Yang, Shengyu; Chang, Wei-Chiao

    2014-01-01

    Tumor metastasis is the major cause of death among cancer patients, with more than 90% of cancer-related death attributable to the spreading of metastatic cells to secondary organs. Store-operated Ca2+ entry (SOCE) is the predominant Ca2+ entry mechanism in most cancer cells, and STIM1 is the endoplasmic reticulum (ER) Ca2+ sensor for store-operated channels (SOC). Here we reported that the STIM1 was overexpressed in colorectal cancer (CRC) patients. STIM1 overexpression in CRC was significantly associated with tumor size, depth of invasion, lymphnode metastasis status and serum levels of carcinoembryonic antigen. Furthermore, ectopic expression of STIM1 promoted CRC cell motility, while depletion of STIM1 with shRNA inhibited CRC cell migration. Our data further suggested that STIM1 promoted CRC cell migration through increasing the expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2). Importantly, ectopically expressed COX-2 or exogenous PGE2 were able to rescue migration defect in STIM1 knockdown CRC cells, and inhibition of COX-2 with ibuprofen and indomethacin abrogated STIM1-mediated CRC cell motility. In short, our data provided clinicopathological significance for STIM1 and store-operated Ca2+ entry in CRC progression, and implicated a role for COX-2 in STIM1-mediated CRC metastasis. Our studies also suggested a new approach to inhibit STIM1-mediated metastasis with COX-2 inhibitors. PMID:25381814

  20. STIM1 as a key regulator for Ca2+ homeostasis in skeletal-muscle development and function

    PubMed Central

    2011-01-01

    Stromal interaction molecules (STIM) were identified as the endoplasmic-reticulum (ER) Ca2+ sensor controlling store-operated Ca2+ entry (SOCE) and Ca2+-release-activated Ca2+ (CRAC) channels in non-excitable cells. STIM proteins target Orai1-3, tetrameric Ca2+-permeable channels in the plasma membrane. Structure-function analysis revealed the molecular determinants and the key steps in the activation process of Orai by STIM. Recently, STIM1 was found to be expressed at high levels in skeletal muscle controlling muscle function and properties. Novel STIM targets besides Orai channels are emerging. Here, we will focus on the role of STIM1 in skeletal-muscle structure, development and function. The molecular mechanism underpinning skeletal-muscle physiology points toward an essential role for STIM1-controlled SOCE to drive Ca2+/calcineurin/nuclear factor of activated T cells (NFAT)-dependent morphogenetic remodeling programs and to support adequate sarcoplasmic-reticulum (SR) Ca2+-store filling. Also in our hands, STIM1 is transiently up-regulated during the initial phase of in vitro myogenesis of C2C12 cells. The molecular targets of STIM1 in these cells likely involve Orai channels and canonical transient receptor potential (TRPC) channels TRPC1 and TRPC3. The fast kinetics of SOCE activation in skeletal muscle seem to depend on the triad-junction formation, favoring a pre-localization and/or pre-formation of STIM1-protein complexes with the plasma-membrane Ca2+-influx channels. Moreover, Orai1-mediated Ca2+ influx seems to be essential for controlling the resting Ca2+ concentration and for proper SR Ca2+ filling. Hence, Ca2+ influx through STIM1-dependent activation of SOCE from the T-tubule system may recycle extracellular Ca2+ losses during muscle stimulation, thereby maintaining proper filling of the SR Ca2+ stores and muscle function. Importantly, mouse models for dystrophic pathologies, like Duchenne muscular dystrophy, point towards an enhanced Ca2+ influx

  1. Relocalization of STIM1 in mouse oocytes at fertilization: early involvement of store-operated calcium entry.

    PubMed

    Gómez-Fernández, Carolina; Pozo-Guisado, Eulalia; Gañán-Parra, Miguel; Perianes, Mario J; Alvarez, Ignacio S; Martín-Romero, Francisco Javier

    2009-08-01

    Calcium waves represent one of the most important intracellular signaling events in oocytes at fertilization required for the exit from metaphase arrest and the resumption of the cell cycle. The molecular mechanism ruling this signaling has been described in terms of the contribution of intracellular calcium stores to calcium spikes. In this work, we considered the possible contribution of store-operated calcium entry (SOCE) to this signaling, by studying the localization of the protein STIM1 in oocytes. STIM1 has been suggested to play a key role in the recruitment and activation of plasma membrane calcium channels, and we show here that mature mouse oocytes express this protein distributed in discrete clusters throughout their periphery in resting cells, colocalizing with the endoplasmic reticulum marker calreticulin. However, immunolocalization of the endogenous STIM1 showed considerable redistribution over larger areas or patches covering the entire periphery of the oocyte during Ca(2+) store depletion induced with thapsigargin or ionomycin. Furthermore, pharmacological activation of endogenous phospholipase C induced a similar pattern of redistribution of STIM1 in the oocyte. Finally, fertilization of mouse oocytes revealed a significant and rapid relocalization of STIM1, similar to that found after pharmacological Ca(2+) store depletion. This particular relocalization supports a role for STIM1 and SOCE in the calcium signaling during early stages of fertilization.

  2. Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest

    PubMed Central

    2013-01-01

    Background Depletion of calcium (Ca2+) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca2+ entry (SOCE) pathway which sustains long-term Ca2+ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca2+ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear. Methods Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo. Results We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model. Conclusion Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma. PMID:23578185

  3. Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest.

    PubMed

    Li, Guilin; Zhang, Zhenxing; Wang, Renzhi; Ma, Wenbin; Yang, Ying; Wei, Junji; Wei, Yanping

    2013-04-11

    Depletion of calcium (Ca2+) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca2+ entry (SOCE) pathway which sustains long-term Ca2+ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca2+ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear. Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo. We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21Waf1/Cip1, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model. Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.

  4. STIM1fl/fl Ksp-Cre Mouse has Impaired Renal Water Balance

    PubMed Central

    Cebotaru, Liudmila; Cebotaru, Valeriu; Wang, Hua; Arend, Lois J.; Guggino, William B.

    2016-01-01

    Background/AIM STIM1 is as an essential component in store operated Ca2+entry. However give the paucity of information on the role of STIM1 in kidney, the aim was to study the function of STIM1 in the medulla of the kidney. Methods we crossed a Ksp-cre mouse with another mouse containing two loxP sites flanking Exon 6 of STIM1. The Ksp-cre mouse is based upon the Ksp-cadherin gene promoter which expresses cre recombinase in developing nephrons, collecting ducts (SD) and thick ascending limbs (TAL) of the loop of Henle. Results The offspring of these mice are viable without gross morphological changes, however, we noticed that the STIM1 Ksp-cre knockout mice produced more urine compared to control. To examine this more carefully, we fed mice low (LP) and high protein (HP) diets respectively. When mice were fed HP diet STIM1 ko mice had significantly increased urinary volume and lower specific gravity compared to wt mice. In STIM1 ko mice fed HP diet urine creatinine and urea were significantly lower compared to wt mice fed HP diet, however the fractional excretion was the same. Conclusion These data support the idea that STIM1 ko mice have impaired urinary concentrating ability when challenged with HP diet is most likely caused by impaired Ca2+-dependent signal transduction through the vasopressin receptor cascade. PMID:27336410

  5. STIM1- and Orai1-dependent store-operated calcium entry regulates human myoblast differentiation.

    PubMed

    Darbellay, Basile; Arnaudeau, Serge; König, Stéphane; Jousset, Hélène; Bader, Charles; Demaurex, Nicolas; Bernheim, Laurent

    2009-02-20

    Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca(2+)](in) involving store-operated Ca(2+) entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca(2+) sensor activating Orai Ca(2+) channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca(2+)](in) and intracellular Ca(2+) store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K(+) channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.

  6. To die or not to die SGK1-sensitive ORAI/STIM in cell survival.

    PubMed

    Lang, Florian; Pelzl, Lisann; Hauser, Stefan; Hermann, Andreas; Stournaras, Christos; Schöls, Ludger

    2018-05-03

    The pore forming Ca 2+ release activated Ca 2+ channel (CRAC) isoforms ORAI1-3 and their regulators STIM1,2 accomplish store operated Ca 2+ entry (SOCE). Activation of SOCE may lead to cytosolic Ca 2+ oscillations, which in turn support cell proliferation and cell survival. ORAI/STIM and thus SOCE are upregulated by the serum and glucocorticoid inducible kinase SGK1, a kinase under powerful genomic regulation and activated by phosphorylation via the phosphoinositol-3-phosphate pathway. SGK1 enhances ORAI1 abundance partially by phosphorylation of Nedd4-2, an ubiquitin ligase priming the channel protein for degradation. The SGK1-phosphorylated Nedd4-2 binds to the protein 14-3-3 and is thus unable to ubiquinate ORAI1. SGK1 further increases the ORAI1 and STIM1 protein abundance by activating nuclear factor kappa B (NF-κB), a transcription factor upregulating the expression of STIM1 and ORAI1. SGK1-sensitive upregulation of ORAI/STIM and thus SOCE is triggered by a wide variety of hormones and growth factors, as well as several cell stressors including ischemia, radiation, and cell shrinkage. SGK1 dependent upregulation of ORAI/STIM confers survival of tumor cells and thus impacts on growth and therapy resistance of cancer. On the other hand, SGK1-dependent upregulation of ORAI1 and STIM1 may support survival of neurons and impairment of SGK1-dependent ORAI/STIM activity may foster neurodegeneration. Clearly, further experimental effort is needed to define the mechanisms linking SGK1-dependent upregulation of ORAI1 and STIM1 to cell survival and to define the impact of SGK1-dependent upregulation of ORAI1 and STIM1 on malignancy and neurodegenerative disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown.

    PubMed

    Shalygin, A V; Vigont, V A; Glushankova, L N; Zimina, O A; Kolesnikov, D O; Skopin, A Yu; Kaznacheeva, E V

    2017-07-01

    An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

  8. Stromal interaction molecule 1 (STIM1) silencing inhibits tumor growth and promotes cell cycle arrest and apoptosis in hypopharyngeal carcinoma.

    PubMed

    Sun, Yuanhao; Cui, Xiaobo; Wang, Jun; Wu, Shuai; Bai, Yunfei; Wang, Yaping; Wang, Boqian; Fang, Jugao

    2015-05-01

    As an important pathway maintaining the balance of intracellular calcium (Ca(2+)), store-operated Ca(2+) entry (SOCE) is critical for cellular functions. Stromal interaction molecule 1 (STIM1), a key component of SOCE, plays a dual role as an endoplasmic reticulum Ca(2+) receptor and an SOCE exciter. Aberrant expression of STIM1 could be discovered in several human cancer cells. However, the role of STIM1 in regulating human hypopharyngeal carcinoma still remains unclear. Real-time polymerase chain reaction (PCR) was used to detect expression of STIM1 in human hypopharyngeal carcinoma cell line FaDu. STIM1 on FaDu cells was knocked down by lentiviral transduction method. The biological impacts after knocking down of STIM1 on FaDu cells were investigated in vitro and in vivo. The result of real-time PCR showed that STIM1 was expressed in FaDu cells. Lentiviral transduction efficiently downregulated the expression of STIM1 in FaDu cells at both mRNA and protein levels. Significant downregulation of STIM1 on FaDu cells inhibited cell proliferation, induced cell cycle arrest in G0/G1 phase, promoted cell apoptosis, and restrained cell growth rate. The antigrowth effect of STIM1 silencing was also discovered in FaDu hypopharyngeal tumor model. Our findings indicate that STIM1 is likely to become a new therapeutic target for hypopharyngeal carcinoma treatment.

  9. Roles of platelet STIM1 and Orai1 in glycoprotein VI- and thrombin-dependent procoagulant activity and thrombus formation.

    PubMed

    Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A H; Stegner, David; van der Meijden, Paola E J; Kuijpers, Marijke J E; Varga-Szabo, David; Heemskerk, Johan W M; Nieswandt, Bernhard

    2010-07-30

    In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

  10. Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation*

    PubMed Central

    Gilio, Karen; van Kruchten, Roger; Braun, Attila; Berna-Erro, Alejandro; Feijge, Marion A. H.; Stegner, David; van der Meijden, Paola E. J.; Kuijpers, Marijke J. E.; Varga-Szabo, David; Heemskerk, Johan W. M.; Nieswandt, Bernhard

    2010-01-01

    In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca2+ entry (SOCE) with Orai1 as principal Ca2+ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca2+ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1−/− and Orai1−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca2+ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca2+ signals of Stim1−/− and Orai1−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1−/− and Orai1−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca2+ entry, inhibited Ca2+ and procoagulant responses even in Stim1−/− and Orai1−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca2+ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca2+ entry and PS exposure, only one relying on STIM1-Orai1 interaction. PMID:20519511

  11. Impaired spatial memory and enhanced long-term potentiation in mice with forebrain-specific ablation of the Stim genes

    PubMed Central

    Garcia-Alvarez, Gisela; Shetty, Mahesh S.; Lu, Bo; Yap, Kenrick An Fu; Oh-Hora, Masatsugu; Sajikumar, Sreedharan; Bichler, Zoë; Fivaz, Marc

    2015-01-01

    Recent findings point to a central role of the endoplasmic reticulum-resident STIM (Stromal Interaction Molecule) proteins in shaping the structure and function of excitatory synapses in the mammalian brain. The impact of the Stim genes on cognitive functions remains, however, poorly understood. To explore the function of the Stim genes in learning and memory, we generated three mouse strains with conditional deletion (cKO) of Stim1 and/or Stim2 in the forebrain. Stim1, Stim2, and double Stim1/Stim2 cKO mice show no obvious brain structural defects or locomotor impairment. Analysis of spatial reference memory in the Morris water maze revealed a mild learning delay in Stim1 cKO mice, while learning and memory in Stim2 cKO mice was indistinguishable from their control littermates. Deletion of both Stim genes in the forebrain resulted, however, in a pronounced impairment in spatial learning and memory reflecting a synergistic effect of the Stim genes on the underlying neural circuits. Notably, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses was markedly enhanced in Stim1/Stim2 cKO mice and was associated with increased phosphorylation of the AMPA receptor subunit GluA1, the transcriptional regulator CREB and the L-type Voltage-dependent Ca2+ channel Cav1.2 on protein kinase A (PKA) sites. We conclude that STIM1 and STIM2 are key regulators of PKA signaling and synaptic plasticity in neural circuits encoding spatial memory. Our findings also reveal an inverse correlation between LTP and spatial learning/memory and suggest that abnormal enhancement of cAMP/PKA signaling and synaptic efficacy disrupts the formation of new memories. PMID:26236206

  12. STIM1 signaling controls store operated calcium entry required for development and contractile function in skeletal muscle

    PubMed Central

    Stiber, Jonathan; Hawkins, April; Zhang, Zhu-Shan; Wang, Sunny; Burch, Jarrett; Graham, Victoria; Ward, Cary C.; Seth, Malini; Finch, Elizabeth; Malouf, Nadia; Williams, R. Sanders; Eu, Jerry P.; Rosenberg, Paul

    2009-01-01

    It is now well established that stromal interaction molecule 1 (STIM1) is the calcium sensor of endoplasmic reticulum (ER) stores required to activate store-operated calcium entry (SOC) channels at the surface of non-excitable cells. Yet little is known about STIM1 in excitable cells such as striated muscle where the complement of calcium regulatory molecules is rather disparate from that of non-excitable cells. Here, we show that STIM1 is expressed in both myotubes and adult skeletal muscle. Myotubes lacking functional STIM1 fail to exhibit SOC and fatigue rapidly. Moreover, mice lacking functional STIM1 die perinatally from a skeletal myopathy. In addition, STIM1 haploinsufficiency confers a contractile defect only under conditions where rapid refilling of stores would be needed. These findings provide novel insight to the role of STIM1 in skeletal muscle and suggest that STIM1 has a universal role as an ER/SR calcium sensor in both excitable and non-excitable cells. PMID:18488020

  13. An aromatic amino acid in the coiled-coil 1 domain plays a crucial role in the auto-inhibitory mechanism of STIM1.

    PubMed

    Yu, Junwei; Zhang, Haining; Zhang, Mingshu; Deng, Yongqiang; Wang, Huiyu; Lu, Jingze; Xu, Tao; Xu, Pingyong

    2013-09-15

    STIM1 (stromal interaction molecule 1) is one of the key elements that mediate store-operated Ca²⁺ entry via CRAC (Ca²⁺- release-activated Ca²⁺) channels in immune and non-excitable cells. Under physiological conditions, the intramolecular auto-inhibitions in STIM1 C- and STIM1 N-termini play essential roles in keeping STIM1 in an inactive state. However, the auto-inhibitory mechanism of the STIM1 C-terminus is still unclear. In the present study, we first predicted a short inhibitory domain (residues 310-317) in human STIM1 that might determine the different localizations of human STIM1 from Caenorhabditis elegans STIM1 in resting cells. Next, we confirmed the prediction and further identified an aromatic amino acid residue, Tyr³¹⁶, that played a crucial role in maintaining STIM1 in a closed conformation in quiescent cells. Full-length STIM1-Y316A formed constitutive clusters near the plasma membrane and activated the CRAC channel in the resting state when co-expressed with Orai1. The introduction of a Y316A mutation caused the higher-order oligomerization of the in vitro purified STIM1 fragment containing both the auto-inhibitory domain and CAD(CRAC-activating domain).We propose that the Tyr³¹⁶ residue may be involved in the auto-inhibitory mechanism of the STIM1 C-terminus in the quiescent state. This inhibition could be achieved either by interacting with the CAD using hydrogen and/or hydrophobic bonds, or by an intermolecular interaction using repulsive forces, which maintained a dimeric STIM1.

  14. Overexpression of SARAF Ameliorates Pressure Overload-Induced Cardiac Hypertrophy Through Suppressing STIM1-Orai1 in Mice.

    PubMed

    Dai, Fengdan; Zhang, Yan; Wang, Qiang; Li, De; Yang, Yongjian; Ma, Shuangtao; Yang, Dachun

    2018-01-01

    Activation of stromal interaction molecule 1 (STIM1) and Orai1 participates in the development of cardiac hypertrophy. Store-operated Ca2+ entry-associated regulatory factor (SARAF) is an intrinsic inhibitor of STIM1-Orai1 interaction. Thus, we hypothesized that SARAF could prevent cardiac hypertrophy. Male C57BL/6 mice, aged 8 weeks, were randomly divided into sham and abdominal aortic constriction surgery groups and were infected with lentiviruses expressing SARAF and GFP (Lenti-SARAF) or GFP alone (Lenti-GFP) via intramyocardial injection. At 4 weeks after aortic constriction, left ventricular structure and function were assessed by echocardiography and hemodynamic assays. The gene and protein expressions of SARAF, STIM1, and Orai1 were measured by quantitative PCR and Western blot, respectively. Gene and protein expressions of SARAF were significantly decreased, while STIM1 and Orai1 were increased in the heart tissue compared with sham group. Overexpression of SARAF in the heart prevented the upregulation of STIM1 and Orai1, and importantly, attenuated aortic constriction-induced decrease in maximal rate of left ventricular pressure decay and increases in thickness of interventricular septum and left ventricular posterior wall, heart weight/body weight ratio, and size of cardiomyocytes. Blood pressure detected through the carotid artery and left ventricular systolic function were not affected by SARAF overexpression. In addition, overexpression of SARAF also attenuated angiotensin II-induced upregulation of STIM1 and Orai1 and hypertrophy of cultured cardiomyocytes. Overexpression of SARAF in the heart prevents cardiac hypertrophy, probably through suppressing the upregulation of STIM1/Orai1. © 2018 The Author(s). Published by S. Karger AG, Basel.

  15. Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1).

    PubMed

    Hoover, Paul J; Lewis, Richard S

    2011-08-09

    Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ∼2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.

  16. Molecular basis of activation of the arachidonate-regulated Ca2+ (ARC) channel, a store-independent Orai channel, by plasma membrane STIM1

    PubMed Central

    Thompson, Jill L; Shuttleworth, Trevor J

    2013-01-01

    Currently, Orai proteins are known to encode two distinct agonist-activated, highly calcium-selective channels: the store-operated Ca2+ release-activated Ca2+ (CRAC) channels, and the store-independent, arachidonic acid-activated ARC channels. Surprisingly, whilst the trigger for activation of these channels is entirely different, both depend on stromal interacting molecule 1 (STIM1). However, whilst STIM1 in the endoplasmic reticulum membrane is the critical sensor for the depletion of this calcium store that triggers CRAC channel activation, it is the pool of STIM1 constitutively resident in the plasma membrane that is essential for activation of the ARC channels. Here, using a variety of approaches, we show that the key domains within the cytosolic part of STIM1 identified as critical for the activation of CRAC channels are also key for activation of the ARC channels. However, examination of the actual steps involved in such activation reveal marked differences between these two Orai channel types. Specifically, loss of calcium from the EF-hand of STIM1 that forms the key initiation point for activation of the CRAC channels has no effect on ARC channel activity. Secondly, in marked contrast to the dynamic and labile nature of interactions between STIM1 and the CRAC channels, STIM1 in the plasma membrane appears to be constitutively associated with the ARC channels. Finally, specific mutations in STIM1 that induce an extended, constitutively active, conformation for the CRAC channels actually prevent activation of the ARC channels by arachidonic acid. Based on these findings, we propose that the likely role of arachidonic acid lies in inducing the actual gating of the channel. PMID:23690558

  17. Higher plasma level of STIM1, OPG are correlated with stent restenosis after PCI.

    PubMed

    Li, Haibin; Jiang, Zhian; Liu, Xiangdong; Yang, Zhihui

    2015-01-01

    Percutaneous Coronary Intervention (PCI) is one of the most effective treatments for Coronary Heart Disease (CHD), but the high rate of In Stent Restenosis (ISR) has plagued clinicians after PCI. We aim to investigate the correlation of plasma Stromal Interaction Molecular 1 (STIM1) and Osteoprotegerin (OPG) level with stent restenosis after PCI. A total of 100 consecutive patients with Coronary Heart Disease (CHD) received PCI procedure were recruited. Coronary angiography was performed 8 months after their PCI. Then patients were divided into 2 groups: observation group was composed by patients who existing postoperative stenosis after intervention; Control group was composed by patients with no postoperative stenosis. The plasma levels of STIM, OPG in all patients were tested before and after intervention. Pearson correlation and multiple linear regression analysis were performed to analysis the correlation between STIM, OPG level and postoperative stenosis. 35 cases were divided into observation group and other 65 were divided into control group. The plasma levels of STIM, OPG have no statistical difference before their PCI procedure, but we observed higher level of High-sensitivity C-reactive protein (Hs-CRP) existed in observation group. We observed higher level of plasma STIM, OPG in observation group when compared with control group after PCI procedure (P < 0.05). Regression analysis demonstrated that Hs-CRP, STIM1, OPG are independent risk factors for ISR. Elevated levels of plasma STIM1, OPG are independent risk factors for ISR in patients received PCI, which could provide useful information for the restenosis control after PCI.

  18. Orai1 and STIM1 are critical for cell migration and proliferation of clear cell renal cell carcinoma

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

    Kim, Ji-Hee; Lkhagvadorj, Sayamaa; Lee, Mi-Ra

    2014-05-23

    Highlights: • Orai1 channel is highly expressed in clear cell renal cell carcinoma (ccRCC) tissues. • Orai1 and STIM1 constitute a native store-operated Ca{sup 2+} entry in ccRCC cells. • Orai1 and STIM1 promote cell migration and proliferation of ccRCC cells. - Abstract: The intracellular Ca{sup 2+} regulation has been implicated in tumorigenesis and tumor progression. Notably, store-operated Ca{sup 2+} entry (SOCE) is a major Ca{sup 2+} entry mechanism in non-excitable cells, being involved in cell proliferation and migration in several types of cancer. However, the expression and biological role of SOCE have not been investigated in clear cell renalmore » cell carcinoma (ccRCC). Here, we demonstrate that Orai1 and STIM1, not Orai3, are crucial components of SOCE in the progression of ccRCC. The expression levels of Orai1 in tumor tissues were significantly higher than those in the adjacent normal parenchymal tissues. In addition, native SOCE was blunted by inhibiting SOCE or by silencing Orai1 and STIM1. Pharmacological blockade or knockdown of Orai1 or STIM1 also significantly inhibited RCC cell migration and proliferative capability. Taken together, Orai1 is highly expressed in ccRCC tissues illuminating that Orai1-mediated SOCE may play an important role in ccRCC development. Indeed, Orai1 and STIM1 constitute a native SOCE pathway in ccRCC by promoting cell proliferation and migration.« less

  19. Orai, STIM1 and iPLA2β: a view from a different perspective

    PubMed Central

    Bolotina, Victoria M

    2008-01-01

    The mechanism of store-operated Ca2+ entry (SOCE) remains one of the intriguing mysteries in the field of Ca2+ signalling. Recent discoveries have resulted in the molecular identification of STIM1 as a Ca2+ sensor in endoplasmic reticulum, Orai1 (CRACM1) as a plasma membrane channel that is activated by the store-operated pathway, and iPLA2β as an essential component of signal transduction from the stores to the plasma membrane channels. Numerous studies have confirmed that molecular knock-down of any one of these three molecules impair SOCE in a wide variety of cell types, but their mutual relations are far from being understood. This report will focus on the functional roles of Orai1, STIM1 and iPLA2β, and will address some specific questions about Orai1 and TRPC1, and their relation to SOC channels in excitable and non-excitable cells. Also, it will analyse the novel role of STIM1 as a trigger for CIF production, and the complex relationship between STIM1 and Orai1 expression, puncta formation and SOCE activation. It will highlight some of the most recent findings that may challenge simple conformational coupling models of SOCE, and will offer some new perspectives on the complex relationships between Orai1, STIM1 and iPLA2β in the SOCE pathway. PMID:18499724

  20. STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts.

    PubMed

    Alicia, Sampieri; Angélica, Zepeda; Carlos, Saldaña; Alfonso, Salgado; Vaca, Luis

    2008-11-01

    While the role of members from the TRPC family of channels as receptor-operated channels (ROC) is well established and supported by numerous studies, the role of this family of channels as store-operated channels (SOC) has been the focus of a heated controversy over the last few years. In the present study, we have explored the modulation of STIM1 on human TRPC1 channel. We show that the association of STIM1 to TRPC1 favors the insertion of TRPC1 into lipid rafts, where TRPC1 functions as a SOC. In the absence of STIM1, TRPC1 associates to other members from the TRPC family of channels to form ROCs. A novel TIRFM-FRET method illustrates the relevance of the dynamic association between STIM1 and TRPC1 for the activation of SOC and the lipid raft localization of the STIM1-TRPC1 complex. This study provides new evidence about the dual activity of TRPC1 (forming ROC or SOC) and the partners needed to determine TRPC1 functional fate. It highlights also the role of plasma membrane microdomains and ER-PM junctions in modulating TRPC1 channel function and its association to STIM1.

  1. SOX2-mediated inhibition of miR-223 contributes to STIM1 activation in phenylephrine-induced hypertrophic cardiomyocytes.

    PubMed

    Zhao, Zhi-Hong; Luo, Jun; Li, Hai-Xia; Wang, Sai-Hua; Li, Xin-Ming

    2018-06-01

    Stromal interaction molecule 1 (STIM1) is the key molecule responsible for store-operated Ca 2+ entry (SOCE). Numerous studies have demonstrated that STIM1 levels appeared to be enhanced during cardiac hypertrophy. However, the mechanism underlining this process remains to be clarified. In this study, phenylephrine (PE) was employed to establish a model of hypertrophic neonatal rat cardiomyocytes (HNRCs) in vitro, and low expression of primary and mature miR-223 was detected in PE-induced HNRCs. Our results have revealed that downregulation of miR-223 by PE contributed to the increase of STIM1, which in turn induced cardiac hypertrophy. As expected, overexpression of miR-223 could prevent the increase in cell surface and reduce the mRNA levels of ANF and BNP in cardiomyocytes. To address the mechanism triggering downregulation of miR-223 under PE, we demonstrated that PE-induced inhibition of GSK-3β activity led to the activation of β-catenin, which initiates the transcription of SOX2. Increased expression of SOX2 occupied the promoter region of primary miR-223 and suppressed its transcription. Therefore, miR-223 appears to be a promising candidate for inhibiting cardiomyocyte hypertrophy, and miR-223/STIM1 axis might be one of interesting targets for the clinical treatment of hypertrophy.

  2. miR-4725-3p targeting Stim1 signaling is involved in xanthohumol inhibition of glioma cell invasion.

    PubMed

    Ho, Kuo-Hao; Chang, Cheng-Kuei; Chen, Peng-Hsu; Wang, Yu-Jia; Chang, Wei-Chiao; Chen, Ku-Chung

    2018-05-10

    Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Due to its highly invasive nature, it is not easy to treat, resulting in high mortality rates. Stromal interacting molecule 1 (Stim1) plays important roles in regulating store-operated Ca 2+ entry (SOCE), and controls invasion by cancer cells. However, the mechanisms and functions of Stim1 in glioma progression are still unclear. In this study, we investigated the effects of targeting Stim1 expression on glioma cell invasion. By analyzing profiles of GBM patients from RNA-sequencing data in The Cancer Genome Atlas (TCGA), higher expression levels of STIM1 were correlated with the poor survival. Furthermore, signaling pathways associated with tumor malignancy, including the epithelial-to-mesenchymal transition (EMT), were activated in patients with high STIM1 expression according to gene set enrichment analyses. Higher Stim1 levels were found in glioma cells compared to human astrocytes, and these higher levels enhanced glioma cell invasion. Xanthohumol (XN), a prenylated flavonoid extracted from the hop plant Humulus lupulus L. (Cannabaceae), significantly reduced cell invasion through inhibiting Stim1 expression. From an micro(mi)RNA array analysis, miR-4725-3p was upregulated by XN treatment. Overexpression of miR-4725-3p inhibited glioma cell invasion via directly targeting the 3'-untranslated region of STIM1. The extracellular signal-regulated kinase/c-Fos pathway was also validated to participate in XN-upregulated miR-4725-3p expression according to promoter and chromatin immunoprecipitation assays. These results emphasize that miR-4725-3p-inhibited STIM1 signaling is involved in XN-attenuated glioma cell invasion. These findings may provide insights into novel therapeutic strategies for future glioblastoma therapy and drug development. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. The Orai-1 and STIM-1 Complex Controls Human Dendritic Cell Maturation

    PubMed Central

    Félix, Romain; Crottès, David; Delalande, Anthony; Fauconnier, Jérémy; Lebranchu, Yvon; Le Guennec, Jean-Yves; Velge-Roussel, Florence

    2013-01-01

    Ca2+ signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca2+ signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca2+ stores that results in a store-operated Ca2+ entry (SOCE). This Ca2+ influx was inhibited by 2-APB and exhibited a Ca2+permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca2+ entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins. PMID:23700407

  4. [Interaction between TRPC1 and STIM1 in calcium sensing receptor mediated calcium influx and nitric oxide production in human umbilical vein endothelial cells].

    PubMed

    Wang, L M; Zhong, H; Tang, N; Pang, L J; Zhang, C J; He, F

    2017-11-24

    Objective: To investigate the interaction of Ca(2+) protein TRPC1 and STIM1 in extracellular Ca(2+) -sensing receptor (CaR)-induced extracellular Ca(2+) influx and the production of nitric oxide (NO). Methods: Human umbilical vein endothelial cells (HUVECs) were cultured and incubated with CaR agonist spermine (activating store-operates cation channels (SOC) and receptor-operated channels (ROC)), CaR negative allosteric modulator Calhex231 (blocking SOC, activating ROC) and ROC analogue TPA (activating ROC, blocking SOC), protein kinase C (PKC) inhibitor Ro31-8220, PKCs and PKCμ inhibitor Go6967(activate SOC, blocking ROC), respectively. The interaction of TRPC1 and STIM1 was determined using the immunofluorescence methods. The interaction between TRPC1 and STIM1 were examined by Co-immuno precipitation. The HUVECs were divided into: TRPC1 and STIM1 short hairpin RNA group (shTRPC1+ shSTIM1 group), vehicle-TRPC1+ vehicle-STIM1 group and control group. The cells were incubated with four different treatments under the action of above mentioned interventions, intracellular Ca(2+) concentration ([Ca(2+) ](i)) was detected using the fluorescence Ca(2+) indicator Fura-2/AM, the production of NO was determined by DAF-FM. Results: (1) The expression of TRPC1 and STIM1 proteins levels in HUVECs: Under the confocal microscope, TRPC1 and STIM1 protein expression showed masculine gender, both located in cytoplasm in the normal control group. Post incubation with Calhex231+ TPA, Ro31-8220 and Go6967, TRPC1 and STIM1 positioned in cytoplasm was significantly reduced, and the combined TRPC1 and STIM1 was also significantly reduced. (2) The interaction of TRPC1 and STIM1 in HUVECs: The relative ratios of Calhex231+ TPA+ Spermine+ Ca(2+) group, Ro31-8220+ Spermine+ Ca(2+) group and Go6976+ Spermine+ Ca(2+) group STIM1/TRPC1 and TRPC1/STIM1 were as follows: (25.98±2.17)% and (44.10±4.01)%, (20.85±1.01)% and (46.31±3.47)%, (23.88±2.05)% and (39.65±2.91)%, which were

  5. Nano-imaging of single cells using STIM

    NASA Astrophysics Data System (ADS)

    Minqin, Ren; van Kan, J. A.; Bettiol, A. A.; Daina, Lim; Gek, Chan Yee; Huat, Bay Boon; Whitlow, H. J.; Osipowicz, T.; Watt, F.

    2007-07-01

    Scanning transmission ion microscopy (STIM) is a technique which utilizes the energy loss of high energy (MeV) ions passing through a sample to provide structural images. In this paper, we have successfully demonstrated STIM imaging of single cells at the nano-level using the high resolution capability of the proton beam writing facility at the Centre for Ion Beam Applications, National University of Singapore. MCF-7 breast cancer cells (American Type Culture Collection [ATCC]) were seeded on to silicon nitride windows, backed by a Hamamatsu pin diode acting as a particle detector. A reasonable contrast was obtained using 1 MeV protons and excellent contrast obtained using 1 MeV alpha particles. In a further experiment, nano-STIM was also demonstrated using cells seeded on to the pin diode directly, and high quality nano-STIM images showing the nucleus and multiple nucleoli were extracted before the detector was significantly damaged.

  6. CRAC channel activity in C. elegans is mediated by Orai1 and STIM1 homologues and is essential for ovulation and fertility

    PubMed Central

    Lorin-Nebel, Catherine; Xing, Juan; Yan, Xiaohui; Strange, Kevin

    2007-01-01

    The Ca2+ release-activated Ca2+ (CRAC) channel is a plasma membrane Ca2+ entry pathway activated by endoplasmic reticulum (ER) Ca2+ store depletion. STIM1 proteins function as ER Ca2+ sensors and regulate CRAC channel activation. Recent studies have demonstrated that CRAC channels are encoded by the human Orai1 gene and a homologous Drosophila gene. C. elegans intestinal cells express a store-operated Ca2+ channel (SOCC) regulated by STIM-1. We cloned a full-length C. elegans cDNA that encodes a 293 amino acid protein, ORAI-1, homologous to human and Drosophila Orai1 proteins. ORAI-1 GFP reporters are co-expressed with STIM-1 in the gonad and intestine. Inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signalling regulates C. elegans gonad function, fertility and rhythmic posterior body wall muscle contraction (pBoc) required for defecation. RNA interference (RNAi) silencing of orai-1 expression phenocopies stim-1 knockdown and causes sterility and prevents intestinal cell SOCC activation, but has no effect on pBoc or intestinal Ca2+ signalling. Orai-1 RNAi suppresses pBoc defects induced by intestinal expression of a STIM-1 Ca2+-binding mutant, indicating that the proteins function in a common pathway. Co-expression of stim-1 and orai-1 cDNAs in HEK293 cells induces large inwardly rectifying cation currents activated by ER Ca2+ depletion. The properties of this current recapitulate those of the native SOCC current. We conclude that C. elegans expresses bona fide CRAC channels that require the function of Orai1- and STIM1-related proteins. CRAC channels thus arose very early in animal evolution. In C. elegans, CRAC channels do not play obligate roles in all IP3-dependent signalling processes and ER Ca2+ homeostasis. Instead, we suggest that CRAC channels carry out highly specialized and cell-specific signalling roles and that they may function as a failsafe mechanism to prevent Ca2+ store depletion under pathophysiological and stress conditions. PMID:17218360

  7. Functional role of STIM1 and Orai1 in silkmoth (Bombyx mori) sex pheromone production

    USDA-ARS?s Scientific Manuscript database

    Store-operated Ca2+ influx has recently been shown to require the activation of two proteins, stromal interaction molecule 1(STIM1) and Orai1. In mammals the putative channel ion selectivity filter is thought to comprise conserved charged residues in the first and third transmembrane domains of Ora...

  8. Identification of key amino acid residues responsible for internal and external pH sensitivity of Orai1/STIM1 channels.

    PubMed

    Tsujikawa, Hiroto; Yu, Albert S; Xie, Jia; Yue, Zhichao; Yang, Wenzhong; He, Yanlin; Yue, Lixia

    2015-11-18

    Changes of intracellular and extracellular pH are involved in a variety of physiological and pathological processes, in which regulation of the Ca(2+) release activated Ca(2+) channel (I CRAC) by pH has been implicated. Ca(2+) entry mediated by I CRAC has been shown to be regulated by acidic or alkaline pH. Whereas several amino acid residues have been shown to contribute to extracellular pH (pHo) sensitivity, the molecular mechanism for intracellular pH (pHi) sensitivity of Orai1/STIM1 is not fully understood. By investigating a series of mutations, we find that the previously identified residue E106 is responsible for pHo sensitivity when Ca(2+) is the charge carrier. Unexpectedly, we identify that the residue E190 is responsible for pHo sensitivity when Na(+) is the charge carrier. Furthermore, the intracellular mutant H155F markedly diminishes the response to acidic and alkaline pHi, suggesting that H155 is responsible for pHi sensitivity of Orai1/STIM1. Our results indicate that, whereas H155 is the intracellular pH sensor of Orai1/STIM1, the molecular mechanism of external pH sensitivity varies depending on the permeant cations. As changes of pH are involved in various physiological/pathological functions, Orai/STIM channels may be an important mediator for various physiological and pathological processes associated with acidosis and alkalinization.

  9. Activating mutations in STIM1 and ORAI1 cause overlapping syndromes of tubular myopathy and congenital miosis

    PubMed Central

    Nesin, Vasyl; Wiley, Graham; Kousi, Maria; Ong, E-Ching; Lehmann, Thomas; Nicholl, David J.; Suri, Mohnish; Shahrizaila, Nortina; Katsanis, Nicholas; Gaffney, Patrick M.; Wierenga, Klaas J.; Tsiokas, Leonidas

    2014-01-01

    Signaling through the store-operated Ca2+ release-activated Ca2+ (CRAC) channel regulates critical cellular functions, including gene expression, cell growth and differentiation, and Ca2+ homeostasis. Loss-of-function mutations in the CRAC channel pore-forming protein ORAI1 or the Ca2+ sensing protein stromal interaction molecule 1 (STIM1) result in severe immune dysfunction and nonprogressive myopathy. Here, we identify gain-of-function mutations in the cytoplasmic domain of STIM1 (p.R304W) associated with thrombocytopenia, bleeding diathesis, miosis, and tubular myopathy in patients with Stormorken syndrome, and in ORAI1 (p.P245L), associated with a Stormorken-like syndrome of congenital miosis and tubular aggregate myopathy but without hematological abnormalities. Heterologous expression of STIM1 p.R304W results in constitutive activation of the CRAC channel in vitro, and spontaneous bleeding accompanied by reduced numbers of thrombocytes in zebrafish embryos, recapitulating key aspects of Stormorken syndrome. p.P245L in ORAI1 does not make a constitutively active CRAC channel, but suppresses the slow Ca2+-dependent inactivation of the CRAC channel, thus also functioning as a gain-of-function mutation. These data expand our understanding of the phenotypic spectrum of dysregulated CRAC channel signaling, advance our knowledge of the molecular function of the CRAC channel, and suggest new therapies aiming at attenuating store-operated Ca2+ entry in the treatment of patients with Stormorken syndrome and related pathologic conditions. PMID:24591628

  10. STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release.

    PubMed

    Darbellay, Basile; Arnaudeau, Serge; Bader, Charles R; Konig, Stephane; Bernheim, Laurent

    2011-07-25

    Cytosolic Ca(2+) signals encoded by repetitive Ca(2+) releases rely on two processes to refill Ca(2+) stores: Ca(2+) reuptake from the cytosol and activation of a Ca(2+) influx via store-operated Ca(2+) entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca(2+) sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca(2+) channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca(2+) channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca(2+) signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca(2+) sensor for their Ca(2+) homeostasis and intracellular signaling.

  11. STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release

    PubMed Central

    Arnaudeau, Serge; Bader, Charles R.; Bernheim, Laurent

    2011-01-01

    Cytosolic Ca2+ signals encoded by repetitive Ca2+ releases rely on two processes to refill Ca2+ stores: Ca2+ reuptake from the cytosol and activation of a Ca2+ influx via store-operated Ca2+ entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca2+ sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca2+ channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca2+ channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca2+ signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca2+ sensor for their Ca2+ homeostasis and intracellular signaling. PMID:21788372

  12. STIM1 and TRPV4 regulate fluid flow-induced calcium oscillation at early and late stages of osteoclast differentiation.

    PubMed

    Li, Ping; Bian, Xueyan; Liu, Chenglin; Wang, Shurong; Guo, Mengmeng; Tao, Yingjie; Huo, Bo

    2018-05-01

    Bone resorption is mainly mediated by osteoclasts (OCs), whose formation and function are regulated by intracellular Ca 2+ oscillation. Our previous studies demonstrated that fluid shear stress (FSS) lead to Ca 2+ oscillation through mechanosensitive cation-selective channels. However, the specific channels responsible for this FSS-induced Ca 2+ oscillation remain unknown. In the present study, we examined the expression of several Ca 2+ channels in OCs, including STIM1, ORAI1, TRPV1, TRPV4, TRPV5, and TRPV6, by western blotting and reverse transcription-polymerase chain reaction. The results showed that STIM1 was highly expressed in early stage OCs, while TRPV4 was highly expressed in late stage OCs. We observed intracellular Ca 2+ responses in OCs that were mechanically stimulated by FSS. When we blocked STIM1-dependent store-operated Ca 2+ entry or inhibited TRPV4 using siRNA or drug inhibition, FSS-induced Ca 2+ oscillations were almost undetectable in early and late stage OCs, respectively. These results indicate that STIM1 and TRPV4 act as mechanical transduction channels for OCs during the early and late differentiation stages, respectively, suggesting that these calcium channel could serve as markers of osteoclastogenesis or bone resorption. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Radiation inhibits salivary gland function by promoting STIM1 cleavage by caspase-3 and loss of SOCE through a TRPM2-dependent pathway

    PubMed Central

    Liu, Xibao; Gong, Baijuan; de Souza, Lorena Brito; Ong, Hwei Ling; Subedi, Krishna P.; Cheng, Kwong Tai; Swaim, William; Zheng, Changyu; Mori, Yasuo; Ambudkar, Indu S.

    2017-01-01

    Store-operated Ca2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3–mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2+/+, but not those from TRPM2−/− mice, displayed an increase in the concentrations of mitochondrial Ca2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy. PMID:28588080

  14. Visualisation of collagen fibrils in joint cartilage using STIM

    NASA Astrophysics Data System (ADS)

    Reinert, T.; Reibetanz, U.; Vogt, J.; Butz, T.; Werner, A.; Gründer, W.

    2001-07-01

    The scanning transmission ion microscopy (STIM) method was used to investigate the collagen network structure of the articular cartilage from a pig's knee in comparison with high resolution nuclear magnetic resonance imaging (microscopic NMR-tomography) and polarised light microscopy (PLM). Single collagen fibrils down to 200 nm in diameter were visualised. It was proved that the cartilage collagen network consists partly of zones of oriented fibrils as suggested by NMR measurements. Radially oriented fibrils were found in the zone near the calcified zone (hypertrophic zone) of both tibia and femur, and in the tibial radial zone. Tangentially oriented fibrils were found in the femoral and tibial superficial zone and in a second zone of the femoral cartilage. Polarisation light microscopy reveals broader zones of orientation than it was found with STIM.

  15. Function of a STIM1 Homologue in C. elegans: Evidence that Store-operated Ca2+ Entry Is Not Essential for Oscillatory Ca2+ Signaling and ER Ca2+ Homeostasis

    PubMed Central

    Yan, Xiaohui; Xing, Juan; Lorin-Nebel, Catherine; Estevez, Ana Y.; Nehrke, Keith; Lamitina, Todd; Strange, Kevin

    2006-01-01

    1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca2+ entry (SOCE) is activated during endoplasmic reticulum (ER) Ca2+ store depletion and is believed to be an essential and ubiquitous component of Ca2+ signaling pathways. SOCE is thought to function to refill Ca2+ stores and modulate Ca2+ signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca2+ sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530–amino acid protein with ∼21% sequence identity to human STIM1. Green fluorescent protein (GFP)–tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1∷GFP expression, suppresses the EF-hand mutation–induced pBoc arrhythmia, and inhibits intestinal store-operated Ca2+ (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca2+ signaling, in wild type and IP3 signaling mutant worms, and has no effect on intestinal Ca2+ oscillations and waves. Depletion of intestinal Ca2+ stores by RNAi knockdown of the ER Ca2+ pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca2+ signaling processes and for maintenance of store Ca2+ levels in C. elegans. These findings

  16. Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis.

    PubMed

    Yan, Xiaohui; Xing, Juan; Lorin-Nebel, Catherine; Estevez, Ana Y; Nehrke, Keith; Lamitina, Todd; Strange, Kevin

    2006-10-01

    1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels

  17. Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca2+i Mobilization and NFAT2 Activation

    PubMed Central

    Jans, Ralph; Mottram, Laura; Johnson, Darren L; Brown, Anna M; Sikkink, Stephen; Ross, Kehinde; Reynolds, Nick J

    2013-01-01

    Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μℳ LPA in 60 μℳ Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mℳ Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway. PMID:23096711

  18. Physiological roles of STIM1 and Orai1 homologs and CRAC channels in the genetic model organism Caenorhabditis elegans

    PubMed Central

    Strange, Kevin; Yan, Xiaohui; Lorin-Nebel, Catherine; Xing, Juan

    2007-01-01

    Summary The nematode Caenorhabditis elegans provides numerous experimental advantages for developing an integrative molecular understanding of physiological processes and has proven to be a valuable model for characterizing Ca2+ signaling mechanisms. This review will focus on the role of Ca2+ release activated Ca2+ (CRAC) channel activity in function of the worm gonad and intestine. Inositol 1,4,5-trisphosphate (IP3)-dependent oscillatory Ca2+ signaling regulates contractile activity of the gonad and rhythmic posterior body wall muscle contraction (pBoc) required for ovulation and defecation, respectively. The C. elegans genome contains a single homolog of both STIM1 and Orai1, proteins required for CRAC channel function in mammalian and Drosophila cells. C. elegans STIM-1 and ORAI-1 are coexpressed in the worm gonad and intestine and give rise to robust CRAC channel activity when coexpressed in HEK293 cells. STIM-1 or ORAI-1 knockdown causes complete sterility demonstrating that the genes are essential components of gonad Ca2+ signaling. Knockdown of either protein dramatically inhibits intestinal cell CRAC channel activity, but surprisingly has no effect on pBoc, intestinal Ca2+ oscillations or intestinal ER Ca2+ store homeostasis. CRAC channels thus do not play obligate roles in all IP3-dependent signaling processes in C. elegans. Instead, we suggest that CRAC channels carry out highly specialized and cell specific signaling roles and that they may function as a failsafe mechanism to prevent Ca2+ store depletion under pathophysiological and stress conditions. PMID:17376526

  19. StimTrack: An open-source software for manual transcranial magnetic stimulation coil positioning.

    PubMed

    Ambrosini, Emilia; Ferrante, Simona; van de Ruit, Mark; Biguzzi, Stefano; Colombo, Vera; Monticone, Marco; Ferriero, Giorgio; Pedrocchi, Alessandra; Ferrigno, Giancarlo; Grey, Michael J

    2018-01-01

    During Transcranial Magnetic Stimulation (TMS) experiments researchers often use a neuronavigation system to precisely and accurately maintain coil position and orientation. This study aimed to develop and validate an open-source software for TMS coil navigation. StimTrack uses an optical tracker and an intuitive user interface to facilitate the maintenance of position and orientation of any type of coil within and between sessions. Additionally, online access to navigation data is provided, hereby adding e.g. the ability to start or stop the magnetic stimulator depending on the distance to target or the variation of the orientation angles. StimTrack allows repeatable repositioning of the coil within 0.7mm for translation and <1° for rotation. Stimulus-response (SR) curves obtained from 19 healthy volunteers were used to demonstrate that StimTrack can be effectively used in a typical experiment. An excellent intra and inter-session reliability (ICC >0.9) was obtained on all parameters computed on SR curves acquired using StimTrack. StimTrack showed a target accuracy similar to that of a commercial neuronavigation system (BrainSight, Rogue Research Inc.). Indeed, small differences both in position (∼0.2mm) and orientation (<1°) were found between the systems. These differences are negligible given the human error involved in landmarks registration. StimTrack, available as supplementary material, is found to be a good alternative for commercial neuronavigation systems facilitating assessment changes in corticospinal excitability using TMS. StimTrack allows researchers to tailor its functionality to their specific needs, providing added value that benefits experimental procedures and improves data quality. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. StimDuino: an Arduino-based electrophysiological stimulus isolator.

    PubMed

    Sheinin, Anton; Lavi, Ayal; Michaelevski, Izhak

    2015-03-30

    Electrical stimulus isolator is a widely used device in electrophysiology. The timing of the stimulus application is usually automated and controlled by the external device or acquisition software; however, the intensity of the stimulus is adjusted manually. Inaccuracy, lack of reproducibility and no automation of the experimental protocol are disadvantages of the manual adjustment. To overcome these shortcomings, we developed StimDuino, an inexpensive Arduino-controlled stimulus isolator allowing highly accurate, reproducible automated setting of the stimulation current. The intensity of the stimulation current delivered by StimDuino is controlled by Arduino, an open-source microcontroller development platform. The automatic stimulation patterns are software-controlled and the parameters are set from Matlab-coded simple, intuitive and user-friendly graphical user interface. The software also allows remote control of the device over the network. Electrical current measurements showed that StimDuino produces the requested current output with high accuracy. In both hippocampal slice and in vivo recordings, the fEPSP measurements obtained with StimDuino and the commercial stimulus isolators showed high correlation. Commercial stimulus isolators are manually managed, while StimDuino generates automatic stimulation patterns with increasing current intensity. The pattern is utilized for the input-output relationship analysis, necessary for assessment of excitability. In contrast to StimuDuino, not all commercial devices are capable for remote control of the parameters and stimulation process. StimDuino-generated automation of the input-output relationship assessment eliminates need for the current intensity manually adjusting, improves stimulation reproducibility, accuracy and allows on-site and remote control of the stimulation parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Cardiovascular and Hemostatic Disorders: Role of STIM and Orai Proteins in Vascular Disorders.

    PubMed

    Tanwar, Jyoti; Trebak, Mohamed; Motiani, Rajender K

    2017-01-01

    Store-operated Ca 2+ entry (SOCE) mediated by STIM and Orai proteins is a highly regulated and ubiquitous signaling pathway that plays an important role in various cellular and physiological functions. Endoplasmic reticulum (ER) serves as the major site for intracellular Ca 2+ storage. Stromal Interaction Molecule 1/2 (STIM1/2) sense decrease in ER Ca 2+ levels and transmits the message to plasma membrane Ca 2+ channels constituted by Orai family members (Orai1/2/3) resulting in Ca 2+ influx into the cells. This increase in cytosolic Ca 2+ in turn activates a variety of signaling cascades to regulate a plethora of cellular functions. Evidence from the literature suggests that SOCE dysregulation is associated with several pathophysiologies, including vascular disorders. Interestingly, recent studies have suggested that STIM proteins may also regulate vascular functions independent of their contribution to SOCE. In this updated book chapter, we will focus on the physiological role of STIM and Orai proteins in the vasculature (endothelial cells and vascular smooth muscle cells). We will further retrospect the literature implicating a critical role for these proteins in vascular disease.

  2. Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy.

    PubMed

    Jessica, Sabourin; Angèle, Boet; Catherine, Rucker-Martin; Mélanie, Lambert; Ana-Maria, Gomez; Jean-Pierre, Benitah; Frédéric, Perros; Marc, Humbert; Fabrice, Antigny

    2018-05-01

    Right ventricular (RV) function is the most important prognostic factor for pulmonary arterial hypertension (PAH) patients. The progressive increase of pulmonary vascular resistance induces RV hypertrophy (RVH) and at term RV failure (RVF). However, the molecular mechanisms of RVH and RVF remain understudied. In this study, we gained insights into cytosolic Ca 2+ signaling remodeling in ventricular cardiomyocytes during the pathogenesis of severe pulmonary hypertension (PH) induced in rats by monocrotaline (MCT) exposure, and we further identified molecular candidates responsible for this Ca 2+ remodeling. After PH induction, hypertrophied RV myocytes presented longer action potential duration, higher and faster [Ca 2+ ] i transients and increased sarcoplasmic reticulum (SR) Ca 2+ content, whereas no changes in these parameters were detected in left ventricular (LV) myocytes. These modifications were associated with increased P-Ser 16 -phospholamban pentamer expression without altering SERCA2a (Sarco/Endoplasmic Reticulum Ca 2+ -ATPase) pump abundance. Moreover, after PH induction, Ca 2+ sparks frequency were higher in hypertrophied RV cells, while total RyR2 (Ryanodine Receptor) expression and phosphorylation were unaffected. Together with cellular hypertrophy, the T-tubules network was disorganized. Hypertrophied RV cardiomyocytes from MCT-exposed rats showed decreased expression of classical STIM1 (Stromal Interaction molecule) associated with increased expression of muscle-specific STIM1 Long isoform, glycosylated-Orai1 channel form, and TRPC1 and TRPC4 channels, which was correlated with an enhanced Ca 2+ -release-activated Ca 2+ (CRAC)-like current. Pharmacological inhibition of TRPCs/Orai1 channels in hypertrophied RV cardiomyocytes normalized [Ca 2+ ] i transients amplitude, the SR Ca 2+ content and cell contractility to control levels. Finally, we showed that most of these changes did not appear in LV cardiomyocytes. These new findings demonstrate RV

  3. The value of adding transcutaneous neuromuscular electrical stimulation (VitalStim) to traditional therapy for post-stroke dysphagia: a randomized controlled trial.

    PubMed

    Li, L; Li, Y; Huang, R; Yin, J; Shen, Y; Shi, J

    2015-02-01

    Dysphagia is not uncommon after stroke. Dysphagia may delay the functional recovery and substantially affects the quality of life after stroke, mainly if lest untreated. To detect and treat dysphagia as early as possible is critical for patients' recovery after stroke. Electrical stimulation has been reported as a treatment for pharyngeal dysphagia in recent studies, but the therapeutic effects of neuromuscular electrical stimulation (VitalStim®) therapy lacks convincing supporting evidence, needs further clinical investigation. To investigate the effects of neuromuscular electrical stimulation (VitalStim®) and traditional swallowing therapy on recovery of swallowing difficulties after stroke. Randomized controlled trial. University hospital. 135 stroke patients who had a diagnosis of dysphagia at the age between 50-80. 135 subjects were randomly divided into three groups: traditional swallowing therapy (N. = 45), VitalStim® therapy (N. = 45), and VitalStim® therapy plus traditional swallowing therapy (N. = 45). The traditional swallowing therapy included basic training and direct food intake training. Electrical stimulation was applied by an occupational therapist, using a modified hand-held battery-powered electrical stimulator (VitalStim® Dual Channel Unit and electrodes, Chattanooga Group, Hixson, TN, USA). Surface electromyography (sEMG), the Standardized Swallowing Assessment (SSA), Videofluoroscopic Swallowing Study (VFSS) and visual analog scale (VAS) were used to assess swallowing function before and 4 weeks after the treatment. The study included 118 subjects with dysphagia, 40 in the traditional swallowing therapy group and VitalStim® therapy group, 38 in the VitalStim and traditional swallowing therapy group. There were significant differences in sEMG value, SSA and VFSS scores in each group after the treatment (P < 0.001). After 4-week treatment, sEMG value (917.1 ± 91.2), SSA value (21.8 ± 3.5), oral transit time (0.4 ± 0.1) and pharyngeal

  4. Scanning transmission ion micro-tomography (STIM-T) of biological specimens.

    PubMed

    Schwertner, Micheal; Sakellariou, Arthur; Reinert, Tilo; Butz, Tilman

    2006-05-01

    Computed tomography (CT) was applied to sets of Scanning Transmission Ion Microscopy (STIM) projections recorded at the LIPSION ion beam laboratory (Leipzig) in order to visualize the 3D-mass distribution in several specimens. Examples for a test structure (copper grid) and for biological specimens (cartilage cells, cygospore) are shown. Scanning Transmission Micro-Tomography (STIM-T) at a resolution of 260 nm was demonstrated for the first time. Sub-micron features of the Cu-grid specimen were verified by scanning electron microscopy. The ion energy loss measured during a STIM-T experiment is related to the mass density of the specimen. Typically, biological specimens can be analysed without staining. Only shock freezing and freeze-drying is required to preserve the ultra-structure of the specimen. The radiation damage to the specimen during the experiment can be neglected. This is an advantage compared to other techniques like X-ray micro-tomography. At present, the spatial resolution is limited by beam position fluctuations and specimen vibrations.

  5. STIM and Orai proteins and the non-capacitative ARC channels

    PubMed Central

    Shuttleworth, Trevor J.

    2012-01-01

    The ARC channel is a small conductance, highly Ca2+-selective ion channel whose activation is specifically dependent on low concentrations of arachidonic acid acting at an intracellular site. They are widely distributed in diverse cell types where they provide an alternative, store-independent pathway for agonist-activated Ca2+ entry. Although biophysically similar to the store-operated CRAC channels, these two conductances function under distinct conditions of agonist stimulation, with the ARC channels providing the predominant route of Ca2+ entry during the oscillatory signals generated at low agonist concentrations. Despite these differences in function, like the CRAC channel, activation of the ARC channels is dependent on STIM1, but it is the pool of STIM1 that constitutively resides in the plasma membrane that is responsible. Similarly, both channels are formed by Orai proteins but, whilst the CRAC channel pore is a tetrameric assembly of Orai1 subunits, the ARC channel pore is formed by a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. There is increasing evidence that the activity of these channels plays a critical role a variety of different cellular activities. PMID:22201777

  6. STIM and Orai proteins as novel targets for cancer therapy. A Review in the Theme: Cell and Molecular Processes in Cancer Metastasis.

    PubMed

    Vashisht, Ayushi; Trebak, Mohamed; Motiani, Rajender K

    2015-10-01

    Calcium (Ca(2+)) regulates a plethora of cellular functions including hallmarks of cancer development such as cell cycle progression and cellular migration. Receptor-regulated calcium rise in nonexcitable cells occurs through store-dependent as well as store-independent Ca(2+) entry pathways. Stromal interaction molecules (STIM) and Orai proteins have been identified as critical constituents of both these Ca(2+) influx pathways. STIMs and Orais have emerged as targets for cancer therapeutics as their altered expression and function have been shown to contribute to tumorigenesis. Recent data demonstrate that they play a vital role in development and metastasis of a variety of tumor types including breast, prostate, cervical, colorectal, brain, and skin tumors. In this review, we will retrospect the data supporting a key role for STIM1, STIM2, Orai1, and Orai3 proteins in tumorigenesis and discuss the potential of targeting these proteins for cancer therapy. Copyright © 2015 the American Physiological Society.

  7. Magnetic resonance imaging following InterStim®: an institutional experience with imaging safety and patient satisfaction.

    PubMed

    Chermansky, Christopher J; Krlin, Ryan M; Holley, Thomas D; Woo, Howard H; Winters, J Christian

    2011-11-01

    We retrospectively assessed patient safety and satisfaction after magnetic resonance imaging (MRI) in patients with an InterStim® unit. The records of all patients implanted with InterStim® between 1998 and 2006 were reviewed. Nine of these patients underwent MRI following InterStim® implantation. The patients' neurologists requested the MRI exams for medical reasons. Both 0.6  Tesla (T) and 1.5  T machines were used. Patient safety, interference of implanted pulse generator (IPG) with radiological interpretation, and patient satisfaction were assessed in these patients. The first patient in the series had IPG failure following MRI. For this patient, the voltage amplitude was set to zero, the IPG was turned off, and the IPG magnetic switch was left on. The patient underwent MRI uneventfully; however, the IPG did not function upon reprogramming. The IPG magnetic switch was turned off for the eight subsequent patients, all of whom underwent MRI safely. In addition, all of their IPGs functioned appropriately following reprogramming. Of the 15 MRIs performed, the lumbar spine was imaged in eight studies, the pelvis was imaged in one study, and the remaining examinations involved imaging the brain or cervical spine. Neither the IPG nor the sacral leads interfered with MRI interpretation. None of the eight patients perceived a change in perception or satisfaction following MRI. Although we don't advocate the routine use of MRI following InterStim® implantation, our experience suggests MRI may be feasible under controlled conditions and without adverse events. Copyright © 2011 Wiley Periodicals, Inc.

  8. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

    PubMed

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

    2009-12-01

    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  9. Current practice patterns and knowledge among gynecologic surgeons of InterStim® programming after implantation.

    PubMed

    Hobson, Deslyn T G; Gaskins, Jeremy T; Frazier, LaTisha; Francis, Sean L; Kinman, Casey L; Meriwether, Kate V

    2017-10-03

    The objective of this study was to describe surgeons' current practices in InterStim® programming after initial implantation and their knowledge of programming parameters. We hypothesized that surgeons performing their own reprogramming would have increased knowledge. We administered a written survey to attendees at the Society of Gynecologic Surgeons Scientific Meeting and analyzed those on which surgeons indicated they offer InterStim® care. The survey queried surgeon characteristics, experience with InterStim® implantation and programming, and clinical opinions regarding reprogramming and tested six knowledge-based questions about programming parameters. Correct response to all six questions was the primary outcome. One hundred and thirty-five of 407 (33%) attendees returned the survey, of which 99 met inclusion criteria. Most respondents (88 of 99; 89%) were between 36 and 60 years, 27 (73%) were women, 76 (77%) practiced in a university setting, and 76 (77%) were trained in Female Pelvic Medicine and Reconstructive Surgery (FPMRS). Surgeons who had InterStim® programming training were more likely to perform their own programming [15/46 (32%) vs 6/47 (13%), p = 0.03]. Most answered all knowledge-based questions correctly (62/90, 69%); no surgeon characteristics were significantly associated with this outcome. Most surgeons cited patient comfort (71/80, 89%) and symptom relief (64/80, 80%) as important factors when reprogramming, but no prevalent themes emerged on how and why surgeons change certain programming parameters. Surgeons who had formal InterStim® programming training are more likely to perform programming themselves. No surgeon characteristic was associated with improved programming knowledge. We found that surgeons prioritize patient comfort and symptoms when deciding to reprogram.

  10. 78 FR 37648 - Space Transportation Infrastructure Matching (STIM) Grants Program

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-21

    ... DEPARTMENT OF TRANSPORTATION Federal Aviation Administration Space Transportation Infrastructure...-availability of Space Transportation Infrastructure Matching Grants in FY 2013. SUMMARY: The Office of Commercial Space Transportation (AST) will not solicit or award grants under the STIM program this fiscal...

  11. α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

    PubMed Central

    Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

    2016-01-01

    Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. PMID:27335124

  12. Effects of stromal interacting molecule 1 gene silencing by short hairpin RNA on the biological behavior of human gastric cancer cells.

    PubMed

    Liu, Bin; Yu, Hai-Hong; Ye, Hong-Li; Luo, Zhi-Ying; Xiao, Feng

    2015-08-01

    Gastric cancer is one of the most common types of cancer worldwide. It has been reported that stromal interacting molecule 1 (STIM1) is associated with tumor progression and metastatic spread, including in cervical cancer, breast carcinoma and prostatic cancer. The present study investigated whether STIM1, an endoplasmic reticulum Ca(2+) sensor and activator of store-operated channel entry, contributed to SGC7901 cell progression. The pGPU6-shSTIM1 recombinant plasmid was constructed, and the effects of downregulation of STIM1 on the proliferation, apoptosis, migration and invasion of SGC7901 cells were examined. Western blot analysis revealed that transfection with the pGPU6-shSTIM1 plasmid successfully inhibited the expression of STIM1. STIM1 silencing in the gastric cancer cells significantly inhibited cell proliferation by arresting the cell cycle at the G0/G1 phase, and increasing the apoptotic rate following treatment of the SGC7901 cells with pGPU6-shSTIM1, indicated using an MTT cell viability assay and flow cytometery, respectively. As expected, STIM1 knock down also reduced the migration and invasion of the SGC7901 cells, demonstrated using a Transwell assay. The possible molecular mechanism involved the regulation of several signaling pathways involved in the biological behavior of cell survival, apoptosis, migration and metastasis. Together, these finding suggested that the expression of STIM1 is crucial for the proliferation and invasion of SGC7901 cells, providing a foundation for the development of novel type‑specific diagnostic strategies and treatments for gastric cancer.

  13. Knockdown of stromal interaction molecule 1 inhibits proliferation of colorectal cancer cells by inducing apoptosis.

    PubMed

    Yang, Dong; Dai, Xiaoyu; Li, Keqiang; Xie, Yangyang; Zhao, Jianpei; Dong, Mingjun; Yu, Hua; Kong, Zhenfang

    2018-06-01

    Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca 2+ sensor which has been reported to be overexpressed in numerous types of cancer, and is involved in the cell proliferation, invasion, migration and metastasis frequently observed in cancer. However, the role of STIM1 in colorectal cancer (CRC) remains unknown. The purpose of the present study was to investigate the effect of STIM1 in human CRC. The expression of STIM1 was specifically knocked down using lentivirus-mediated small hairpin RNA (shRNA) interference techniques in the CRC cell lines HCT116 and SW1116. Subsequently, the efficiency of infection was confirmed using green fluorescent protein (GFP)-positive signals. The knockdown efficiency was further determined using the reverse transcription-quantitative polymerase chain reaction and western blotting analysis. As a result, CRC cell lines with STIM1 silenced were successfully constructed and subsequently employed in a series of cell function assays. Knockdown of STIM1 significantly suppressed cell proliferation and colony formation, as revealed by an MTT and colony formation assay. Furthermore, it was identified that STIM1 silencing may promote cell apoptosis through the induction of mitochondria-associated apoptosis, as was identified by increased expression levels of B-cell lymphoma 2 (Bcl-2)-associated death promoter, Bcl-2-associated X protein and poly(ADP-ribose) polymerase cleavage. Therefore, STIM1 may serve a critical role in the progression of CRC by regulating cell proliferation and apoptosis, which may provide a potential therapeutic target for the treatment of CRC.

  14. α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels.

    PubMed

    Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

    2016-08-15

    Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP-deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP-depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. © 2016 Li, Miao, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. Stromal interaction molecules 1 and 2 are key regulators of autoreactive T cell activation in murine autoimmune central nervous system inflammation.

    PubMed

    Schuhmann, Michael K; Stegner, David; Berna-Erro, Alejandro; Bittner, Stefan; Braun, Attila; Kleinschnitz, Christoph; Stoll, Guido; Wiendl, Heinz; Meuth, Sven G; Nieswandt, Bernhard

    2010-02-01

    Calcium (Ca(2+)) signaling in T lymphocytes is essential for a variety of functions, including the regulation of differentiation, gene transcription, and effector functions. A major Ca(2+) entry pathway in nonexcitable cells, including T cells, is store-operated Ca(2+) entry (SOCE), wherein depletion of intracellular Ca(2+) stores upon receptor stimulation causes subsequent influx of extracellular Ca(2+) across the plasma membrane. Stromal interaction molecule (STIM) 1 is the Ca(2+) sensor in the endoplasmic reticulum, which controls this process, whereas the other STIM isoform, STIM2, coregulates SOCE. Although the contribution of STIM molecules and SOCE to T lymphocyte function is well studied in vitro, their significance for immune processes in vivo has remained largely elusive. In this study, we studied T cell function in mice lacking STIM1 or STIM2 in a model of myelin-oligodendrocyte glycoprotein (MOG(35-55))-induced experimental autoimmune encephalomyelitis (EAE). We found that STIM1 deficiency significantly impaired the generation of neuroantigen-specific T cell responses in vivo with reduced Th1/Th17 responses, resulting in complete protection from EAE. Mice lacking STIM2 developed EAE, but the disease course was ameliorated. This was associated with a reduced clinical peak of disease. Deficiency of STIM2 was associated with an overall reduced proliferative capacity of lymphocytes and a reduction of IFN-gamma/IL-17 production by neuroantigen-specific T cells. Neither STIM1 nor STIM2 deficiency altered the phenotype or function of APCs. These findings reveal a crucial role of STIM-dependent pathways for T cell function and activation under autoimmune inflammatory conditions, establishing them as attractive new molecular therapeutic targets for the treatment of inflammatory and autoimmune disorders.

  16. Dysphagia management: an analysis of patient outcomes using VitalStim therapy compared to traditional swallow therapy.

    PubMed

    Kiger, Mary; Brown, Catherine S; Watkins, Lynn

    2006-10-01

    This study compares the outcomes using VitalStim therapy to outcomes using traditional swallowing therapy for deglutition disorders. Twenty-two patients had an initial and a followup videofluoroscopic swallowing study or fiberoptic endoscopic evaluation of swallowing and were divided into an experimental group that received VitalStim treatments and a control group that received traditional swallowing therapy. Outcomes were analyzed for changes in oral and pharyngeal phase dysphagia severity, dietary consistency restrictions, and progression from nonoral to oral intake. Results of chi(2) analysis showed no statistically significant difference in outcomes between the experimental and control groups.

  17. Transcutaneous neuromuscular electrical stimulation (VitalStim) curative therapy for severe dysphagia: myth or reality?

    PubMed

    Shaw, Gary Y; Sechtem, Phillip R; Searl, Jeff; Keller, Kristina; Rawi, Taib A; Dowdy, Emily

    2007-01-01

    VitalStim therapy was approved by the US Food and Drug Administration in 2001 for the treatment of dysphagia through the application of neuromuscular electrical stimulation to cervical swallowing muscles. This approval was based upon submission of data on more than 800 patients who received this therapy collected by the principal developer and patent-holder of the device. The therapy is marketed as successful in restoring long-term swallowing function in 97.5% of dysphagic patients past the point of requiring a feeding tube and as significantly better than existing therapies. More than 2,500 speech-language pathologists have taken the certification course, and thousands of devices have been sold. To date, however, aside from the developer's own studies, there are no peer-reviewed publications supporting these claims. We sought to evaluate the effectiveness of VitalStim therapy in a heterogeneous group of dysphagic patients. We performed a retrospective analysis of 18 patients who received this therapy at an urban tertiary referral center. All patients underwent pretherapy evaluation by speech-language pathologists, including modified barium swallow and/or functional endoscopic evaluation of swallowing and clinical evaluation of swallowing that included assessment of laryngeal elevation, diet tolerance, and swallowing delay, and were then assigned an overall dysphagia severity score. After therapy, all patients underwent the same assessments. Twelve of the 18 also underwent a functional swallowing telephone survey months (range, 1 to 21 months) after their therapy to assess whether the improvement was worthwhile and sustained. Eleven of the 18 patients (61%) demonstrated some improvement in their swallowing. Six of the 18 patients (33%) were improved enough to no longer require a feeding tube. However, of the 5 patients categorized as having "severe dysphagia" before therapy, only 2 showed any improvement, and these patients still required a feeding tube for

  18. Latest technologic and surgical developments in using InterStim Therapy for sacral neuromodulation: impact on treatment success and safety.

    PubMed

    Spinelli, Michele; Sievert, Karl-Dietrich

    2008-12-01

    This article accompanies a "surgery in motion" DVD on sacral neuromodulation (SNM) with InterStim Therapy, which visualizes the implantation of the InterStim II system. The article describes the technical and surgical developments of SNM and their impact on treatment success, safety, and patient's quality of life (QoL). Relevant literature on SNM with regard to technical changes and related clinical outcomes has been reviewed. Since its introduction in the early 1990s, SNM has proven useful in the treatment of several types of chronic urinary (and bowel) dysfunction. Recent technical improvements in devices and, in particular, the introduction of the tined lead 5 yr ago made SNM progress from an elaborate, open-surgery, general anesthesia, one-stage implant procedure to a minimally invasive, local anesthesia, percutaneous technique in two stages. The permanent tined lead implant enables a longer patient testing period (minimum of 14 d recommended) and less lead migration. This has considerably reduced technical failures and improved the success rate of the test phase; the response rate was almost doubled to approximately 80%. These improvements also affected tolerability, resulting in increased QoL for the patient. The use of the recently introduced smaller implantable neurostimulator InterStim II seems to further improve patient comfort and makes the implant procedure for the physician easier and shorter. However, this must be further addressed in clinical studies. SNM with InterStim Therapy using the tined lead offers an efficient treatment modality for patients in whom conservative treatment has failed.

  19. 49 CFR 172.411 - EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 2 2011-10-01 2011-10-01 false EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels..., EMERGENCY RESPONSE INFORMATION, TRAINING REQUIREMENTS, AND SECURITY PLANS Labeling § 172.411 EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label. (a) Except for size and color...

  20. Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells*

    PubMed Central

    Sabourin, Jessica; Le Gal, Loïc; Saurwein, Lisa; Haefliger, Jacques-Antoine; Raddatz, Eric; Allagnat, Florent

    2015-01-01

    Store-operated Ca2+ channels (SOCs) are voltage-independent Ca2+ channels activated upon depletion of the endoplasmic reticulum Ca2+ stores. Early studies suggest the contribution of such channels to Ca2+ homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca2+ depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca2+ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca2+ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes. PMID:26494622

  1. 49 CFR 172.411 - EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels..., 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label. (a) Except for size and color....5 and EXPLOSIVE 1.6 labels must be as follows: EXPLOSIVE 1.4: EC02MR91.016 EXPLOSIVE 1.5: EC02MR91...

  2. Bacterially activated B-cells drive T cell differentiation towards Tr1 through PD-1/PD-L1 expression.

    PubMed

    Said, Sawsan Sudqi; Barut, Guliz Tuba; Mansur, Nesteren; Korkmaz, Asli; Sayi-Yazgan, Ayca

    2018-04-01

    Regulatory B cells (Bregs) play a crucial role in immunological tolerance primarily through the production of IL-10 in many diseases including autoimmune disorders, allergy, infectious diseases, and cancer. To date, various Breg subsets with overlapping phenotypes have been identified. However, the roles of Bregs in Helicobacter infection are largely unknown. In the present study, we investigate the phenotype and function of Helicobacter -stimulated B cells. Our results demonstrate that Helicobacter felis -stimulated IL-10- producing B cells (Hf stim - IL-10 + B) are composed of B10 and Transitional 2 Marginal Zone Precursor (T2-MZP) cells with expression of CD9, Tim-1, and programmed death 1 (PD-1). On the other hand, Helicobacter felis -stimulated IL-10- nonproducing B (Hf stim - IL-10 - B) cells are mainly marginal zone (MZ) B cells that express PD-L1 and secrete TGF-β, IL-6, and TNF-α, and IgM and IgG2b. Furthermore, we show that both Hf stim - IL-10 + B cells and Hf stim - IL-10 - B cells induce CD49b + LAG-3 + Tr1 cells. Here, we describe a novel mechanism for PD-1/PD-L1- driven B cell-dependent Tr1 cell differentiation. Finally, we explore the capability of Hf stim - IL-10 - B cells to induce Th17 cell differentiation, which we find to be dependent on TGF-β. Taken together, the current study demonstrates that Hf stim - B cells induce Tr1 cells through the PD-1/PD-L1 axis and Th17 cells by secreting TGF-β. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Comprehensive Corticospinal Labeling with mu-crystallin Transgene Reveals Axon Regeneration after Spinal Cord Trauma in ngr1−/− Mice

    PubMed Central

    Fink, Kathren L.

    2015-01-01

    -green fluorescent protein (GFP) transgenic mice as a comprehensive and specific tool with which to study the primary descending motor tract, the corticospinal tract (CST). CST labeling with crym-GFP is 10 times more efficient compared with BDA. The enhanced sensitivity afforded by crym-GFP revealed significant CST regeneration in NgR1 knock-out mice. Therefore, crym-GFP can be used as a standardized tool for future CST spinal cord injury studies. PMID:26586827

  4. Label Review Training: Module 1: Label Basics, Page 29

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is a quiz on Module 1.

  5. MIB-1 labeling index, Ki-67, is an indicator of invasive intraductal papillary mucinous neoplasm.

    PubMed

    Shimura, Tatsuo; Kofunato, Yasuhide; Okada, Ryo; Yashima, Rei; Okada, Koji; Araki, Kenichiro; Hosouchi, Yasuo; Kuwano, Hiroyuki; Takenoshita, Seiichi

    2016-08-01

    Despite strict criteria for the observation of intraductal papillary mucinous neoplasm (IPMN), it remains difficult to distinguish invasive IPMN from non-invasive IPMN. The aim of the present study was to identify an indicator of invasive IPMN. The present study retrospectively evaluated 53 patients (28 with non-invasive and 25 with invasive IPMN) who underwent resection of IPMN, and examined the usefulness of the MIB-1 labeling index as an indicator of invasive IPMN. The MIB-1 labeling indexes in patients with invasive IPMN were significantly higher compared with those with non-invasive IPMN (P<0.001). A receiver operating characteristic curve revealed that the area under the curve was 0.822. These results suggested that a cut-off level for the MIB-1 labeling index should be set to 15.5% to distinguish invasive from non-invasive IPMN. A multivariate analysis using a logistic regression model revealed the MIB-1 labeling index (hazard ratio, 18.692; 95% confidential interval, 4.171-83.760; P<0.001) and the existence of mural nodules (hazard ratio, 6.187, 95% confidential interval, 1.039-36.861; P=0.045) were predictive factors for invasive IPMN. However, no statistically significant differences were observed between patients with a lower MIB-1 labeling index and patients with a higher MIB-1 labeling index (P=0.798). The MIB-1 labeling index must be considered as a candidate for the classification of IPMN.

  6. STIM proteins and the endoplasmic reticulum-plasma membrane junctions.

    PubMed

    Carrasco, Silvia; Meyer, Tobias

    2011-01-01

    Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

  7. Store-operated Ca2+ entry controls ameloblast cell function and enamel development

    PubMed Central

    Eckstein, Miriam; Vaeth, Martin; Fornai, Cinzia; Vinu, Manikandan; Bromage, Timothy G.; Nurbaeva, Meerim K.; Sorge, Jessica L.; Coelho, Paulo G.; Idaghdour, Youssef; Feske, Stefan; Lacruz, Rodrigo S.

    2017-01-01

    Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release–activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel. PMID:28352661

  8. Targeting the Ca(2+) Sensor STIM1 by Exosomal Transfer of Ebv-miR-BART13-3p is Associated with Sjögren's Syndrome.

    PubMed

    Gallo, Alessia; Jang, Shyh-Ing; Ong, Hwei Ling; Perez, Paola; Tandon, Mayank; Ambudkar, Indu; Illei, Gabor; Alevizos, Ilias

    2016-08-01

    Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease that is associated with inflammation and dysfunction of salivary and lacrimal glands. The molecular mechanism(s) underlying this exocrinopathy is not known, although the syndrome has been associated with viruses, such as the Epstein Barr Virus (EBV). We report herein that an EBV-specific microRNA (ebv-miR-BART13-3p) is significantly elevated in salivary glands (SGs) of pSS patients and we show that it targets stromal interacting molecule 1 (STIM1), a primary regulator of the store-operated Ca(2+) entry (SOCE) pathway that is essential for SG function, leading to loss of SOCE and Ca(2+)-dependent activation of NFAT. Although EBV typically infects B cells and not salivary epithelial cells, ebv-miR-BART13-3p is present in both cell types in pSS SGs. Importantly, we further demonstrate that ebv-miR-BART13-3p can be transferred from B cells to salivary epithelial cells through exosomes and it recapitulates its functional effects on calcium signaling in a model system. Published by Elsevier B.V.

  9. Label Review Training: Module 1: Label Basics, Page 21

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about types of labels.

  10. Label Review Training: Module 1: Label Basics, Page 20

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section focuses on supplemental labeling.

  11. Label Review Training: Module 1: Label Basics, Page 22

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about what labels require review.

  12. Label Review Training: Module 1: Label Basics, Page 19

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section covers supplemental distributor labeling.

  13. Label-free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis.

    PubMed

    Uckermann, Ortrud; Galli, Roberta; Leupold, Susann; Coras, Roland; Meinhardt, Matthias; Hallmeyer-Elgner, Susanne; Mayer, Thomas; Storch, Alexander; Schackert, Gabriele; Koch, Edmund; Blümcke, Ingmar; Steiner, Gerald; Kirsch, Matthias

    2017-01-01

    The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti-Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two-photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG-active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co-localized with positive albumin immunohistochemistry and might indicate degeneration-associated vascular leakage. We present a label-free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label-free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

  14. Label Review Training: Module 1: Label Basics, Page 18

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section discusses the types of labels.

  15. Label Review Training: Module 1: Label Basics, Page 26

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about mandatory and advisory label statements.

  16. Label Review Training: Module 1: Label Basics, Page 15

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the consequences of improper labeling.

  17. Label Review Training: Module 1: Label Basics, Page 14

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about positive effects from proper labeling.

  18. Label Review Training: Module 1: Label Basics, Page 24

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is about which labels require review.

  19. Label Review Training: Module 1: Label Basics, Page 17

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See an overview of the importance of labels.

  20. Label Review Training: Module 1: Label Basics, Page 27

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See examples of mandatory and advisory label statements.

  1. Label Review Training: Module 1: Label Basics, Page 23

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Lists types of labels that do not require review.

  2. Label Review Training: Module 1: Label Basics, Page 16

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the importance of labels and the role in enforcement.

  3. Magnetic Labeling of Activated Microglia in Experimental Gliomas1

    PubMed Central

    Fleige, Gerrit; Nolte, Christiane; Synowitz, Michael; Seeberger, Florian; Kettenmann, Helmut; Zimmer, Claus

    2001-01-01

    Abstract Microglia, as intrinsic immunoeffector cells of the central nervous system (CNS), play a very sensitive, crucial role in the response to almost any brain pathology where they are activated to a phagocytic state. Based on the characteristic features of activated microglia, we investigated whether these cells can be visualized with magnetic resonance imaging (MRI) using ultrasmall superparamagnetic iron oxides (USPIOs). The hypothesis of this study was that MR microglia visualization could not only reveal the extent of the tumor, but also allow for assessing the status of immunologic defense. Using USPIOs in cell culture experiments and in a rat glioma model, we showed that microglia can be labeled magnetically. Labeled microglia are detected by confocal microscopy within and around tumors in a typical border-like pattern. Quantitative in vitro studies revealed that microglia internalize amounts of USPIOs that are significantly higher than those incorporated by tumor cells and astrocytes. Labeled microglia can be detected and quantified with MRI in cell phantoms, and the extent of the tumor can be seen in glioma-bearing rats in vivo. We conclude that magnetic labeling of microglia provides a potential tool for MRI of gliomas, which reflects tumor morphology precisely. Furthermore, the results suggest that MRI may yield functional data on the immunologic reaction of the CNS. PMID:11774031

  4. Innervation of taste buds revealed with Brainbow-labeling in mouse.

    PubMed

    Zaidi, Faisal N; Cicchini, Vanessa; Kaufman, Daniel; Ko, Elizabeth; Ko, Abraham; Van Tassel, Heather; Whitehead, Mark C

    2016-12-01

    Nerve fibers that surround and innervate the taste bud were visualized with inherent fluorescence using Brainbow transgenic mice that were generated by mating the founder line L with nestin-cre mice. Multicolor fluorescence revealed perigemmal fibers as branched within the non-taste epithelium and ending in clusters of multiple rounded swellings surrounding the taste pore. Brainbow-labeling also revealed the morphology and branching pattern of single intragemmal fibers. These taste bud fibers frequently innervated both the peripheral bud, where immature gemmal cells are located, and the central bud, where mature, differentiated cells are located. The fibers typically bore preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs. These results establish an anatomical substrate for taste nerve fibers to contact and remodel among receptor cells at all stages of their differentiation, an interpretation that was supported by staining with GAP-43, a marker for growing fibers and growth cones. © 2016 Anatomical Society.

  5. Arterial spin labelling reveals an abnormal cerebral perfusion pattern in Parkinson's disease.

    PubMed

    Melzer, Tracy R; Watts, Richard; MacAskill, Michael R; Pearson, John F; Rüeger, Sina; Pitcher, Toni L; Livingston, Leslie; Graham, Charlotte; Keenan, Ross; Shankaranarayanan, Ajit; Alsop, David C; Dalrymple-Alford, John C; Anderson, Tim J

    2011-03-01

    There is a need for objective imaging markers of Parkinson's disease status and progression. Positron emission tomography and single photon emission computed tomography studies have suggested patterns of abnormal cerebral perfusion in Parkinson's disease as potential functional biomarkers. This study aimed to identify an arterial spin labelling magnetic resonance-derived perfusion network as an accessible, non-invasive alternative. We used pseudo-continuous arterial spin labelling to measure cerebral grey matter perfusion in 61 subjects with Parkinson's disease with a range of motor and cognitive impairment, including patients with dementia and 29 age- and sex-matched controls. Principal component analysis was used to derive a Parkinson's disease-related perfusion network via logistic regression. Region of interest analysis of absolute perfusion values revealed that the Parkinson's disease pattern was characterized by decreased perfusion in posterior parieto-occipital cortex, precuneus and cuneus, and middle frontal gyri compared with healthy controls. Perfusion was preserved in globus pallidus, putamen, anterior cingulate and post- and pre-central gyri. Both motor and cognitive statuses were significant factors related to network score. A network approach, supported by arterial spin labelling-derived absolute perfusion values may provide a readily accessible neuroimaging method to characterize and track progression of both motor and cognitive status in Parkinson's disease.

  6. Spin-labeled 1-alkyl-1-nitrosourea synergists of antitumor antibiotics.

    PubMed

    Gadjeva, V; Koldamova, R

    2001-01-01

    A new method for synthesis of four spin-labeled structural analogues of the antitumor drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), using ethyl nitrite for nitrosation of the intermediate spin-labeled ureas has been described. In vitro synergistic effects of 1-ethyl-3-[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)]-1-nitrosourea (3b) on the cytotoxicity of bleomycin and farmorubicin were found in human lymphoid leukemia tumor cells. We measured the tissue distribution of 3b in organ homogenates of C57BL mice by an electron paramagnetic resonance method. The spin-labeled nitrosourea was mainly localized in the lungs. Our results strongly support the development and validation of a new approach for synthesis of less toxic nitrosourea derivatives as potential synergists of antitumor drugs.

  7. Label Review Training: Module 1: Label Basics, Page 25

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review: clarity, accuracy, consistency with EPA policy, and enforceability.

  8. Phospholipid bilayer relaxation dynamics as revealed by the pulsed electron-electron double resonance of spin labels

    NASA Astrophysics Data System (ADS)

    Syryamina, V. N.; Dzuba, S. A.

    2012-10-01

    Electron paramagnetic resonance (EPR) spectroscopy in the form of pulsed electron-electron double resonance (ELDOR) was applied to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid bilayers containing lipids that were spin-labeled at different carbon positions along the lipid acyl chain. Pulsed ELDOR detects motionally induced spin flips of nitrogen nuclei in the nitroxide spin labels, which manifests itself as magnetization transfer (MT) in the nitroxide EPR spectrum. The MT effect was observed over a wide temperature range (100-225 K) on a microsecond time scale. In line with a previous study on molecular glasses [N. P. Isaev and S. A. Dzuba, J. Chem. Phys. 135, 094508 (2011), 10.1063/1.3633241], the motions that induce MT effect were suggested to have the same nature as those in dielectric secondary (β) Johari-Goldstein fast relaxation. The results were compared with literature dielectric relaxation data for POPC bilayers, revealing some common features. Molecular motions resulting in MT are faster for deeper spin labels in the membrane interior. The addition of cholesterol to the bilayer suppresses the lipid motions near the steroid nucleus and accelerates the lipid motions beyond the steroid nucleus, in the bilayer interior. This finding was attributed to the lipid acyl chains being more ordered near the steroid nucleus and less ordered in the bilayer interior. The motions are absent in dry lipids, indicating that the motions are determined by intermolecular interactions in the bilayer.

  9. A review of the design and clinical evaluation of the ShefStim array-based functional electrical stimulation system.

    PubMed

    Kenney, Laurence P; Heller, Ben W; Barker, Anthony T; Reeves, Mark L; Healey, Jamie; Good, Timothy R; Cooper, Glen; Sha, Ning; Prenton, Sarah; Liu, Anmin; Howard, David

    2016-11-01

    Functional electrical stimulation has been shown to be a safe and effective means of correcting foot drop of central neurological origin. Current surface-based devices typically consist of a single channel stimulator, a sensor for determining gait phase and a cuff, within which is housed the anode and cathode. The cuff-mounted electrode design reduces the likelihood of large errors in electrode placement, but the user is still fully responsible for selecting the correct stimulation level each time the system is donned. Researchers have investigated different approaches to automating aspects of setup and/or use, including recent promising work based on iterative learning techniques. This paper reports on the design and clinical evaluation of an electrode array-based FES system for the correction of drop foot, ShefStim. The paper reviews the design process from proof of concept lab-based study, through modelling of the array geometry and interface layer to array search algorithm development. Finally, the paper summarises two clinical studies involving patients with drop foot. The results suggest that the ShefStim system with automated setup produces results which are comparable with clinician setup of conventional systems. Further, the final study demonstrated that patients can use the system without clinical supervision. When used unsupervised, setup time was 14min (9min for automated search plus 5min for donning the equipment), although this figure could be reduced significantly with relatively minor changes to the design. Copyright © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.

  10. Using a Regression Method for Estimating Performance in a Rapid Serial Visual Presentation Target-Detection Task

    DTIC Science & Technology

    2017-12-01

    values designating each stimulus as a target ( true ) or nontarget (false). Both stim_time and stim_label should have length equal to the number of...position unless so designated by other authorized documents. Citation of manufacturer’s or trade names does not constitute an official endorsement or...depend strongly on the true values of hit rate and false-alarm rate. Based on its better estimation of hit rate and false-alarm rate, the regression

  11. An essential and NSF independent role for α-SNAP in store-operated calcium entry.

    PubMed

    Miao, Yong; Miner, Cathrine; Zhang, Lei; Hanson, Phyllis I; Dani, Adish; Vig, Monika

    2013-07-16

    Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca(2+) sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE. DOI:http://dx.doi.org/10.7554/eLife.00802.001.

  12. Metabolic labeling reveals proteome dynamics of mouse mitochondria.

    PubMed

    Kim, Tae-Young; Wang, Ding; Kim, Allen K; Lau, Edward; Lin, Amanda J; Liem, David A; Zhang, Jun; Zong, Nobel C; Lam, Maggie P Y; Ping, Peipei

    2012-12-01

    Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water ((2)H(2)O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with (2)H(2)O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level (2)H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d(-1) and 0.163 d(-1), respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (≈ 60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.

  13. Covalent Label Transfer between Peroxisomal Importomer Components Reveals Export-driven Import Interactions.

    PubMed

    Bhogal, Moninder S; Lanyon-Hogg, Thomas; Johnston, Katherine A; Warriner, Stuart L; Baker, Alison

    2016-01-29

    Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal malate dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes because of high degrees of protease susceptibility or resistance, respectively. Here we present a means for analysis of in vitro import through a covalent biotin label transfer and employ this method to the import of PEX5C. Label transfer demonstrates that the PEX5C construct is monomeric under the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labeling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and, strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Label Review Training: Module 1: Label Basics, Page 7

    EPA Pesticide Factsheets

    Page 7, Label Training, Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human he

  15. Neurog1 Genetic Inducible Fate Mapping (GIFM) Reveals the Existence of Complex Spatiotemporal Cyto-Architectures in the Developing Cerebellum.

    PubMed

    Obana, Edwin A; Lundell, Travis G; Yi, Kevin J; Radomski, Kryslaine L; Zhou, Qiong; Doughty, Martin L

    2015-06-01

    Neurog1 is a pro-neural basic helix-loop-helix (bHLH) transcription factor expressed in progenitor cells located in the ventricular zone and subsequently the presumptive white matter tracts of the developing mouse cerebellum. We used genetic inducible fate mapping (GIFM) with a transgenic Neurog1-CreER allele to characterize the contributions of Neurog1 lineages to cerebellar circuit formation in mice. GIFM reveals Neurog1-expressing progenitors are fate-mapped to become Purkinje cells and all GABAergic interneuron cell types of the cerebellar cortex but not glia. The spatiotemporal sequence of GIFM is unique to each neuronal cell type. GIFM on embryonic days (E) 10.5 to E12.5 labels Purkinje cells with different medial-lateral settling patterns depending on the day of tamoxifen delivery. GIFM on E11.5 to P7 labels interneurons and the timing of tamoxifen administration correlates with the final inside-to-outside resting position of GABAergic interneurons in the cerebellar cortex. Proliferative status and long-term BrdU retention of GIFM lineages reveals Purkinje cells express Neurog1 around the time they become post-mitotic. In contrast, GIFM labels mitotic and post-mitotic interneurons. Neurog1-CreER GIFM reveals a correlation between the timing of Neurog1 expression and the spatial organization of GABAergic neurons in the cerebellar cortex with possible implications for cerebellar circuit assembly.

  16. Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors.

    PubMed

    Rios, Adan; Quesada, Jorge; Anderson, Dallas; Goldstein, Allan; Fossum, Theresa; Colby-Germinario, Susan; Wainberg, Mark A

    2011-01-01

    We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. 5-a-day fruit and vegetable food product labels: reduced fruit and vegetable consumption following an exaggerated compared to a modest label.

    PubMed

    Appleton, K M; Pidgeon, H J

    2018-05-15

    Food product labels based on the WHO 5-a-day fruit and vegetable (FV) message are becoming increasingly common, but these labels may impact negatively on complementary or subsequent FV consumption. This study aimed to investigate the impact of a '3 of your 5-a-day' versus a '1 of your 5-a-day' smoothie product label on subsequent FV consumption. Using an acute experimental design, 194 participants (90 males, 104 females) were randomised to consume a smoothie labelled as either '3 of your 5-a-day' (N = 97) or '1 of your 5-a-day' (N = 97) in full, following a usual breakfast. Subsequent FV consumption was measured for the rest of the day using 24-h recall. Usual FV consumption was also assessed via 24-h recall for the day before the study. Regression analyses revealed a significantly lower subsequent FV consumption following smoothies displaying the '3 of your 5-a-day' label compared to the '1 of your 5-a-day' label (Beta = - 0.15, p = 0.04). Secondary analyses revealed these effects to be driven mainly by changes to consumption in usual high FV consumers, in females and in vegetable as opposed to fruit consumption. These findings demonstrate a role for label information in food intake, and the potential negative impacts of an exaggerated food product label on healthy food consumption and healthy dietary profiles.

  18. Incorporation of {sup 13}C-labeled intermediates into developing lignin revealed by analytical pyrolysis and CuO oxidation in combination with IRM-GC-MS

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

    Eglinton, T.I.; Goni, M.A.; Boon, J.J.

    1995-12-31

    Tissue samples from Ginkgo shoots (Ginkgo biloba L.) and Rice grass (Oryzasitiva sp.) incubated in the presence of {sup 13}C-labeled substrates such as coniferin (postulated to be biosynthetic intermediates in lignin biosynthesis) were studied using thermal and chemical dissociation methods in combination with molecular-level isotopic measurements. The aim of the study was (1) to investigate dissociation mechanisms, and (2) to examine and quantify the proportions of labeled material incorporated within each sample. Isotopic analysis of specific dissociation products revealed the presence of the label in its original positions, and only within lignin-derived (phenolic) products. Moreover, the distribution and isotopic compositionmore » of the dissociation products strongly suggest an origin from newly-formed lignin. These results clearly indicate that there is no {open_quotes}scrambling{close_quotes} of carbon atoms as a result of the dissociation process, thereby lending support to this analytical approach. In addition, the data provide confidence in the selective labeling approach for elucidation of the structure and biosynthesis of lignin.« less

  19. Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

    PubMed

    Cooke, R J; Björnestedt, R; Douglas, K T; McKie, J H; King, M D; Coles, B; Ketterer, B; Mannervik, B

    1994-09-01

    The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

  20. Arterial spin labelling reveals prolonged arterial arrival time in idiopathic Parkinson's disease.

    PubMed

    Al-Bachari, Sarah; Parkes, Laura M; Vidyasagar, Rishma; Hanby, Martha F; Tharaken, Vivek; Leroi, Iracema; Emsley, Hedley C A

    2014-01-01

    Idiopathic Parkinson's disease (IPD) is the second most common neurodegenerative disease, yet effective disease modifying treatments are still lacking. Neurodegeneration involves multiple interacting pathological pathways. The extent to which neurovascular mechanisms are involved is not well defined in IPD. We aimed to determine whether novel magnetic resonance imaging (MRI) techniques, including arterial spin labelling (ASL) quantification of cerebral perfusion, can reveal altered neurovascular status (NVS) in IPD. Fourteen participants with IPD (mean ± SD age 65.1 ± 5.9 years) and 14 age and cardiovascular risk factor matched control participants (mean ± SD age 64.6 ± 4.2 years) underwent a 3T MRI scan protocol. ASL images were collected before, during and after a 6 minute hypercapnic challenge. FLAIR images were used to determine white matter lesion score. Quantitative images of cerebral blood flow (CBF) and arterial arrival time (AAT) were calculated from the ASL data both at rest and during hypercapnia. Cerebrovascular reactivity (CVR) images were calculated, depicting the change in CBF and AAT relative to the change in end-tidal CO2. A significant (p = 0.005) increase in whole brain averaged baseline AAT was observed in IPD participants (mean ± SD age 1532 ± 138 ms) compared to controls (mean ± SD age 1335 ± 165 ms). Voxel-wise analysis revealed this to be widespread across the brain. However, there were no statistically significant differences in white matter lesion score, CBF, or CVR between patients and controls. Regional CBF, but not AAT, in the IPD group was found to correlate positively with Montreal cognitive assessment (MoCA) scores. These findings provide further evidence of alterations in NVS in IPD.

  1. Using mass spectrometry to study the photo-affinity labeling of protein tyrosine phosphatase 1B

    NASA Astrophysics Data System (ADS)

    Leriche, Tammy; Skorey, Kathryn; Roy, Patrick; McKay, Dan; Bateman, Kevin P.

    2004-11-01

    Protein tyrosine phosphatase 1B (PTP1B) is a potential target for the treatment of Type II diabetes and several companies are developing small molecule inhibitors of this enzyme. Part of the characterization of these compounds as PTP1B inhibitors is the understanding of how they bind in the enzyme active site. The use of photo-activated inhibitors that target the active site can provide such insight. This paper describes the characterization of a photoprobe directed at the active site of PTP1B. Mass spectrometry revealed the specific binding of the probe to the intact protein. Digestion of the labeled protein followed by LC-MS and LC-MS/MS was used to show that the photoprobe binds to a specific active site amino acid. This was confirmed by comparison with the X-ray structure of PTP1B with a PTP1B inhibitor. The probe labels a conserved acidic residue (Asp) that is required for catalytic activity. This photoprobe may prove to be a useful tool for the development of a PTP1B inhibitor or for the study of PTPs in general.

  2. Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

    PubMed

    Vazquez Reyes, Carolina; Tangprasertchai, Narin S; Yogesha, S D; Nguyen, Richard H; Zhang, Xiaojun; Rajan, Rakhi; Qin, Peter Z

    2017-06-01

    In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

  3. Preparation of 99mTc-TRODAT-1 with high labeling yield in boiling water bath: a new formulation.

    PubMed

    Erfani, Mostafa; Shafiei, Mohammad

    2014-04-01

    A new formulation for preparation of (99m)Tc-labeled tropane derivative, (99m)Tc-TRODAT-1, which is useful as a potential CNS dopamine transporter imaging agent, was evaluated and characterized. Preparation of (99m)Tc-TRODAT-1 was attained previously by a formulation in which vial has to be autoclaved at 121 °C for 30 min. It is highly desirable to further improve the preparation method by developing a simplified one vial formulation which will be labeled in boiling water bath (95 °C) for 15 min and a high labeling yield will be achieved. A formulation contained 10 μg of TRODAT-1, 20 μg tricine, 40 μg SnCl2 and 20mg manitol was prepared. Labeling was performed at 95 °C for 15 min and radiochemical analysis involved ITLC and HPLC methods. The stability of radioconjugate was checked in the presence of human serum at 37 °C up to 24h. (99m)Tc-TRODAT-1 was prepared with a radiochemical purity of more than 95% and specific activity of 64.3 MBq/nmol. Biodistribution studies of this new formulation in rats revealed similar regional brain distribution as compared with those obtained with the previous preparation in which brain uptake was high in striatum and striatum to cerebellum ratio was high. Requiring no autoclave facility for labeling, this new formulation will significantly improve the using feasibility of this radiopharmaceutical in clinic. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Mechanistic Studies of Hafnium-Pyridyl Amido-Catalyzed 1-Octene Polymerization and Chain Transfer Using Quench-Labeling Methods.

    PubMed

    Cueny, Eric S; Johnson, Heather C; Anding, Bernie J; Landis, Clark R

    2017-08-30

    Chromophore quench-labeling applied to 1-octene polymerization as catalyzed by hafnium-pyridyl amido precursors enables quantification of the amount of active catalyst and observation of the molecular weight distribution (MWD) of Hf-bound polymers via UV-GPC analysis. Comparison of the UV-detected MWD with the MWD of the "bulk" (all polymers, from RI-GPC analysis) provides important mechanistic information. The time evolution of the dual-detection GPC data, concentration of active catalyst, and monomer consumption suggests optimal activation conditions for the Hf pre-catalyst in the presence of the activator [Ph 3 C][B(C 6 F 5 ) 4 ]. The chromophore quench-labeling agents do not react with the chain-transfer agent ZnEt 2 under the reaction conditions. Thus, Hf-bound polymeryls are selectively labeled in the presence of zinc-polymeryls. Quench-labeling studies in the presence of ZnEt 2 reveal that ZnEt 2 does not influence the rate of propagation at the Hf center, and chain transfer of Hf-bound polymers to ZnEt 2 is fast and quasi-irreversible. The quench-label techniques represent a means to study commercial polymerization catalysts that operate with high efficiency at low catalyst concentrations without the need for specialized equipment.

  5. Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

    PubMed Central

    Nelson, P S; Kent, M; Muthini, S

    1992-01-01

    Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared. PMID:1475185

  6. Isotope Label-Aided Mass Spectrometry Reveals the Influence of Environmental Factors on Metabolism in Single Eggs of Fruit Fly

    PubMed Central

    Tseng, Te-Wei; Wu, June-Tai; Chen, Yu-Chie; Urban, Pawel L.

    2012-01-01

    In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar (13C6-glucose) for 12 h – either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate – possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism. PMID:23185587

  7. On-target labeling of intracellular metabolites combined with chemical mapping of individual hyphae revealing cytoplasmic relocation of isotopologues.

    PubMed

    Hu, Jie-Bi; Chen, Yu-Chie; Urban, Pawel L

    2012-06-05

    A microscale analytical platform integrating microbial cell culture, isotopic labeling, along with visual and mass spectrometric imaging with single-cell resolution has been developed and applied in the monitoring of cellular metabolism in fungal mycelium. The method implements open chips with a two-dimensional surface pattern composed of hydrophobic and hydrophilic zones. Two hydrophilic islands are used as medium reservoirs, while the hydrophobic area constitutes the support for the growing aerial hyphae, which do not have direct contact with the medium. The first island, containing (12)C(6)-glucose medium, was initially inoculated with the mycelium (Neurospora crassa), and following the initial incubation period, the hyphae progressed toward the second medium island, containing an isotopically labeled substrate ((13)C(6)-glucose). The (13)C atoms were gradually incorporated into cellular metabolites, which was revealed by MALDI-MS. The fate of the chitin-biosynthesis precursor, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), was monitored by recording mass spectra with characteristic isotopic patterns, which indicated the presence of various (12)C/(13)C isotopologues. The method enabled mapping the (13)C-labeled UDP-GlcNAc in fungal mycelium and recording its redistribution in hyphae, directly on the chip.

  8. Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging

    PubMed Central

    2014-01-01

    The molecular mechanisms underlying skeletal muscle aging and associated sarcopenia have been linked to an altered oxidative status of redox-sensitive proteins. Reactive oxygen and reactive nitrogen species (ROS/RNS) generated by contracting skeletal muscle are necessary for optimal protein function, signaling, and adaptation. To investigate the redox proteome of aging gastrocnemius muscles from adult and old male mice, we developed a label-free quantitative proteomic approach that includes a differential cysteine labeling step. The approach allows simultaneous identification of up- and downregulated proteins between samples in addition to the identification and relative quantification of the reversible oxidation state of susceptible redox cysteine residues. Results from muscles of adult and old mice indicate significant changes in the content of chaperone, glucose metabolism, and cytoskeletal regulatory proteins, including Protein DJ-1, cAMP-dependent protein kinase type II, 78 kDa glucose regulated protein, and a reduction in the number of redox-responsive proteins identified in muscle of old mice. Results demonstrate skeletal muscle aging causes a reduction in redox-sensitive proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response. Data is available via ProteomeXchange with identifier PXD001054. PMID:25181601

  9. A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis

    PubMed Central

    Liechti, G.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, Y. V.; VanNieuwenhze, M.; Maurelli, A. T.

    2014-01-01

    Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is consistent with the presence of chlamydial PG modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence to date that chlamydial species possess functional PG. PMID:24336210

  10. Effects of autoshaping procedures on 3H-8-OH-DPAT-labeled 5-HT1a binding and 125I-LSD-labeled 5-HT2a binding in rat brain.

    PubMed

    Tomie, Arthur; Di Poce, Jason; Aguado, Allison; Janes, Amy; Benjamin, Daniel; Pohorecky, Larissa

    2003-06-13

    Effects of experience with Pavlovian autoshaping procedures on lever-press autoshaping conditioned response (CR) performance and 3H-8-OH-DPAT-labeled binding of 5-HT(1a) receptors as well as 125I-LSD-labeled binding of 5-HT(2a) receptors were evaluated in four groups of male Long-Evans hooded rats. Two groups of rats (Group Paired High CR and Group Paired Low CR) received Pavlovian autoshaping procedures wherein the presentation of a lever (conditioned stimulus, CS) was followed by the response-independent presentation of food (unconditioned stimulus, US). Rats in Group Paired High CR (n=12) showed more rapid CR acquisition and higher asymptotic levels of lever-press autoshaping CR performance relative to rats in Group Low CR (n=12). Group Omission (n=9) received autoshaping with an omission contingency, such that performing the lever-press autoshaping CR resulted in the cancellation the food US, while Group Random (n=9) received presentations of lever CS and food US randomly with respect to one another. Though Groups Omission and Random did not differ in lever-press autoshaping CR performance, Group Omission showed significantly lower levels of 3H-8-OH-DPAT-labeled 5-HT(1a) binding in post-synaptic areas (frontal cortex, septum, caudate putamen), as well as significantly higher plasma corticosterone levels than Group Random. In addition, Group Random showed higher levels of 3H-8-OH-DPAT-labeled 5-HT(1a) binding in pre-synaptic somatodendritic autoreceptors on dorsal raphe nucleus relative to each of the other three groups. Autoradiographic analysis of 125I-LSD-labeled 5-HT(2a) receptor binding revealed no significant differences between Groups Paired High CR and Paired Low CR or between Groups Omission and Random in any brain regions.

  11. Isomeric forms of specifically. beta. -subunit labeled mitochondrial F/sub 1/-adenosinetriphosphatase with different properties

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

    Wang, J.H.; Wu, J.C.; Joshi, V.

    1986-05-01

    Treatment of the mitochondrial F/sub 1/-ATPase (MF/sub 1/) containing 1 specific 7-(4-nitro-2,1,3-(/sup 14/C)benzoxadiazolyl)-label (NBD) per enzyme molecule with acetylcysteine (AC) shows that the ratio r of specific ATPase activity of (O-NBD)/sub n/MF/sub 1/ to that of the control MF/sub 1/ increases linearly with the number of labels removed by AC from r < 0.1 to r > 0.9 and that dr/dn approx. = -1 as expected from specific labeling of an essential Tyr in the catalytic ..beta..' subunit. The r value of this labeled enzyme can also be increased 10-fold by LiCl-induced rearrangement of its subunits without removing any ofmore » the label. Similar treatment of the rearranged (O-NBD)/sub n/MF/sub 1/ shows that only a fraction of its radioactive labels can be removed at the normal rate by AC with dr/dn approx. = -1. The remaining labels have little inhibitory effect and are removed at much slower rates by AC with dr/dn approx. = 0. If the reaction with the rearranged (O-NBD)/sub n/MF/sub 1/ is terminated by gel-filtration when most of the labels on ..beta..' have been removed, an isomeric form of the covalently labeled enzyme is obtained with n > 0.5 but r approx. = 1, indicating that its labels are on the subunits (..beta..'') which do not catalyze directly. Incubation of O-..beta..'-NBD-MF/sub 1/ and O-BETA''-NBD-MF/sub 1/ at pH 8.95 gives N-..beta..'-NBD-MF/sub 1/ and N-..beta..''-NBD-MF/sub 1/ respectively with different fluorescence quenching characteristics.« less

  12. TRPC1 is involved in Ca²⁺ influx and cytotoxicity following Pb²⁺ exposure in human embryonic kidney cells.

    PubMed

    Zhang, Hongmei; Li, Wenjun; Xue, Yong; Zou, Fei

    2014-08-17

    Lead (Pb(2+)) is a divalent heavy metal ion which causes severe damage to almost all life forms and is therefore considered a notorious toxicant. Exposure to Pb(2+) is associated with poor cognitive development in children at relatively low levels that previously were thought to be safe. The mechanism through which Pb(2+) enters cells, however, is unclear. Previous studies have showed that Ca(2+) release-activated Ca(2+) protein 1 (Orai1), a component of store-operated Ca(2+) channels (SOCs), contributes to Pb(2+) cellular entry. Canonical transient receptor potential (TRPC1) channel 1 is a transient receptor potential (TRP) channel which is sometimes referred to as a SOC. The present study was designed to investigate the role of TRPC1 in Pb(2+) entry and toxicity in human embryonic kidney cells (HEK293). Additionally, changes in intracellular Ca(2+) concentration were determined through Fluo-4 and Mag-fluo-4 fluorescent Ca(2+) imaging. Following Pb(2+) exposure, there was a dose-dependent decrease in cell viability. Overexpression of TRPC1 increased Pb(2+)-induced cell death, while knockdown of this channel attenuated cell death. There was increased entry of Pb(2+), as measured by inductively coupled plasma mass spectrometry (ICP-MS), following overexpression of TRPC1. Conversely, knockdown of TRPC1 led to a decrease in Pb(2+) influx. Down-regulation of STIM1 by RNA interference attenuated the Pb(2+) influx, and transfection with a mutant STIM1, which could not gate TRPC1, had a similar effect. Co-transfection of mutant STIM1 and mutant TRPC1, which restore the electrostatic interaction between STIM1 and TRPC1, resumed Pb(2+) entry in HEK293 cells. Down-regulation of TRPC1 by RNA interference decreased Ca(2+) influx whilst its overexpression increased Ca(2+) entry in HEK293 cells. These results suggest that TRPC1 is involved in the cytotoxicity and entry of Pb(2+) through molecular interactions with STIM1 and subsequent Ca(2+) influx in HEK293 cells. Copyright

  13. Photoaffinity Labeling Studies on a Promoter of Dendritic Spine Formation

    NASA Astrophysics Data System (ADS)

    Sibucao, Kevin Carlo Abril

    The small molecule BTA-EG4 has been shown to be a promoter of dendritic spine formation. The mechanism behind this phenomenon, however, is not well understood. The work in this dissertation is motivated by this gap in knowledge. The first part of this dissertation focuses on photoaffinity labeling studies to identify the cellular targets of BTA-EG4. Chapter 1 provides a summary of Alzheimer's disease, the rational design of BTA-EG 4, and methods to determine targets of small molecules. In Chapter 2, the synthesis of a BTA-EG4-based photoaffinity labeling probe and photodegradation studies are presented. Kinetic studies demonstrate that the probe photolyzes rapidly under UV light. In Chapter 3, photoaffinity labeling studies and subsequent protein identification experiments are reported. Competition experiments with the photoaffinity labeling probe and BTA-EG4 demonstrate that the probe labels a 55-kDa protein specifically. Tandem mass spectrometry revealed that the 55-kDa protein is the actin binding protein fascin 1. The second part of this dissertation focuses on the major protein identified from photoaffinity labeling studies, fascin 1. Chapter 4 provides a brief survey of the structure and function of fascin 1. In Chapter 5, characterizations of the interaction between BTA-EG4 and fascin 1 are reported. Isothermal titration calorimetry confirms the physical binding between fascin 1 and BTA-EG6, a BTA-EG4 analog. Slow speed sedimentation assays reveal that BTA-EG4 does not affect the actin-bundling activity of fascin 1. However, GST pull-down experiments show that BTA-EG4 inhibits the binding of fascin 1 with the GTPase Rab35. In addition, this work demonstrates that BTA-EG4 may be mechanistically distinct from the known fascin inhibitor G2.

  14. Interleukin 1 increases thymidine labeling index of normal tissues of mic but not the tumor

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

    Zaghloul, M.S.; Dorie, M.J.; Kallman, R.F.

    1994-07-01

    This study was conducted to investigate the action of human recombinant interleukin 1 as a radioprotector for different mouse normal cells other than bone marrow cells. Semi-continuous injections of tritiated thymidine were administered every 6 h, over 24 h to determine thymidine labeling index. Mice were injected with recombinant human interleukin 1 24 h prior to tritiated thymidine and were compared to control animals that did not receive interleukin 1. Mice were killed 1 h after the last thymidine injection. The 24 h thymidine labeling index for normal tissues and RIF-1 tumor was determined. Labeling indices were also determined 1-14more » days after a series of fractionated irradiations with or without pretreatment with a single dose of interleukin 1 administered 24 h prior to the first radiation. The thymidine labeling index of normal tissues was higher following the injection of recombinant human interleukin 1 24 h before radiolabeling. This was found in all normal tissues tested. The thymidine labeling index of RIF-1 fibrosarcoma was not affected by interleukin 1 injection. A single interleukin 1 injection 24 h before the first radiation fraction also increased the thymidine labeling indices of normal tissues after localized fractionated irradiation. The thymidine labeling index of RIF-1 tumor was not increased by interleukin 1 administration except after relatively high radiation doses (20 Gy in five fractions). The ability of interleukin 1 to enhance the thymidine labeling index declined after the first day following the completion of fractionated irradiation. Recombinant human interleukin 1 increased the 24 h thymidine labeling index in normal tissues in mice, but not in RIF-1 tumor. Fractionated irradiation could maintain the effect of a single dose of interleukin 1, administered 24 h prior to the first fraction, up to 24 h after the end of radiation. 25 refs., 3 figs., 1 tab.« less

  15. Activity-induced regulation of myosin isoform distribution - Comparison of two contractile activity programs

    NASA Technical Reports Server (NTRS)

    Diffee, Gary M.; Caiozzo, Vince J.; Mccue, Samuel A.; Herrick, Robert E.; Baldwin, Kenneth M.

    1993-01-01

    This study examined the role of specific types of contractile activity in regulating myosin heavy chain (MHC) isoform expression in rodent soleus. A combination of hindlimb suspension (SN) and two programmed contractile training activity paradigms, either isometric contractile activity (ST-IM) or high-load slowly shortening isovelocity activity, were utilized. Both training paradigms increased muscle mass compared with SN alone. However, only ST-IM resulted in a partial prevention of the suspension-induced decrease in type I MHC. With the use of a fluorescently labeled antibody to type IIa MHC, the distribution of MHCs among fibers was examined immunohistochemically. In SN, the percentage of cells staining positive for type IIa MHC was increased but the staining intensity of the positively staining cells was unchanged compared with control cells. In the ST-IM soleus, the percentage of positively staining fibers was unchanged but the intensity of the positively staining cells was decreased compared with SN values. These results suggest that 1) isometric contractile activity is more effective than isovelocity activity in preventing suspension-induced shifts in soleus MHC distribution and 2) changes associated with both suspension and training occur in only a small number of fibers, with the majority of fibers apparently unresponsive to these interventions.

  16. Label Review Training: Module 1: Label Basics, Page 8

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human he

  17. PQQ: Biosynthetic studies in Methylobacterium AM1 and Hyphomicrobium X using specific TC labeling and NMR. [Pyrroloquinoline quinones

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

    Houck, D.R.; Hanners, J.L.; Unkefer, C.J.

    Using TC labeling and NMR spectroscopy we have determined biosynthetic precursors of pyrroloquinoline quinone (PQQ) in two closely related serine-type methylotrophs, Methylobacterium AM1 and Hyphomicrobium X. Analysis of the TC-labeling data revealed that PQQ is constructed from two amino acids: the portion containing N-6, C-7,8,9 and the two carboxylic acid groups, C-7' and 9', is derived-intact-from glutamate. The remaining portion is derived from tyrosine; the phenol side chain provides the six carbons of the ring containing the orthoquinone, whereas internal cyclization of the amino acid backbone forms the pyrrole-2-carboxylic acid moiety. This is analogous to the cyclization of dopaquinone tomore » form dopachrome. Dopaquinone is a product of the oxidation of tyrosine (via dopa) in reactions catalyzed by monophenol monooxygenase (EC 1.14.18.1). Starting with tyrosine and glutamate, we will discuss possible biosynthetic routes to PQQ. 29 refs., 4 figs., 2 tabs.« less

  18. Verbal Labels Modulate Perceptual Object Processing in 1-Year-Old Children

    ERIC Educational Resources Information Center

    Gliga, Teodora; Volein, Agnes; Csibra, Gergely

    2010-01-01

    Whether verbal labels help infants visually process and categorize objects is a contentious issue. Using electroencephalography, we investigated whether possessing familiar or novel labels for objects directly enhances 1-year-old children's neural processes underlying the perception of those objects. We found enhanced gamma-band (20-60 Hz)…

  19. Label Review Training: Module 1: Label Basics, Page 2

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  20. Label Review Training: Module 1: Label Basics, Page 9

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  1. Label Review Training: Module 1: Label Basics, Page 5

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  2. Label Review Training: Module 1: Label Basics, Page 4

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  3. Label Review Training: Module 1: Label Basics, Page 3

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  4. Label Review Training: Module 1: Label Basics, Page 6

    EPA Pesticide Factsheets

    Page 6, Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment

  5. Calcium-activated K(+) channel (K(Ca)3.1) activity during Ca(2+) store depletion and store-operated Ca(2+) entry in human macrophages.

    PubMed

    Gao, Ya-dong; Hanley, Peter J; Rinné, Susanne; Zuzarte, Marylou; Daut, Jurgen

    2010-07-01

    STIM1 'senses' decreases in endoplasmic reticular (ER) luminal Ca(2+) and induces store-operated Ca(2+) (SOC) entry through plasma membrane Orai channels. The Ca(2+)/calmodulin-activated K(+) channel K(Ca)3.1 (previously known as SK4) has been implicated as an 'amplifier' of the Ca(2+)-release activated Ca(2+) (CRAC) current, especially in T lymphocytes. We have previously shown that human macrophages express K(Ca)3.1, and here we used the whole-cell patch-clamp technique to investigate the activity of these channels during Ca(2+) store depletion and store-operated Ca(2+) influx. Using RT-PCR, we found that macrophages express the elementary CRAC channel components Orai1 and STIM1, as well as Orai2, Orai3 and STIM2, but not the putatively STIM1-activated channels TRPC1, TRPC3-7 or TRPV6. In whole-cell configuration, a robust Ca(2+)-induced outwardly rectifying K(+) current inhibited by clotrimazole and augmented by DC-EBIO could be detected, consistent with K(Ca)3.1 channel current (also known as intermediate-conductance IK1). Introduction of extracellular Ca(2+) following Ca(2+) store depletion via P2Y(2) receptors induced a robust charybdotoxin (CTX)- and 2-APB-sensitive outward K(+) current and hyperpolarization. We also found that SOC entry induced by thapsigargin treatment induced CTX-sensitive K(+) current in HEK293 cells transiently expressing K(Ca)3.1. Our data suggest that SOC and K(Ca)3.1 channels are tightly coupled, such that a small Ca(2+) influx current induces a much large K(Ca)3.1 channel current and hyperpolarization, providing the necessary electrochemical driving force for prolonged Ca(2+) signaling and store repletion. Copyright 2010 Elsevier Ltd. All rights reserved.

  6. Site-specific orientation of an α-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

    PubMed

    Ding, Bei; Laaser, Jennifer E; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T; Chen, Zhan

    2013-11-27

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

  7. Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

    PubMed Central

    Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

    2013-01-01

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

  8. Open Sesame: treasure in store-operated calcium entry pathway for cancer therapy

    PubMed Central

    Zui, PAN; JianJie, MA

    2016-01-01

    Store-operated Ca2+ entry (SOCE) controls intracellular Ca2+ homeostasis and regulates a wide range of cellular events including proliferation, migration and invasion. The discovery of STIM proteins as Ca2+ sensors and Orai proteins as Ca2+ channel pore forming units provided molecular tools to understand the physiological function of SOCE. Many studies have revealed the pathophysiological roles of Orai and STIM in tumor cells. This review focuses on recent advances in SOCE and its contribution to tumorigenesis. Altered Orai and/or STIM functions may serve as biomarkers for cancer prognosis, and targeting the SOCE pathway may provide a novel means for cancer treatment. PMID:25481035

  9. Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.

    PubMed

    Favazza, M; Lerho, M; Houssier, C

    1990-06-01

    Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.

  10. Serial post-surgical stimulated and unstimulated highly sensitive thyroglobulin measurements in low- and intermediate-risk papillary thyroid carcinoma patients not receiving radioactive iodine.

    PubMed

    Kashat, Lawrence; Orlov, Steven; Orlov, David; Assi, Jasmeet; Salari, Farnaz; Walfish, Paul G

    2016-11-01

    The purpose of this study was to determine the natural temporal trends of serial thyroglobulin (Tg) among low/intermediate-risk PTC patients not receiving radioactive iodine (RAI) using TSH-stimulated Tg (Stim-Tg) and unstimulated highly sensitive Tg (u-hsTg). We prospectively analyzed serial Stim-Tg measurements after total thyroidectomy ± therapeutic central neck dissection among 121 consecutive low/intermediate-risk PTC patients who did not receive RAI, of whom 104 also had serial u-hsTg measurements available. Median follow-up was 6.5 years with Stim-Tg measurements commencing 3 months after surgery and u-hsTg commencing 1.8 years after surgery (when the assay became available). TSH stimulation was performed with 9-day T3 withdrawal, 22-day T4 withdrawal, or using recombinant human TSH (rhTSH). To account for within-patient correlations of repeated Tg measurements, temporal trends in Stim-Tg and u-hsTg were assessed using Generalized Estimating Equations. Stim-Tg models were adjusted for the method of TSH stimulation, whereas the u-hsTg models were adjusted for concurrent TSH level. Linear regression modeling was used to assess the trend in serial Stim-Tg and u-hsTg measurements as a function time from time of surgery throughout the duration of follow-up. The main outcome measured was the change in u-hsTg and Stim-Tg measurements over time. A total of 337 Stim-Tg (2.8/patient) and 602 u-hsTg (5.8/patient) measurements were analyzed. Among the 337 Stim-Tg measurements, Stim-Tg was assessed using rhTSH in 202 (60 %), T4 withdrawal in 41 (12 %), and T3 withdrawal in 94 (28 %) measurements. The overall mean ± 1SD for Stim-Tg and u-hsTg measured was 1.0 ± 1.2 and 0.2 ± 0.1 μg/L, respectively. When adjusted for method of TSH stimulation, serial Stim-Tg measurements did not significantly change over time (all p = NS). The estimated changes in Stim-Tg per year for rhTSH, T4 withdrawal, and T3 withdrawal were 0.01, -0.08, and 0.04

  11. L(2,1)-Labeling of the Strong Product of Paths and Cycles

    PubMed Central

    2014-01-01

    An L(2,1)-labeling of a graph G = (V, E) is a function f from the vertex set V(G) to the set of nonnegative integers such that the labels on adjacent vertices differ by at least two and the labels on vertices at distance two differ by at least one. The span of f is the difference between the largest and the smallest numbers in f(V). The λ-number of G, denoted by λ(G), is the minimum span over all L(2,1)-labelings of G. We consider the λ-number of P n⊠C m and for n ≤ 11 the λ-number of C n⊠C m. We determine λ-numbers of graphs of interest with the exception of a finite number of graphs and we improve the bounds on the λ-number of C n⊠C m, m ≥ 24 and n ≥ 26. PMID:24711734

  12. Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish

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

    Toraya, Tetsuo; Kida, Tetsuo; Kuyama, Atsushi

    Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N{sup 6}-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised againstmore » a 1-MeAde hapten, a single band with M{sub r} of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors. - Highlights: • Synthesis of photoaffinity labeling reagents for 1-methyladenine binders of starfish. • Immunochemical detection of photoaffinity-labeled 1-methyladenine binders. • Immunophotoaffinity labeling of a 47.5 K 1-methyladenine binder in oocytes and testis. • A possible candidate of oocyte maturation-inducing hormone receptors of starfish.« less

  13. Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR.

    PubMed

    Gupta, Sebanti; Tycko, Robert

    2018-02-01

    Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716-730, 2010; J Mol Biol 426:1109-1127, 2014; J Biol Chem 291:13098-13112, 2016; J Am Chem Soc 138:8538-8546, 2016; J Am Chem Soc 138:12029-12032, 2016; J Am Chem Soc 134:6455-6466, 2012; J Am Chem Soc 132:1976-1987, 2010; J Am Chem Soc 135:17793-17803, 2013; Proc Natl Acad Sci USA 112:14617-14622, 2015; J Am Chem Soc 138:14066-14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements. Nonetheless, the relatively high molecular weight of HIV-1 CA leads to congestion of solid state NMR spectra of fully isotopically labeled assemblies that has been an impediment to further progress. Here we describe an efficient protocol for production of segmentally labeled HIV-1 CA samples in which either the N-terminal domain (NTD) or the C-terminal domain (CTD) is uniformly 15 N, 13 C-labeled. Segmental labeling is achieved by trans-splicing, using the DnaE split intein. Comparisons of two-dimensional solid state NMR spectra of fully labeled and segmentally labeled tubular CA assemblies show substantial improvements in spectral resolution. The molecular structure of HIV-1 assemblies is not significantly perturbed by the single Ser-to-Cys substitution that we introduce between NTD and CTD segments, as required for trans-splicing.

  14. Store-Operated Calcium Channels

    PubMed Central

    Lewis, Richard S.

    2015-01-01

    Store-operated calcium channels (SOCs) are a major pathway for calcium signaling in virtually all metozoan cells and serve a wide variety of functions ranging from gene expression, motility, and secretion to tissue and organ development and the immune response. SOCs are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER), triggered physiologically through stimulation of a diverse set of surface receptors. Over 15 years after the first characterization of SOCs through electrophysiology, the identification of the STIM proteins as ER Ca2+ sensors and the Orai proteins as store-operated channels has enabled rapid progress in understanding the unique mechanism of store-operate calcium entry (SOCE). Depletion of Ca2+ from the ER causes STIM to accumulate at ER-plasma membrane (PM) junctions where it traps and activates Orai channels diffusing in the closely apposed PM. Mutagenesis studies combined with recent structural insights about STIM and Orai proteins are now beginning to reveal the molecular underpinnings of these choreographic events. This review describes the major experimental advances underlying our current understanding of how ER Ca2+ depletion is coupled to the activation of SOCs. Particular emphasis is placed on the molecular mechanisms of STIM and Orai activation, Orai channel properties, modulation of STIM and Orai function, pharmacological inhibitors of SOCE, and the functions of STIM and Orai in physiology and disease. PMID:26400989

  15. 10 CFR Appendix C to Part 20 - Quantities 1 of Licensed Material Requiring Labeling

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 1 2014-01-01 2014-01-01 false Quantities 1 of Licensed Material Requiring Labeling C Appendix C to Part 20 Energy NUCLEAR REGULATORY COMMISSION STANDARDS FOR PROTECTION AGAINST RADIATION Pt. 20, App. C Appendix C to Part 20—Quantities 1 of Licensed Material Requiring Labeling Radionuclide...

  16. 10 CFR Appendix C to Part 20 - Quantities 1 of Licensed Material Requiring Labeling

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 1 2013-01-01 2013-01-01 false Quantities 1 of Licensed Material Requiring Labeling C Appendix C to Part 20 Energy NUCLEAR REGULATORY COMMISSION STANDARDS FOR PROTECTION AGAINST RADIATION Pt. 20, App. C Appendix C to Part 20—Quantities 1 of Licensed Material Requiring Labeling Radionuclide...

  17. 10 CFR Appendix C to Part 20 - Quantities 1 of Licensed Material Requiring Labeling

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Quantities 1 of Licensed Material Requiring Labeling C Appendix C to Part 20 Energy NUCLEAR REGULATORY COMMISSION STANDARDS FOR PROTECTION AGAINST RADIATION Pt. 20, App. C Appendix C to Part 20—Quantities 1 of Licensed Material Requiring Labeling Radionuclide...

  18. 10 CFR Appendix C to Part 20 - Quantities 1 of Licensed Material Requiring Labeling

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 1 2011-01-01 2011-01-01 false Quantities 1 of Licensed Material Requiring Labeling C Appendix C to Part 20 Energy NUCLEAR REGULATORY COMMISSION STANDARDS FOR PROTECTION AGAINST RADIATION Pt. 20, App. C Appendix C to Part 20—Quantities 1 of Licensed Material Requiring Labeling Radionuclide...

  19. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

  20. Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy.

    PubMed

    Nestor, Gustav; Anderson, Taigh; Oscarson, Stefan; Gronenborn, Angela M

    2017-05-03

    NMR of a uniformly 13 C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the 13 C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the 13 C spectral dispersion of 13 C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.

  1. Carbocyanine dye labeling reveals a new motor nucleus in octopus brain.

    PubMed

    Robertson, J D; Schwartz, O M; Lee, P

    1993-02-22

    This work aims at a better understanding of the organization of the brain of Octopus vulgaris, emphasizing the touch and visual learning centers. We injected the carbocyanine dye, DiI, into the cerebrobrachial connectives and, separately, into the brachial nerves of living octopuses. In both experiments, retrogradely transported granules of DiI appeared in motor neurons in the superior buccal, posterior buccal and subvertical lobes and in a hitherto unsuspected motor nucleus of several hundred neurons in the posterior dorsal basal and median basal lobes. In addition we labeled afferent fibers by injecting DiI into the caudal (sensory) division of the cerebrobrachial connective on one side; the label spread throughout the superior buccal, posterior buccal and the lateral and median inferior frontal lobes mainly on the injected side. It extended through the cerebral tract into the subvertical lobe, into the superior frontal lobe through the interfrontal tract, through the posterior buccal commissure into the opposite posterior buccal lobe and into the median inferior frontal lobe. The work suggests a new function for the posterior dorsal and median basal lobes, which are shown for the first time to project through the inferior frontal lobe system into the brachial nerves. In addition it represents the first full report of the successful use of the carbocyanine dyes DiI and DiO for labeling nerve tissue in a live invertebrate animal.

  2. Potent and selective CC chemokine receptor 1 antagonists labeled with carbon-13, carbon-14, and tritium.

    PubMed

    Latli, Bachir; Hrapchak, Matt; Cheveliakov, Maxim; Reeves, Jonathan T; Marsini, Maurice; Busacca, Carl A; Senanayake, Chris H

    2018-05-15

    1-(4-Fluorophenyl)-1H-pyrazolo[3,4-c]pyridine-4-carboxylic acid (2-methanesulfonyl-pyridin-4-ylmethyl)-amide (1) and its analogs (2) and (3) are potent CCR1 antagonists intended for the treatment of rheumatoid arthritis. The detailed syntheses of these 3 compounds labeled with carbon-13 as well as the preparation of (1) and (2) labeled with carbon-14, and (1) labeled with tritium, are described. Copyright © 2018 John Wiley & Sons, Ltd.

  3. Magnetic Particle Spectroscopy Reveals Dynamic Changes in the Magnetic Behavior of Very Small Superparamagnetic Iron Oxide Nanoparticles During Cellular Uptake and Enables Determination of Cell-Labeling Efficacy.

    PubMed

    Poller, Wolfram C; Löwa, Norbert; Wiekhorst, Frank; Taupitz, Matthias; Wagner, Susanne; Möller, Konstantin; Baumann, Gert; Stangl, Verena; Trahms, Lutz; Ludwig, Antje

    2016-02-01

    In vivo tracking of nanoparticle-labeled cells by magnetic resonance imaging (MRI) crucially depends on accurate determination of cell-labeling efficacy prior to transplantation. Here, we analyzed the feasibility and accuracy of magnetic particle spectroscopy (MPS) for estimation of cell-labeling efficacy in living THP-1 cells incubated with very small superparamagnetic iron oxide nanoparticles (VSOP). Cell viability and proliferation capacity were not affected by the MPS measurement procedure. In VSOP samples without cell contact, MPS enabled highly accurate quantification. In contrast, MPS constantly overestimated the amount of cell associated and internalized VSOP. Analyses of the MPS spectrum shape expressed as harmonic ratio A₅/A₃ revealed distinct changes in the magnetic behavior of VSOP in response to cellular uptake. These changes were proportional to the deviation between MPS and actual iron amount, therefore allowing for adjusted iron quantification. Transmission electron microscopy provided visual evidence that changes in the magnetic properties correlated with cell surface interaction of VSOP as well as with alterations of particle structure and arrangement during the phagocytic process. Altogether, A₅/A₃-adjusted MPS enables highly accurate, cell-preserving VSOP quantification and furthermore provides information on the magnetic characteristics of internalized VSOP.

  4. 9 CFR 317.1 - Labels required; supervision by Program employee.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., DEPARTMENT OF AGRICULTURE AGENCY ORGANIZATION AND TERMINOLOGY; MANDATORY MEAT AND POULTRY PRODUCTS INSPECTION... to bear such a label. (1) Wrappings of dressed carcasses and primal parts in an unprocessed state...

  5. Deep brain stimulation of the subthalamic nucleus enhances emotional processing in Parkinson disease.

    PubMed

    Schneider, Frank; Habel, Ute; Volkmann, Jens; Regel, Sabine; Kornischka, Jürgen; Sturm, Volker; Freund, Hans-Joachim

    2003-03-01

    High-frequency electrical stimulation of the subthalamic nucleus is a new and highly effective therapy for complications of long-term levodopa therapy and motor symptoms in advanced Parkinson disease (PD). Clinical observations indicate additional influence on emotional behavior. Electrical stimulation of deep brain nuclei with pulse rates above 100 Hz provokes a reversible, lesioning-like effect. Here, the effect of deep brain stimulation of the subthalamic nucleus on emotional, cognitive, and motor performance in patients with PD (n = 12) was examined. The results were compared with the effects of a suprathreshold dose of levodopa intended to transiently restore striatal dopamine deficiency. Patients were tested during medication off/stimulation off (STIM OFF), medication off/stimulation on (STIM ON), and during the best motor state after taking levodopa without deep brain stimulation (MED). More positive self-reported mood and an enhanced mood induction effect as well as improvement in emotional memory during STIM ON were observed, while during STIM OFF, patients revealed reduced emotional performance. Comparable effects were revealed by STIM ON and MED. Cognitive performance was not affected by the different conditions and treatments. Deep brain stimulation of the subthalamic nucleus selectively enhanced affective processing and subjective well-being and seemed to be antidepressive. Levodopa and deep brain stimulation had similar effects on emotion. This finding may provide new clues about the neurobiologic bases of emotion and mood disorders, and it illustrates the important role of the basal ganglia and the dopaminergic system in emotional processing in addition to the well-known motor and cognitive functions.

  6. The multispecific thyroid hormone transporter OATP1C1 mediates cell-specific sulforhodamine 101-labeling of hippocampal astrocytes.

    PubMed

    Schnell, Christian; Shahmoradi, Ali; Wichert, Sven P; Mayerl, Steffen; Hagos, Yohannes; Heuer, Heike; Rossner, Moritz J; Hülsmann, Swen

    2015-01-01

    Sulforhodamine 101 (SR101) is widely used for astrocyte identification, though the labeling mechanism remains unknown and the efficacy of labeling in different brain regions is heterogeneous. By combining region-specific isolation of astrocytes followed by transcriptome analysis, two-photon excitation microscopy, and mouse genetics, we identified the thyroid hormone transporter OATP1C1 as the SR101-uptake transporter in hippocampus and cortex.

  7. Orai1 enhances muscle endurance by promoting fatigue-resistant type I fiber content but not through acute store-operated Ca2+ entry

    PubMed Central

    Carrell, Ellie M.; Coppola, Aundrea R.; McBride, Helen J.; Dirksen, Robert T.

    2016-01-01

    Orai1 is a transmembrane protein that forms homomeric, calcium-selective channels activated by stromal interaction molecule 1 (STIM1) after depletion of intracellular calcium stores. In adult skeletal muscle, depletion of sarcoplasmic reticulum calcium activates STIM1/Orai1-dependent store-operated calcium entry. Here, we used constitutive and inducible muscle-specific Orai1-knockout (KO) mice to determine the acute and long-term developmental effects of Orai1 ablation on muscle structure and function. Skeletal muscles from constitutive, muscle-specific Orai-KO mice exhibited normal postnatal growth and fiber type differentiation. However, a significant reduction in fiber cross-sectional area occurred by 3 mo of age, with the most profound reduction observed in oxidative, fatigue-resistant fiber types. Soleus muscles of constitutive Orai-KO mice exhibited a reduction in unique type I fibers, concomitant with an increase in hybrid fibers expressing both type I and type IIA myosins. Additionally, ex vivo force measurements showed reduced maximal specific force and in vivo exercise assays revealed reduced endurance in constitutive muscle-specific Orai-KO mice. Using tamoxifen-inducible, muscle-specific Orai-KO mice, these functional deficits were found to be the result of the delayed fiber changes resulting from an early developmental loss of Orai1 and not the result of an acute loss of Orai1-dependent store-operated calcium entry.—Carrell, E. M., Coppola, A. R., McBride, H. J., Dirksen, R. T. Orai1 enhances muscle endurance by promoting fatigue-resistant type I fiber content but not through acute store-operated Ca2+ entry. PMID:27587568

  8. Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function.

    PubMed

    Concepcion, Axel R; Vaeth, Martin; Wagner, Larry E; Eckstein, Miriam; Hecht, Lee; Yang, Jun; Crottes, David; Seidl, Maximilian; Shin, Hyosup P; Weidinger, Carl; Cameron, Scott; Turvey, Stuart E; Issekutz, Thomas; Meyts, Isabelle; Lacruz, Rodrigo S; Cuk, Mario; Yule, David I; Feske, Stefan

    2016-11-01

    Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

  9. Effects of radiolysis on yttrium-90-labeled Lym-1 antibody preparations.

    PubMed

    Salako, Q A; O'Donnell, R T; DeNardo, S J

    1998-04-01

    The physical half-life of 2.6 days and 2.2 MeV beta emissions of 90Y provide excellent properties for radioimmunotherapy applications. However, the clinically useful beta particles may be a source of radiation-induced damage of 90Y-labeled immunoconjugate radiopharmaceuticals during preparation or short-term storage. The stability of 90Y-labeled Lym-1 antibody was studied in standard radiopharmacy conditions to establish a formulation at which radiolysis is not a problem. Lym-1-21T-BAD immunoconjugate intermediate was prepared according to our standard procedure, then labeled with 90Y at 1, 2, 4 and 9.4 mCi/mg Lym-1 using 0.5 M tetramethylammonium acetate, pH 7, labeling buffer. Each mixture was challenged in diethylenetriaminepentaacetic acid to remove nonspecifically bound 90Y. The 90Y-21T-BAD-Lym-1 products were purified by centrifuged molecular sieving column chromatography. The radiochemical purity and immunoreactivity of each preparation was monitored daily by high-performance liquid chromatography (HPLC) and solid-phase radioimmunoassay, respectively, for 3 days. The preparation at 2 mCi/mg was also formulated in 4% (wt/vol) human serum albumin (HSA) overall and at 9.4 mCi/mg in five-fold water, 4 and 10% (wt/vol) HSA overall; all were monitored as above. The monomeric quality and purity profile of products at 1 and 2 mCi/mg were retained (> or = 80%) as was their immunoreactivity (> or = 75%) over 3 days. The radiochemical purity and immunoreactivity of the product at 4 mCi/mg declined to 65% and 28%, respectively, by 3 days after preparation and in just 48 hr, the product at 9.4 mCi/mg had degraded to 21% in radiochemical purity with only 3% immunoreactivity. The current HPLC data and earlier published chromatographic evidence did not support a compromised radiochemical integrity of 90Y-DOTA complexes by loss of 90Y from the DOTA chelate. Radiolysis of 90Y-labeled antibody preparations did not appear to be a problem at 90Y-21T-BAD-Lym-1 products < or = 2 m

  10. Cumulative effects of anodal and priming cathodal tDCS on pegboard test performance and motor cortical excitability.

    PubMed

    Christova, Monica; Rafolt, Dietmar; Gallasch, Eugen

    2015-01-01

    Transcranial direct current stimulation (tDCS) protocols applied over the primary motor cortex are associated with changes in motor performance. This transcranial magnetic stimulation (TMS) study examines whether cathodal tDCS prior to motor training, combined with anodal tDCS during motor training improves motor performance and off-line learning. Three study groups (n=36) were trained on the grooved pegboard test (GPT) in a randomized, between-subjects design: SHAM-sham stimulation prior and during training, STIM1-sham stimulation prior and atDCS during training, STIM2-ctDCS stimulation prior and atDCS during training. Motor performance was assessed by GPT completion time and retested 14 days later to determine off-line learning. Cortical excitability was assessed via TMS at baseline (T0), prior training (T1), after training (T2), and 60 min after training (T3). Motor evoked potentials (MEP) were recorded from m. abductor pollicis brevis of the active left hand. GPT completion time was reduced for both stimulated groups compared to SHAM. For STIM2 this reduction in time was significantly higher than for STIM1 and further off-line learning occurred after STIM2. After ctDCS at T1, MEP amplitude and intracortical facilitation was decreased and intracortical inhibition was increased. After atDCS at T2, an opposite effect was observed for STIM1 and STIM2. For STIM2 these neuromodulatory effects were retained until T3. It is concluded that application of atDCS during the training improves pegboard performance and that additional priming with ctDCS has a positive effect on off-line learning. These cumulative behavioral gains were indicated by the preceding neuromodulatory changes. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Decreased store operated Ca2+ entry in dendritic cells isolated from mice expressing PKB/SGK-resistant GSK3.

    PubMed

    Schmid, Evi; Yan, Jing; Nurbaeva, Meerim K; Russo, Antonella; Yang, Wenting; Faggio, Caterina; Shumilina, Ekaterina; Lang, Florian

    2014-01-01

    Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca(2+)-concentration ([Ca(2+)]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3(KI) ). Factors affecting [Ca(2+)]i include Ca(2+)-release from intracellular stores (CRIS), store-operated Ca(2+)-entry (SOCE) through STIM1/STIM2-regulated Orai1, K(+)-dependent Na(+)/Ca(2+)-exchangers (NCKX), K(+)-independent Na(+)/Ca(2+)-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3(KI) mice and respective wild-type mice (gsk3(WT) ), [Ca(2+)]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1-10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3(WT) than in gsk3(KI) DCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3(KI) than in gsk3(WT) DCs. Activity of NCKX and NCX was significantly higher in gsk3(KI) than in gsk3(WT) DCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3(WT) DCs with SB216763 (1 µM, 4-24 h) or GSK-XIII (10 µM, 4-24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca(2+) in DCs. Acute inhibition of GSK3 blunted the increase of [Ca(2+)]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB

  12. Decreased Store Operated Ca2+ Entry in Dendritic Cells Isolated from Mice Expressing PKB/SGK-Resistant GSK3

    PubMed Central

    Nurbaeva, Meerim K.; Russo, Antonella; Yang, Wenting; Faggio, Caterina; Shumilina, Ekaterina; Lang, Florian

    2014-01-01

    Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca2+-concentration ([Ca2+]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3KI). Factors affecting [Ca2+]i include Ca2+-release from intracellular stores (CRIS), store-operated Ca2+-entry (SOCE) through STIM1/STIM2-regulated Orai1, K+-dependent Na+/Ca2+-exchangers (NCKX), K+-independent Na+/Ca2+-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3KI mice and respective wild-type mice (gsk3WT), [Ca2+]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1–10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3WT than in gsk3KIDCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3KI than in gsk3WTDCs. Activity of NCKX and NCX was significantly higher in gsk3KI than in gsk3WTDCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3WT DCs with SB216763 (1 µM, 4–24 h) or GSK-XIII (10 µM, 4–24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca2+ in DCs. Acute inhibition of GSK3 blunted the increase of [Ca2+]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase

  13. Carbon Metabolism of Soil microorganisms at Low Temperatures: Position-Specific 13C Labeled Glucose Reveals the Story

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Bore, E. K.; Halicki, S.; Kuzyakov, Y.; Dippold, M.

    2015-12-01

    Metabolic pathway activities at low temperature are not well understood, despite the fact that the processes are relevant for many soils globally and seasonally. To analyze soil metabolism at low temperature, isotopomeres of position-specifically 13C labeled glucose were applied at three temperature levels; +5, -5 -20 oC. In additon, one sterilization treatment with sodium azide at +5 oC was also performed. Soils were incubated for 1, 3 and 10 days while soil samples at -20 oC were additionally sampled after 30 days. The 13C from individual molecule position in respired CO2 was quantifed. Incorporation of 13C in bulk soil, extractable microbial biomass by chloroform fumigation extraction (CFE) and cell membranes of different microbial communities classified by 13C phospholipid fatty acid analysis (PLFA) was carried out. Our 13CO2 data showed a dominance of C-1 respiration at +5 °C for treatments with and without sodium azide, but total respiration for sodium azide inhibited treatments increased by 14%. In contrast, at -5 and -20 oC metabolic behavior showed intermingling of preferential respiration of the glucose C-4 and C-1 positions. Therefore, at +5 °C, pentose phosphate pathway activity is a dominant metabolic pathway used by microorganisms to metabolize glucose. The respiration increase due to NaN3 inhibition was attributed to endoenzymes released from dead organisms that are stabilized at the soil matrix and have access to suitable substrate and co-factors to permit their funtions. Our PLFA analysis showed that incorporation of glucose 13C was higher in Gram negative bacteria than other microbial groups as they are most competitive for LMWOS. Only a limited amount of microbial groups maintained their glucose utilizing activity at -5 and -20 °C and they strongly shifted towards a metabolization of glucose via both glycolysis and pentose phosphate pathways indicating both growth and cellular maintenance. This study revealed a remarkable microbial acitivity

  14. Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

    PubMed

    Kaspar-Schoenefeld, Stephanie; Merx, Kathleen; Jozefowicz, Anna Maria; Hartmann, Anja; Seiffert, Udo; Weschke, Winfriede; Matros, Andrea; Mock, Hans-Peter

    2016-06-30

    Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage

  15. Temperature optimum of insulin-stimulated 2-deoxy-D-glucose uptake in rat adipocytes. Correlation of cellular transport with membrane spin-label and fluorescence-label data.

    PubMed Central

    Hyslop, P A; Kuhn, C E; Sauerheber, R D

    1984-01-01

    The effects of temperature alterations between 22 degrees C and 48 degrees C on basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose uptake were examined in isolated rat adipocytes. A distinct optimum was found near physiological temperature for uptake in the presence of maximally effective insulin concentrations where insulin stimulation and hexose uptake were both conducted at each given assay temperature. Basal uptake was only subtly affected. Control and maximally insulin-stimulated cells incubated at 35 degrees C subsequently exhibited minimal temperature-sensitivity of uptake measured between 30 and 43 degrees C. The data are mostly consistent with the concept that insulin-sensitive glucose transporters are, after stimulation by insulin, functionally similar to basal transporters. Adipocyte plasma membranes were labelled with various spin- and fluorescence-label probes in lipid structural studies. The temperature-dependence of the order parameter S calculated from membranes labelled with 5-nitroxide stearate indicated the presence of a lipid phase change at approx. 33 degrees C. Membranes labelled with the fluorescence label 1,6-diphenylhexa-1,3,5-triene, or the cholesterol-like spin label nitroxide cholestane, reveal sharp transitions at lower temperatures. We suggest that a thermotropic lipid phase separation occurs in the adipocyte membrane that may be correlated with the temperature-dependence of hexose transport and insulin action in the intact cells. PMID:6324752

  16. Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

    PubMed Central

    Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

    2013-01-01

    Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light

  17. Kinesthetic stimulation for obstructive sleep apnea syndrome: An "on-off" proof of concept trial.

    PubMed

    Hernández, Alfredo I; Pérez, Diego; Feuerstein, Delphine; Loiodice, Corinne; Graindorge, Laurence; Guerrero, Gustavo; Limousin, Nadège; Gagnadoux, Frédéric; Dauvilliers, Yves; Tamisier, Renaud; Prigent, Arnaud; Mabo, Philippe; Amblard, Amel; Senhadji, Lotfi; Pépin, Jean-Louis

    2018-02-15

    Obstructive sleep apnea (OSA) occurs when the upper airway narrows or collapses due to the loss of upper airway muscle activation at sleep onset. This study investigated the effectiveness of triggered kinesthetic stimulation in patients with OSA. This proof-of-concept, open-label, multicenter prospective study was conducted on 24 patients with severe OSA. During a one night evaluation, kinesthetic stimulation was intermittently delivered in 30 minute periods. The duration of apneas and hypopneas during Stim on and Stim off periods were compared. Five hospital-based university centers in France participated. Sleep studies were evaluated by a single scorer at a core laboratory (CHU Grenoble). Results show that during the Stim on phases, statistically significant decreases in durations of apneas and hypopneas were observed in 56% and 46% of patients, respectively. Overall, 75% of patients showed an improvement in apneas or hypopneas durations. The mean reduction in durations for patients with a significant decrease was 4.86 seconds for apneas and 6.00 seconds for hypopneas. This proof of concept study is the first to identify kinesthetic stimulation as a potentially effective therapy for OSA. These data justify evaluation in a controlled study.

  18. Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum—Phytophthora capsici Phytopathosystem

    PubMed Central

    Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G.; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

    2016-01-01

    Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset

  19. Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum-Phytophthora capsici Phytopathosystem.

    PubMed

    Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

    2016-01-01

    Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset

  20. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ... container of a misleading form or size.\\1\\ FSIS has interpreted these provisions as requiring that the...-evaluating-labeling . Labels submitted as an extraordinary circumstance are given the highest priority for... submissions to FSIS headquarters, thus increasing the availability of FSIS labeling staff. Upon publication of...

  1. Review of nutrition labeling formats.

    PubMed

    Geiger, C J; Wyse, B W; Parent, C R; Hansen, R G

    1991-07-01

    This article examines nutrition labeling history as well as the findings of nine research studies of nutrition labeling formats. Nutrition labeling regulations were announced in 1973 and have been periodically amended since then. In response to requests from consumers and health care professionals for revision of the labeling system, the Food and Drug Administration initiated a three-phase plan for reform of nutrition labeling in 1990. President Bush signed the Nutrition Labeling and Education Act in November 1990. Literature analysis revealed that only nine studies with an experimental design have focused on nutrition labeling since 1971. Four were conducted before 1975, which was the year that nutrition labeling was officially implemented, two were conducted in 1980, and three were conducted after 1986. Only two of the nine studies supported the traditional label format mandated by the Code of Federal Regulations, and one study partially supported it. Four of the nine studies that evaluated graphic presentations of nutrition information found that consumer comprehension of nutrition information was improved with a graphic format for nutrition labeling: three studies supported the use of bar graphs and one study supported the use of a pie chart. Full disclosure (ie, complete nutrient and ingredient labeling) was preferred by consumers in two of the three studies that examined this variable. The third study supported three types of information disclosure dependent upon socioeconomic class. In those studies that tested graphics, a bar graph format was significantly preferred and showed better consumer comprehension than the traditional format.

  2. Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

    PubMed

    Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

    2014-07-16

    We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence

  3. Determination of inadvertent atrial capture during para-Hisian pacing.

    PubMed

    Obeyesekere, Manoj; Leong-Sit, Peter; Skanes, Allan; Krahn, Andrew; Yee, Raymond; Gula, Lorne J; Bennett, Matthew; Klein, George J

    2011-08-01

    Inadvertent capture of the atrium will lead to spurious results during para-Hisian pacing. We sought to establish whether the stimulation-to-atrial electrogram interval at the proximal coronary sinus (stim-PCS) or high right atrium (stim-HRA) could signal inadvertent atrial capture. Para-Hisian pacing with and without intentional atrial capture was performed in 31 patients. Stim-HRA and stim-PCS intervals were measured with atrial capture, His plus para-Hisian ventricular (H+V) capture, and para-Hisian ventricular (V) capture alone. The mean stim-HRA interval was significantly shorter with atrial capture (66 ± 18 ms) than with H+V (121 ± 27 ms, P < 0.001) or V capture alone (174 ± 38 ms, P < 0.001). The mean stim-PCS interval was significantly shorter with atrial capture (51 ± 16 ms) than with H+V (92 ± 22 ms, P<0.001) or V capture alone (146 ± 33 ms, P < 0.001). A stim-PCS < 60 ms (stim-HRA < 70 ms) was observed only with atrial capture. A stim-PCS >90 ms (stim-HRA >100 ms) was observed only in the absence of atrial capture. A stim-HRA of < 85 ms was highly specific and stim-PCS of < 85 ms highly sensitive at identifying atrial capture. Stim-HRA intervals of 75 to 97 ms and stim-PCS intervals of 65 to 88 ms were observed with either atrial, His, or para-Hisian ventricular capture without atrial capture. In this overlap zone, all patients demonstrated a stim-PCS or stim-HRA interval prolongation of at least 20 ms when the catheter was advanced to avoid deliberate atrial pacing. The QRS morphology was of limited value in distinguishing atrial capture due to concurrent ventricular or H+V capture, as observed in 20 of 31 (65%) patients. Stim-PCS and stim-HRA intervals can be used to monitor for inadvertent atrial capture during para-Hisian pacing. A stim-PCS < 60 ms (or stim-HRA < 70 ms) and stim-PCS > 90 ms (or stim-HRA > 100 ms) were observed only with and without atrial capture, respectively, but there was significant overlap between these values. Deliberate

  4. Reflexes in the shoulder muscles elicited from the human coracoacromial ligament.

    PubMed

    Diederichsen, Louise Pyndt; Nørregaard, Jesper; Krogsgaard, Michael; Fischer-Rasmussen, Torsten; Dyhre-Poulsen, Poul

    2004-09-01

    Morphological studies have demonstrated mechanoreceptors in the capsuloligamentous structures of the shoulder joint, however knowledge of the role these joint receptors play in the control of shoulder stability is limited. We therefore investigated the effect of electrically induced afferent activity from mechanoreceptors in the coracoacromial ligament (CAL) on the activity of voluntary activated shoulder muscles in healthy humans. In study I, wire electrodes, for electrical stimulation, were inserted into the CAL in eight normal shoulders. In study II, a needle electrode was inserted into the CAL in seven normal shoulders. Electric activity was recorded from eight shoulder muscles by surface and intramuscular electrodes. During isometric contractions, electrical stimulation was applied to the CAL at two different stimulus intensities, a weak stimulus (stim-1) and a stronger stimulus (stim-2). In both experiments, electrical stimulation of the CAL elicited a general inhibition in the voluntary activated shoulder muscles. In study I the average latencies (mean+/-SE) of the muscular inhibition were 66+/-4 ms (stim-1) and 62+/-4 ms (stim-2) during isometric flexion and 73+/-3 ms (stim-1) and 73+/-5 ms (stim-2) during isometric extension. In study II the average latency (mean+/-SE) of the response was 66+/-4 ms (stim-1) during isometric flexion. Our results demonstrated a response, probably of reflex origin, from mechanoreceptors in the CAL to the shoulder muscles. The existence of this synaptic connection between mechanoreceptors in CAL and the shoulder muscles suggest a role of these receptors in muscle coordination and in the functional joint stability.

  5. Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function

    PubMed Central

    Concepcion, Axel R.; Vaeth, Martin; Wagner, Larry E.; Eckstein, Miriam; Hecht, Lee; Yang, Jun; Crottes, David; Seidl, Maximilian; Shin, Hyosup P.; Weidinger, Carl; Cameron, Scott; Turvey, Stuart E.; Issekutz, Thomas; Meyts, Isabelle; Lacruz, Rodrigo S.; Cuk, Mario; Yule, David I.

    2016-01-01

    Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release–activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel–deficient patients and mice with ectodermal tissue–specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice. PMID:27721237

  6. Shrinkage of X cells in the lateral geniculate nucleus after monocular deprivation revealed by FoxP2 labeling.

    PubMed

    Duffy, Kevin R; Holman, Kaitlyn D; Mitchell, Donald E

    2014-05-01

    The parallel processing of visual features by distinct neuron populations is a central characteristic of the mammalian visual system. In the A laminae of the cat dorsal lateral geniculate nucleus (dLGN), parallel processing streams originate from two principal neuron types, called X and Y cells. Disruption of visual experience early in life by monocular deprivation has been shown to alter the structure and function of Y cells, but the extent to which deprivation influences X cells remains less clear. A transcription factor, FoxP2, has recently been shown to selectively label X cells in the ferret dLGN and thus provides an opportunity to examine whether monocular deprivation alters the soma size of X cells. In this study, FoxP2 labeling was examined in the dLGN of normal and monocularly deprived cats. The characteristics of neurons labeled for FoxP2 were consistent with FoxP2 being a marker for X cells in the cat dLGN. Monocular deprivation for either a short (7 days) or long (7 weeks) duration did not alter the density of FoxP2-positive neurons between nondeprived and deprived dLGN layers. However, for each deprived animal examined, measurement of the cross-sectional area of FoxP2-positive neurons (X cells) revealed that within deprived layers, X cells were smaller by approximately 20% after 7 days of deprivation, and by approximately 28% after 7 weeks of deprivation. The observed alteration to the cross-sectional area of X cells indicates that perturbation of this major pathway contributes to the functional impairments that develop from monocular deprivation.

  7. Methylphenidate, cognition, and epilepsy: A 1-month open-label trial.

    PubMed

    Adams, Jesse; Alipio-Jocson, Valerie; Inoyama, Katherine; Bartlett, Victoria; Sandhu, Saira; Oso, Jemima; Barry, John J; Loring, David W; Meador, Kimford J

    2017-12-01

    Cognitive difficulties are common in epilepsy. Beyond reducing seizures and adjusting antiepileptic medications, no well-validated treatment exists in adults. Methylphenidate is used effectively in children with epilepsy and attention-deficit/hyperactivity disorder, but its effects in adults have not been systematically evaluated. We hypothesized that methylphenidate can safely improve cognition in adults with epilepsy. We detail here the open-label follow-up to a double-blind, placebo-controlled, single-dose study. Thirty epilepsy patients entered a 1-month open-label methylphenidate trial after a double-blind phase. Doses were titrated according to clinical practice and patient tolerance, ranging 20-40 mg/day. Primary measures included: Conners' Continuous Performance Test (CPT), Symbol-Digit Modalities Test (SDMT), and Medical College of Georgia Memory Test (MCG). Secondary measures were: Beck Depression Inventory, 2nd Edition (BDI-II), Beck Anxiety Inventory, Apathy Evaluation Scale (AES), Stimulant Side-Effect Checklist, Adverse Events Profile, Quality of Life in Epilepsy-89 (QOLIE-89), and seizure frequency. Fourteen healthy, nonmedicated controls were tested concurrently. Twenty-eight participants with epilepsy (13 men/15 women) completed the trial. Withdrawals occurred due to anxiety (n = 1) and fatigue (n = 1). Mean age was 36.4 years (range = 20-60). Epilepsy types were: focal (n = 21), generalized (n = 6), or unclassified (n = 1). Mean epilepsy duration was 12.3 years. Mean baseline seizure frequency was 2.8/month. There were significant improvements on methylphenidate for SDMT, MCG, CPT (the ability to discriminate between targets and nontargets [d'] hits, hit reaction time standard deviation, omissions, and commissions), and QOLIE subscales (energy/fatigue, attention/concentration, memory, and language; paired t tests; p ≤ 0.002). BDI-II and additional subscales also improved, at a lower level of statistical significance. Effect

  8. Genetics Home Reference: Stormorken syndrome

    MedlinePlus

    ... Genetic Changes Stormorken syndrome is caused by a mutation in the STIM1 gene. The protein produced from ... and division, and immune function. The STIM1 gene mutation involved in Stormorken syndrome leads to production of ...

  9. Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

    PubMed

    Venkataramani, Varun; Kardorff, Markus; Herrmannsdörfer, Frank; Wieneke, Ralph; Klein, Alina; Tampé, Robert; Heilemann, Mike; Kuner, Thomas

    2018-04-03

    With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

  10. FocusStack and StimServer: a new open source MATLAB toolchain for visual stimulation and analysis of two-photon calcium neuronal imaging data

    PubMed Central

    Muir, Dylan R.; Kampa, Björn M.

    2015-01-01

    Two-photon calcium imaging of neuronal responses is an increasingly accessible technology for probing population responses in cortex at single cell resolution, and with reasonable and improving temporal resolution. However, analysis of two-photon data is usually performed using ad-hoc solutions. To date, no publicly available software exists for straightforward analysis of stimulus-triggered two-photon imaging experiments. In addition, the increasing data rates of two-photon acquisition systems imply increasing cost of computing hardware required for in-memory analysis. Here we present a Matlab toolbox, FocusStack, for simple and efficient analysis of two-photon calcium imaging stacks on consumer-level hardware, with minimal memory footprint. We also present a Matlab toolbox, StimServer, for generation and sequencing of visual stimuli, designed to be triggered over a network link from a two-photon acquisition system. FocusStack is compatible out of the box with several existing two-photon acquisition systems, and is simple to adapt to arbitrary binary file formats. Analysis tools such as stack alignment for movement correction, automated cell detection and peri-stimulus time histograms are already provided, and further tools can be easily incorporated. Both packages are available as publicly-accessible source-code repositories1. PMID:25653614

  11. RFP tags for labeling secretory pathway proteins

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

    Han, Liyang; Zhao, Yanhua; Zhang, Xi

    2014-05-09

    Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to anmore » environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.« less

  12. Reliable Genetic Labeling of Adult-Born Dentate Granule Cells Using Ascl1 CreERT2 and Glast CreERT2 Murine Lines.

    PubMed

    Yang, Sung M; Alvarez, Diego D; Schinder, Alejandro F

    2015-11-18

    Newly generated dentate granule cells (GCs) are relevant for input discrimination in the adult hippocampus. Yet, their precise contribution to information processing remains unclear. To address this question, it is essential to develop approaches to precisely label entire cohorts of adult-born GCs. In this work, we used genetically modified mice to allow conditional expression of tdTomato (Tom) in adult-born GCs and characterized their development and functional integration. Ascl1(CreERT2);CAG(floxStopTom) and Glast(CreERT2);CAG(floxStopTom) mice resulted in indelible expression of Tom in adult neural stem cells and their lineage upon tamoxifen induction. Whole-cell recordings were performed to measure intrinsic excitability, firing behavior, and afferent excitatory connectivity. Developing GCs were also staged by the expression of early and late neuronal markers. The slow development of adult-born GCs characterized here is consistent with previous reports using retroviral approaches that have revealed that a mature phenotype is typically achieved after 6-8 weeks. Our findings demonstrate that Ascl1(CreERT2) and Glast(CreERT2) mouse lines enable simple and reliable labeling of adult-born GC lineages within restricted time windows. Therefore, these mice greatly facilitate tagging new neurons and manipulating their activity, required for understanding adult neurogenesis in the context of network remodeling, learning, and behavior. Our study shows that Ascl1(CreERT2) and Glast(CreERT2) mice lines can be used to label large cohorts of adult-born dentate granule cells with excellent time resolution. Neurons labeled in this manner display developmental and functional profiles that are in full agreement with previous findings using thymidine analogs and retroviral labeling, thus providing an alternative approach to tackle fundamental questions on circuit remodeling. Because of the massive neuronal targeting and the simplicity of this method, genetic labeling will

  13. Measuring the labeling efficiency of pseudocontinuous arterial spin labeling.

    PubMed

    Chen, Zhensen; Zhang, Xingxing; Yuan, Chun; Zhao, Xihai; van Osch, Matthias J P

    2017-05-01

    Optimization and validation of a sequence for measuring the labeling efficiency of pseudocontinuous arterial spin labeling (pCASL) perfusion MRI. The proposed sequence consists of a labeling module and a single slice Look-Locker echo planar imaging readout. A model-based algorithm was used to calculate labeling efficiency from the signal acquired from the main brain-feeding arteries. Stability of the labeling efficiency measurement was evaluated with regard to the use of cardiac triggering, flow compensation and vein signal suppression. Accuracy of the measurement was assessed by comparing the measured labeling efficiency to mean brain pCASL signal intensity over a wide range of flip angles as applied in the pCASL labeling. Simulations show that the proposed algorithm can effectively calculate labeling efficiency when correcting for T1 relaxation of the blood spins. Use of cardiac triggering and vein signal suppression improved stability of the labeling efficiency measurement, while flow compensation resulted in little improvement. The measured labeling efficiency was found to be linearly (R = 0.973; P < 0.001) related to brain pCASL signal intensity over a wide range of pCASL flip angles. The optimized labeling efficiency sequence provides robust artery-specific labeling efficiency measurement within a short acquisition time (∼30 s), thereby enabling improved accuracy of pCASL CBF quantification. Magn Reson Med 77:1841-1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine Magn Reson Med 77:1841-1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

  14. Electrical stimulation therapy for dysphagia: a follow-up survey of USA dysphagia practitioners.

    PubMed

    Barikroo, Ali; Carnaby, Giselle; Crary, Michael

    2017-12-01

    The aim of this study was to compare current application, practice patterns, clinical outcomes, and professional attitudes of dysphagia practitioners regarding electrical stimulation (e-stim) therapy with similar data obtained in 2005. A web-based survey was posted on the American Speech-Language-Hearing Association Special Interest Group 13 webpage for 1 month. A total of 271 survey responses were analyzed and descriptively compared with the archived responses from the 2005 survey. Results suggested that e-stim application increased by 47% among dysphagia practitioners over the last 10 years. The frequency of weekly e-stim therapy sessions decreased while the reported total number of treatment sessions increased between the two surveys. Advancement in oral diet was the most commonly reported improvement in both surveys. Overall, reported satisfaction levels of clinicians and patients regarding e-stim therapy decreased. Still, the majority of e-stim practitioners continue to recommend this treatment modality to other dysphagia practitioners. Results from the novel items in the current survey suggested that motor level e-stim (e.g. higher amplitude) is most commonly used during dysphagia therapy with no preferred electrode placement. Furthermore, the majority of clinicians reported high levels of self-confidence regarding their ability to perform e-stim. The results of this survey highlight ongoing changes in application, practice patterns, clinical outcomes, and professional attitudes associated with e-stim therapy among dysphagia practitioners.

  15. A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement

    NASA Astrophysics Data System (ADS)

    Yang, Zhongyu; Bridges, Michael D.; López, Carlos J.; Rogozhnikova, Olga Yu.; Trukhin, Dmitry V.; Brooks, Evan K.; Tormyshev, Victor; Halpern, Howard J.; Hubbell, Wayne L.

    2016-08-01

    Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu2+ bound to an engineered Cu2+ binding site, enhanced T1 relaxation of TAM should enable measurement of interspin distances up to 50 Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins.

  16. Consumer preferences for food allergen labeling.

    PubMed

    Marra, Carlo A; Harvard, Stephanie; Grubisic, Maja; Galo, Jessica; Clarke, Ann; Elliott, Susan; Lynd, Larry D

    2017-01-01

    Food allergen labeling is an important tool to reduce risk of exposure and prevent anaphylaxis for individuals with food allergies. Health Canada released a Canadian food allergen labeling regulation (2008) and subsequent update (2012) suggesting that research is needed to guide further iterations of the regulation to improve food allergen labeling and reduce risk of exposure. The primary objective of this study was to examine consumer preferences in food labeling for allergy avoidance and anaphylaxis prevention. A secondary objective was to identify whether different subgroups within the consumer population emerged. A discrete choice experiment using a fractional factorial design divided into ten different versions with 18 choice-sets per version was developed to examine consumer preferences for different attributes of food labeling. Three distinct subgroups of Canadian consumers with different allergen considerations and food allergen labeling needs were identified. Overall, preferences for standardized precautionary and safety symbols at little or no increased cost emerged. While three distinct groups with different preferences were identified, in general the results revealed that the current Canadian food allergen labeling regulation can be improved by enforcing the use of standardized precautionary and safety symbols and educating the public on the use of these symbols.

  17. 2D-IR Spectroscopy of an AHA Labeled Photoswitchable PDZ2 Domain.

    PubMed

    Stucki-Buchli, Brigitte; Johnson, Philip J M; Bozovic, Olga; Zanobini, Claudio; Koziol, Klemens L; Hamm, Peter; Gulzar, Adnan; Wolf, Steffen; Buchenberg, Sebastian; Stock, Gerhard

    2017-12-14

    We explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties. Only one mutant revealed a measurable 2D IR difference signal. In contrast to the commonly observed frequency shifts that report on the degree of solvation, in this case we observe an intensity change. To understand this spectral response, we performed classical MD simulations, evaluating local contacts of the AHA labels to water molecules and protein side chains and calculating the vibrational frequency on the basis of an electrostatic model. Although these simulations revealed in part significant and complex changes of the number of intraprotein and water contacts upon trans-cis photoisomerization, they could not provide a clear explanation of why this one label would stick out. Subsequent quantum-chemistry calculations suggest that the response is the result of an electronic interaction involving charge transfer of the azido group with sulfonate groups from the photoswitch. To the best of our knowledge, such an effect has not been described before.

  18. Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin.

    PubMed

    Nelson, Wendy D; Blakely, Sarah E; Nesmelov, Yuri E; Thomas, David D

    2005-03-15

    We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.

  19. Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

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

    Li, Zhou; Adams, Rachel M; Chourey, Karuna

    2012-01-01

    A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

  20. Optimization and validation of FePro cell labeling method.

    PubMed

    Janic, Branislava; Rad, Ali M; Jordan, Elaine K; Iskander, A S M; Ali, Md M; Varma, N Ravi S; Frank, Joseph A; Arbab, Ali S

    2009-06-11

    Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17

  1. Metabolic Patterns in Spirodela polyrhiza Revealed by 15N Stable Isotope Labeling of Amino Acids in Photoautotrophic, Heterotrophic, and Mixotrophic Growth Conditions

    PubMed Central

    Evans, Erin M.; Freund, Dana M.; Sondervan, Veronica M.; Cohen, Jerry D.; Hegeman, Adrian D.

    2018-01-01

    In this study we describe a [15N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for 17 of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of <100% [15N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies. PMID:29904627

  2. Metabolic Patterns in Spirodela polyrhiza Revealed by 15N Stable Isotope Labeling of Amino Acids in Photoautotrophic, Heterotrophic, and Mixotrophic Growth Conditions.

    PubMed

    Evans, Erin M; Freund, Dana M; Sondervan, Veronica M; Cohen, Jerry D; Hegeman, Adrian D

    2018-01-01

    In this study we describe a [ 15 N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for 17 of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of <100% [ 15 N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies.

  3. Metabolic patterns in Spirodela polyrhiza revealed by 15N stable isotope labeling of amino acids in photoautotrophic, heterotrophic, and mixotrophic growth conditions

    NASA Astrophysics Data System (ADS)

    Evans, Erin M.; Freund, Dana M.; Sondervan, Veronica M.; Cohen, Jerry D.; Hegeman, Adrian D.

    2018-05-01

    In this study we describe a [15N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for fifteen of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of less than 100% [15N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies.

  4. Category labels versus feature labels: category labels polarize inferential predictions.

    PubMed

    Yamauchi, Takashi; Yu, Na-Yung

    2008-04-01

    What makes category labels different from feature labels in predictive inference? This study suggests that category labels tend to make inductive reasoning polarized and homogeneous. In two experiments, participants were shown two schematic pictures of insects side by side and predicted the value of a hidden feature of one insect on the basis of the other insect. Arbitrary verbal labels were shown above the two pictures, and the meanings of the labels were manipulated in the instructions. In one condition, the labels represented the category membership of the insects, and in the other conditions, the same labels represented attributes of the insects. When the labels represented category membership, participants' responses became substantially polarized and homogeneous, indicating that the mere reference to category membership can modify reasoning processes.

  5. Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

    PubMed

    Yano, Hiromi; Muguruma, Naoki; Ito, Susumu; Aoyagi, Eriko; Kimura, Tetsuo; Imoto, Yoshitaka; Cao, Jianxin; Inoue, Shohei; Sano, Shigeki; Nagao, Yoshimitsu; Kido, Hiroshi

    2006-09-01

    In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro

  6. Magnetic resonance imaging of single co-labeled mesenchymal stromal cells after intracardial injection in mice.

    PubMed

    Salamon, J; Wicklein, D; Didié, M; Lange, C; Schumacher, U; Adam, G; Peldschus, K

    2014-04-01

    The aim of this study was to establish co-labeling of mesenchymal stromal cells (MSC) for the detection of single MSC in-vivo by MRI and histological validation. Mouse MSC were co-labeled with fluorescent iron oxide micro-particles and carboxyfluorescein succinimidyl ester (CFSE). The cellular iron content was determined by atomic absorption spectrometry. Cell proliferation and expression of characteristic surface markers were determined by flow cytometry. The chondrogenic differentiation capacity was assessed. Different amounts of cells (n1 = 5000, n2 = 15 000, n3 = 50 000) were injected into the left heart ventricle of 12 mice. The animals underwent sequential MRI on a clinical 3.0 T scanner (Intera, Philips Medical Systems, Best, The Netherlands). For histological validation cryosections were examined by fluorescent microscopy. Magnetic and fluorescent labeling of MSC was established (mean cellular iron content 23.6 ± 3 pg). Flow cytometry showed similar cell proliferation and receptor expression of labeled and unlabeled MSC. Chondrogenic differentiation of labeled MSC was verified. After cell injection MRI revealed multiple signal voids in the brain and fewer signal voids in the kidneys. In the brain, an average of 4.6 ± 1.2 (n1), 9.0 ± 3.6 (n2) and 25.0 ± 1.0 (n3) signal voids were detected per MRI slice. An average of 8.7 ± 3.1 (n1), 22.0 ± 6.1 (n2) and 89.8 ± 6.5 (n3) labeled cells per corresponding stack of adjacent cryosections could be detected in the brain. Statistical correlation of the numbers of MRI signal voids in the brain and single MSC found by histology revealed a correlation coefficient of r = 0.91. The study demonstrates efficient magnetic and fluorescent co-labeling of MSC and their detection on a single cell level in mice by in-vivo MRI and histology. The described techniques may broaden the methods for in-vivo tracking of MSC. • Detection of single magnetically labeled MSC in

  7. A new fluorogenic small molecule labeling tool for surface diffusion analysis and advanced fluorescence imaging of β-site amyloid precursor protein (APP)-cleaving enzyme 1 based on silicone rhodamine: SiR-BACE1.

    PubMed

    Karch, Sandra; Broichhagen, Johannes; Schneider, Julia; Böning, Daniel; Hartmann, Stephanie; Schmid, Benjamin; Tripal, Philipp; Palmisano, Ralf; Alzheimer, Christian; Johnsson, Kai; Huth, Tobias

    2018-06-25

    β-site APP-cleaving enzyme 1 (BACE1) is a major player in the pathogenesis of Alzheimer's disease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques either require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduce SiR-BACE1, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photo-physical and pharmacologic properties, and evaluate its behavior in solution, in over-expression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion (STED), and dynamic single molecule microscopy. First functional studies with SiR-BACE1 on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.

  8. Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging

    NASA Astrophysics Data System (ADS)

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Ma, Tian-Hsiang; Lin, Yi-Chun; Lo, Szecheng J.; Chang, Ta-Chau

    2016-08-01

    Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm-1) and lipid (~2845 cm-1) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans.

  9. Putting the "th" in Tenths: Providing Place-Value Labels Helps Reveal the Structure of Our Base-10 Numeral System

    ERIC Educational Resources Information Center

    Loehr, Abbey M.; Rittle-Johnson, Bethany

    2017-01-01

    Research has demonstrated that providing labels helps children notice key features of examples. Much less is known about how different labels impact children's ability to make inferences about the structure underlying mathematical notation. We tested the impact of labeling decimals such as 0.34 using formal place-value labels ("3 tenths and 4…

  10. Retrograde double-labeling demonstrates convergent afferent innervation of the prostate and bladder.

    PubMed

    Lee, Sanghee; Yang, Guang; Xiang, William; Bushman, Wade

    2016-06-01

    Prostatic inflammation is a common histologic finding in men with lower urinary tract symptoms (LUTS). It has been postulated that prostatic inflammation could sensitize afferent neurons innervating the bladder and thereby produce changes in voiding behavior. In support of this, we demonstrate an anatomic basis for pelvic cross-talk involving the prostate and bladder. Retrograde labeling was performed by an application of a neuro-tracer Fast Blue (FB) to one side of either the anterior prostate (AP), dorsal lateral prostate (DLP)/ventral prostate (VP), bladder, or seminal vesicle (SV). Examination of dorsal root ganglion (DRG) neuron labeling revealed shared afferent innervation of the prostate and bladder at spinal segments of T13, L1, L2, L6, and S1. Dual labeling was performed by an application of FB and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyaine perchlorate (DiI) to the AP and bladder, respectively. We observed double-labeled DRG neurons at T13, L1, L2, L6, and S1--a finding that proves convergent innervation of prostate and bladder. Our observations demonstrate the potential for neural cross-talk between the prostate and bladder and support a postulated mechanism that prostatic inflammation may induce hyper-sensitization of bladder afferents and produce irritative LUTS. © 2016 Wiley Periodicals, Inc.

  11. 9 CFR 412.1 - Label approval.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... foreign language or printing labels that bear a statement of the quantity of contents in accordance with... Policy Book, (except for “natural” and negative claims (e.g., “gluten free”)), health claims, ingredient and processing method claims (e.g., high-pressure processing), structure-function claims, claims...

  12. Epigallocatechin-3-gallate (EGCG) up-regulates miR-15b expression thus attenuating store operated calcium entry (SOCE) into murine CD4+ T cells and human leukaemic T cell lymphoblasts.

    PubMed

    Zhang, Shaqiu; Al-Maghout, Tamer; Bissinger, Rosi; Zeng, Ni; Pelzl, Lisann; Salker, Madhuri S; Cheng, Anchun; Singh, Yogesh; Lang, Florian

    2017-10-27

    CD4 + T cells are key elements in immune responses and inflammation. Activation of T cell receptors in CD4 + T cells triggers cytosolic Ca 2+ release with subsequent store operated Ca 2+ entry (SOCE), which is accomplished by the pore forming Ca 2+ release activated Ca 2+ (CRAC) channel Orai1 and its regulator stromal cell-interaction molecule 2 (STIM2). Green tea polyphenol epigallocatechin-3-gallate (EGCG) acts as a potent anti-inflammatory and anti-oxidant agent for various types of cells including immune cells. However, how post-transcriptional gene regulators such as miRNAs are involved in the regulation of Ca 2+ influx into murine CD4 + T cells and human Jurkat T cells through EGCG is not defined. EGCG treatment of murine CD4 + T cells significantly down-regulated the expression of STIM2 and Orai1 both at mRNA and protein levels. Furthermore, EGCG significantly decreased SOCE in both murine and human T cells. EGCG treatment increased miRNA-15b (miR-15b) abundance in both murine and human T cells. Bioinformatics analysis reveals that miR-15b, which has a STIM2 binding site, is involved in the down-regulation of SOCE. Overexpression of miR-15b significantly decreased the mRNA and protein expression of STIM2 and Orai1 in murine T cells. Treatment of Jurkat T cells with 10 μM EGCG further decreased mTOR and PTEN protein levels. EGCG decreased mitochondrial membrane potential (MMP) in both human and murine T cells. In conclusion, the observations suggest that EGCG inhibits the Ca 2+ entry into murine and human T cells, an effect accomplished at least in part by up-regulation of miR-15b.

  13. ORAI1 mutations abolishing store-operated Ca2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency.

    PubMed

    Lian, Jayson; Cuk, Mario; Kahlfuss, Sascha; Kozhaya, Lina; Vaeth, Martin; Rieux-Laucat, Frédéric; Picard, Capucine; Benson, Melina J; Jakovcevic, Antonia; Bilic, Karmen; Martinac, Iva; Stathopulos, Peter; Kacskovics, Imre; Vraetz, Thomas; Speckmann, Carsten; Ehl, Stephan; Issekutz, Thomas; Unutmaz, Derya; Feske, Stefan

    2017-11-16

    Store-operated Ca 2+ entry (SOCE) through Ca 2+ release-activated Ca 2+ channels is an essential signaling pathway in many cell types. Ca 2+ release-activated Ca 2+ channels are formed by ORAI1, ORAI2, and ORAI3 proteins and activated by stromal interaction molecule (STIM) 1 and STIM2. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. We performed molecular and immunologic analysis of patients with CID, anhidrosis, and ectodermal dysplasia of unknown etiology. We performed DNA sequencing of the ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, SOCE measurements, immunologic analysis of peripheral blood lymphocyte populations by using flow cytometry, and histologic and ultrastructural analysis of patient tissues. We identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis, and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. In addition to impaired T-cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer T and regulatory T (Treg) cells and altered composition of γδ T-cell and natural killer cell subsets. ORAI1 null mutations are associated with reduced numbers of invariant natural killer T and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1-deficient patients have dental enamel defects and anhidrosis, representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency that is distinct from previously reported patients with anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in the nuclear factor κB signaling

  14. IGF-1 in autosomal dominant cerebellar ataxia - open-label trial.

    PubMed

    Sanz-Gallego, Irene; Rodriguez-de-Rivera, Francisco J; Pulido, Irene; Torres-Aleman, Ignacio; Arpa, Javier

    2014-01-01

    The objective of this clinical open-label trial was to test the safety, tolerability and efficacy of IGF-1 therapy for autosomal dominant cerebellar ataxia (ADCA) patients. A total of 19 molecularly confirmed patients with SCA3, 1 patient with SCA6 and 6 patients with SCA7 completed our study. They were 8 females and 18 males, 28 to 74 years of age (average ± SD: 49.3 ± 14.1). Patients were treated with IGF-1 therapy with a dosage of 50 μg/kg twice a day for 12 months. The efficacy of this therapy was assessed by change from baseline on the scale for the assessment and rating of ataxia (SARA). Ten patients, consecutively selected, continued their assigned dosages in a second year open-label extension trial. A statistically significant improvement in SARA scores was observed for patients with SCA3, patients with SCA7 and all patients grouped together after the first year of IGF-1 therapy, while a stabilization of the disease was confirmed during the second year (extension study). The single patient with SCA6 showed 3 improvement points in SARA score after 3 four-month periods of IGF-1 therapy when compared with baseline measurements. Our data indicate that IGF-1 is safe and well tolerated in general. Our data, in comparison with results from previous cohorts, indicate a trend for IGF-1 treatment to stabilize the disease progression for patients with SCA, indicating that IGF-1 therapy is able to decrease the progressivity of ADCA.

  15. 9 CFR 317.1 - Labels required; supervision by Program employee.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Labels required; supervision by... Labels required; supervision by Program employee. (a) When, in an official establishment, any inspected... supervision of a Program employee. ...

  16. 9 CFR 317.1 - Labels required; supervision by Program employee.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Labels required; supervision by... Labels required; supervision by Program employee. (a) When, in an official establishment, any inspected... supervision of a Program employee. ...

  17. Live-cell imaging of ER-PM contact architecture by a novel TIRFM approach reveals extension of junctions in response to store-operated Ca2+-entry.

    PubMed

    Poteser, Michael; Leitinger, Gerd; Pritz, Elisabeth; Platzer, Dieter; Frischauf, Irene; Romanin, Christoph; Groschner, Klaus

    2016-10-19

    Nanometer-spaced appositions between endoplasmic reticulum and plasma membrane (ER-PM junctions) stabilized by membrane-joining protein complexes are critically involved in cellular Ca 2+ -handling and lipid trafficking. ER-PM junctional architecture and plasticity associated with inter-membrane communication are as yet barely understood. Here, we introduce a method to precisely characterize ER-PM junction morphology and dynamics with high temporal resolution and minimal disturbance of junctional intermembrane communication. We show that expression of soluble cytosolic fluorophores in combination with TIRFM enables to delineate ER and PM distance in the range of 10-150 nm. Live-cell imaging of sub-plasmalemmal structures in RBL-2H3 mast cells by this method, designated as fluorescence density mapping (FDM), revealed profound dynamics of ER-PM contact sites in response to store-depletion. We report the existence of a Ca 2+ -dependent process that expands the junctional ER to enlarge its contact surface with the PM, thereby promoting and stabilizing STIM1-Orai1 competent ER-PM junctions.

  18. Cortical effect and functional recovery by the electromyography-triggered neuromuscular stimulation in chronic stroke patients.

    PubMed

    Shin, Hwa Kyung; Cho, Sang Hyun; Jeon, Hye-seon; Lee, Young-Hee; Song, Jun Chan; Jang, Sung Ho; Lee, Chu-Hee; Kwon, Yong Hyun

    2008-09-19

    We investigated the effect of electromyography (EMG)-triggered neuromuscular electrical stimulation (NMES; EMG-stim) on functional recovery of the hemiparetic hand and the related cortical activation pattern in chronic stroke patients. We enrolled 14 stroke patients, who were randomly assigned to the EMG-stim (n=7) or the control groups (n=7). The EMG-stim was applied to the wrist extensor of the EMG-stim group for two sessions (30 min/session) a day, five times per week for 10 weeks. Four functional tests (box and block, strength, the accuracy index, and the on/offset time of muscle contraction) and functional MRI (fMRI) were performed before and after treatment. fMRI was measured at 1.5 T in parallel with timed finger flexion-extension movements at a fixed rate. Following treatment, the EMG-stim group showed a significant improvement in all functional tests. The main cortical activation change with such functional improvement was shifted from the ipsilateral sensorimotor cortex (SMC) to the contralateral SMC. We demonstrated that 10-week EMG-stim can induce functional recovery and change of cortical activation pattern in the hemiparetic hand of chronic stroke patients.

  19. Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs.

    PubMed

    Gao, Jinxu; Mfuh, Adelphe; Amako, Yuka; Woo, Christina M

    2018-03-28

    Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.

  20. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these items: (1) For batts and blankets of any...

  1. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these items: (1) For batts and blankets of any...

  2. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these items: (1) For batts and blankets of any...

  3. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these items: (1) For batts and blankets of any...

  4. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels must contain: (a) The type of insulation. (b) A chart showing these items: (1) For batts and blankets of any...

  5. 21 CFR 895.25 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... eliminated by labeling or a change in labeling, or change in advertising if the device is a restricted device... person(s) responsible for the labeling or advertising of the device specifying: (1) The deception or risk... labeling, or change in advertising if the device is a restricted device, necessary to correct the deception...

  6. Do nutrition labels influence healthier food choices? Analysis of label viewing behaviour and subsequent food purchases in a labelling intervention trial.

    PubMed

    Ni Mhurchu, Cliona; Eyles, Helen; Jiang, Yannan; Blakely, Tony

    2018-02-01

    There are few objective data on how nutrition labels are used in real-world shopping situations, or how they affect dietary choices and patterns. The Starlight study was a four-week randomised, controlled trial of the effects of three different types of nutrition labels on consumer food purchases: Traffic Light Labels, Health Star Rating labels, or Nutrition Information Panels (control). Smartphone technology allowed participants to scan barcodes of packaged foods and receive randomly allocated labels on their phone screen, and to record their food purchases. The study app therefore provided objectively recorded data on label viewing behaviour and food purchases over a four-week period. A post-hoc analysis of trial data was undertaken to assess frequency of label use, label use by food group, and association between label use and the healthiness of packaged food products purchased. Over the four-week intervention, study participants (n = 1255) viewed nutrition labels for and/or purchased 66,915 barcoded packaged products. Labels were viewed for 23% of all purchased products, with decreasing frequency over time. Shoppers were most likely to view labels for convenience foods, cereals, snack foods, bread and bakery products, and oils. They were least likely to view labels for sugar and honey products, eggs, fish, fruit and vegetables, and meat. Products for which participants viewed the label and subsequently purchased the product during the same shopping episode were significantly healthier than products where labels were viewed but the product was not subsequently purchased: mean difference in nutrient profile score -0.90 (95% CI -1.54 to -0.26). In a secondary analysis of a nutrition labelling intervention trial, there was a significant association between label use and the healthiness of products purchased. Nutrition label use may therefore lead to healthier food purchases. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. The use of nutritional labels by college students in a food-court setting.

    PubMed

    Kolodinsky, Jane; Green, Jennifer; Michahelles, Marina; Harvey-Berino, Jean R

    2008-01-01

    Between January and September 2006, the authors examined when, why, if, and how nutrition labels impact food purchase decisions of college students. Participants were 16 college-aged students at a large northeastern university. As part of a larger study undertaken at a large northeastern university on the effect of nutrition labels in restaurant settings on food purchases, the authors held a focus group to look more deeply at when and why nutrition labeling impacted college student food purchases. Although results of the large study are still being discerned, the focus group results reveal that college women and men were interested in the provision of nutrition labels in the food court-like setting found at the university, and that those exposed to labels over the course of the study noticed these labels and often referred to them when making purchase decisions. Additional findings reveal that price and convenience also play a role in food purchases and that, of those items listed on each label, calories and fat were most important to the study population. Although more research is needed, this qualitative study finds that students want nutrition labels and would use them to make food purchasing decisions.

  8. Regulation of store-operated Ca2+ entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells.

    PubMed

    Kito, Hiroaki; Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto; Ohya, Susumu; Asai, Kiyofumi; Imaizumi, Yuji

    2015-04-10

    Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Characterization of Bifunctional Spin Labels for Investigating the Structural and Dynamic Properties of Membrane Proteins Using EPR Spectroscopy.

    PubMed

    Sahu, Indra D; Craig, Andrew F; Dunagum, Megan M; McCarrick, Robert M; Lorigan, Gary A

    2017-10-05

    Site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study structural and dynamic properties of membrane proteins. The most widely used spin label is methanthiosulfonate (MTSL). However, the flexibility of this spin label introduces greater uncertainties in EPR measurements obtained for determining structures, side-chain dynamics, and backbone motion of membrane protein systems. Recently, a newer bifunctional spin label (BSL), 3,4-bis(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy, has been introduced to overcome the dynamic limitations associated with the MTSL spin label and has been invaluable in determining protein backbone dynamics and inter-residue distances due to its restricted internal motion and fewer size restrictions. While BSL has been successful in providing more accurate information about the structure and dynamics of several proteins, a detailed characterization of the spin label is still lacking. In this study, we characterized BSLs by performing CW-EPR spectral line shape analysis as a function of temperature on spin-labeled sites inside and outside of the membrane for the integral membrane protein KCNE1 in POPC/POPG lipid bilayers and POPC/POPG lipodisq nanoparticles. The experimental data revealed a powder pattern spectral line shape for all of the KCNE1-BSL samples at 296 K, suggesting the motion of BSLs approaches the rigid limit regime for these series of samples. BSLs were further utilized to report for the first time the distance measurement between two BSLs attached on an integral membrane protein KCNE1 in POPC/POPG lipid bilayers at room temperature using dipolar line broadening CW-EPR spectroscopy. The CW dipolar line broadening EPR data revealed a 15 ± 2 Å distance between doubly attached BSLs on KCNE1 (53/57-63/67) which is consistent with molecular dynamics modeling and the solution NMR structure of KCNE1 which yielded a

  10. Appearance of acute gouty arthritis on indium-111-labeled leukocyte scintigraphy

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

    Palestro, C.J.; Vega, A.; Kim, C.K.

    1990-05-01

    Indium-111-labeled leukocyte scintigraphy was performed on a 66-yr-old male with polyarticular acute gouty arthritis. Images revealed intense labeled leukocyte accumulation in a pattern indistinguishable from septic arthritis, in both knees and ankles, and the metatarsophalangeal joint of both great toes, all of which were involved in the acute gouty attack. Joint aspirate as well as blood cultures were reported as no growth; the patient was treated with intravenous colchicine and ACTH for 10 days with dramatic improvement noted. Labeled leukocyte imaging, repeated 12 days after the initial study, revealed near total resolution of joint abnormalities, concordant with the patient's clinicalmore » improvement. This case demonstrates that while acute gouty arthritis is a potential pitfall in labeled leukocyte imaging, in the presence of known gout, it may provide a simple, objective, noninvasive method of evaluating patient response to therapy.« less

  11. 130. Post1911. Photograph labeled, 'SEASON 1913. CAPTAIN, 1st MATE, SUPT ...

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

    130. Post-1911. Photograph labeled, 'SEASON 1913. CAPTAIN, 1st MATE, SUPT AND STOREKEEPER, A.P. ASS'N CANNERY, SHIP STAR OF ALASKA.' View forward from mizzenmast, post side. - Ship BALCLUTHA, 2905 Hyde Street Pier, San Francisco, San Francisco County, CA

  12. Visual Search for Wines with a Triangle on the Label in a Virtual Store

    PubMed Central

    Zhao, Hui; Huang, Fuxing; Spence, Charles; Wan, Xiaoang

    2017-01-01

    Two experiments were conducted in a virtual reality (VR) environment in order to investigate participants’ in-store visual search for bottles of wines displaying a prominent triangular shape on their label. The experimental task involved virtually moving along a wine aisle in a virtual supermarket while searching for the wine bottle on the shelf that had a different triangle on its label from the other bottles. The results of Experiment 1 revealed that the participants identified the bottle with a downward-pointing triangle on its label more rapidly than when looking for an upward-pointing triangle on the label instead. This finding replicates the downward-pointing triangle superiority (DPTS) effect, though the magnitude of this effect was more pronounced in the first as compared to the second half of the experiment, suggesting a modulating role of practice. The results of Experiment 2 revealed that the DPTS effect was also modulated by the location of the target on the shelf. Interestingly, however, the results of a follow-up survey demonstrate that the orientation of the triangle did not influence the participants’ evaluation of the wine bottles. Taken together, these findings reveal how in-store the attention of consumers might be influenced by the design elements in product packaging. These results therefore suggest that shopping in a virtual supermarket might offer a practical means of assessing the shelf standout of product packaging, which has important implications for food marketing. PMID:29326624

  13. Visual Search for Wines with a Triangle on the Label in a Virtual Store.

    PubMed

    Zhao, Hui; Huang, Fuxing; Spence, Charles; Wan, Xiaoang

    2017-01-01

    Two experiments were conducted in a virtual reality (VR) environment in order to investigate participants' in-store visual search for bottles of wines displaying a prominent triangular shape on their label. The experimental task involved virtually moving along a wine aisle in a virtual supermarket while searching for the wine bottle on the shelf that had a different triangle on its label from the other bottles. The results of Experiment 1 revealed that the participants identified the bottle with a downward-pointing triangle on its label more rapidly than when looking for an upward-pointing triangle on the label instead. This finding replicates the downward-pointing triangle superiority (DPTS) effect, though the magnitude of this effect was more pronounced in the first as compared to the second half of the experiment, suggesting a modulating role of practice. The results of Experiment 2 revealed that the DPTS effect was also modulated by the location of the target on the shelf. Interestingly, however, the results of a follow-up survey demonstrate that the orientation of the triangle did not influence the participants' evaluation of the wine bottles. Taken together, these findings reveal how in-store the attention of consumers might be influenced by the design elements in product packaging. These results therefore suggest that shopping in a virtual supermarket might offer a practical means of assessing the shelf standout of product packaging, which has important implications for food marketing.

  14. Dual-process theory and consumer response to front-of-package nutrition label formats.

    PubMed

    Sanjari, S Setareh; Jahn, Steffen; Boztug, Yasemin

    2017-11-01

    Nutrition labeling literature yields fragmented results about the effect of front-of-package (FOP) nutrition label formats on healthy food choice. Specifically, it is unclear which type of nutrition label format is effective across different shopping situations. To address this gap, the present review investigates the available nutrition labeling literature through the prism of dual-process theory, which posits that decisions are made either quickly and automatically (system 1) or slowly and deliberately (system 2). A systematically performed review of nutrition labeling literature returned 59 papers that provide findings that can be explained according to dual-process theory. The findings of these studies suggest that the effectiveness of nutrition label formats is influenced by the consumer's dominant processing system, which is a function of specific contexts and personal variables (eg, motivation, nutrition knowledge, time pressure, and depletion). Examination of reported findings through a situational processing perspective reveals that consumers might prefer different FOP nutrition label formats in different situations and can exhibit varying responses to the same label format across situations. This review offers several suggestions for policy makers and researchers to help improve current FOP nutrition label formats. © The Author(s) 2017. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. Menu-labeling usage and its association with diet and exercise: 2011 BRFSS Sugar Sweetened Beverage and Menu Labeling module.

    PubMed

    Bowers, Kelly M; Suzuki, Sumihiro

    2014-01-02

    The primary objective of our study was to investigate the association between menu-labeling usage and healthy behaviors pertaining to diet (consumption of fruits, vegetables, sodas, and sugar-sweetened beverages) and exercise. Data from the 2011 Behavioral Risk Factor Surveillance System, Sugar Sweetened Beverage and Menu-Labeling module, were used. Logistic regression was used to determine the association between menu-labeling usage and explanatory variables that included fruit, vegetable, soda, and sugar-sweetened beverage consumption as well as exercise. Nearly half (52%) of the sample indicated that they used menu labeling. People who used menu labeling were more likely to be female (odds ratio [OR], 2.29; 95% confidence interval [CI], 2.04-2.58), overweight (OR, 1.13; 95% CI, 1.00-1.29) or obese (OR, 1.29; 95% CI, 1.12-1.50), obtain adequate weekly aerobic exercise (OR, 1.18; 95% CI, 1.06-1.32), eat fruits (OR, 1.20; 95% CI, 1.12-1.29) and vegetables (OR, 1.12; 95% CI, 1.05-1.20), and drink less soda (OR, 0.76; 95% CI, 0.69-0.83). Although obese and overweight people were more likely to use menu labeling, they were also adequately exercising, eating more fruits and vegetables, and drinking less soda. Menu labeling is intended to combat the obesity epidemic; however the results indicate an association between menu-labeling usage and certain healthy behaviors. Thus, efforts may be necessary to increase menu-labeling usage among people who are not partaking in such behaviors.

  16. In vitro labelling and detection of mesenchymal stromal cells: a comparison between magnetic resonance imaging of iron-labelled cells and magnetic resonance spectroscopy of fluorine-labelled cells.

    PubMed

    Rizzo, Stefania; Petrella, Francesco; Zucca, Ileana; Rinaldi, Elena; Barbaglia, Andrea; Padelli, Francesco; Baggi, Fulvio; Spaggiari, Lorenzo; Bellomi, Massimo; Bruzzone, Maria Grazia

    2017-01-01

    Among the various stem cell populations used for cell therapy, adult mesenchymal stromal cells (MSCs) have emerged as a major new cell technology. These cells must be tracked after transplantation to monitor their migration within the body and quantify their accumulation at the target site. This study assessed whether rat bone marrow MSCs can be labelled with superparamagnetic iron oxide (SPIO) nanoparticles and perfluorocarbon (PFC) nanoemulsion formulations without altering cell viability and compared magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) results from iron-labelled and fluorine-labelled MSCs, respectively. Of MSCs, 2 × 10 6 were labelled with Molday ION Rhodamine-B (MIRB) and 2 × 10 6 were labelled with Cell Sense. Cell viability was evaluated by trypan blue exclusion method. Labelled MSCs were divided into four samples containing increasing cell numbers (0.125 × 10 6 , 0.25 × 10 6 , 0.5 × 10 6 , 1 × 10 6 ) and scanned on a 7T MRI: for MIRB-labelled cells, phantoms and cells negative control, T1, T2 and T2* maps were acquired; for Cell Sense labelled cells, phantoms and unlabelled cells, a 19 F non-localised single-pulse MRS sequence was acquired. In total, 86.8% and 83.6% of MIRB-labelled cells and Cell Sense-labelled cells were viable, respectively. MIRB-labelled cells were visible in all samples with different cell numbers; pellets containing 0.5 × 10 6 and 1 × 10 6 of Cell Sense-labelled cells showed a detectable 19 F signal. Our data support the use of both types of contrast material (SPIO and PFC) for MSCs labelling, although further efforts should be dedicated to improve the efficiency of PFC labelling.

  17. PRN 95-1: Effluent Discharge Labeling Statements

    EPA Pesticide Factsheets

    This notice exempts certain pesticide products from bearing effluent discharge labeling statements specified by P.R. Notice 93-10 for manufacturing use products and end use products that may be discharged to waters of the U.S. or municipal sewer systems.

  18. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a) Be...

  19. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a) Be...

  20. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a) Be...

  1. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a) Be...

  2. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator must make a label, the label must— (a) Be...

  3. Capacitive label reader

    DOEpatents

    Arlowe, H. Duane

    1985-01-01

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  4. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1983-07-15

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  5. Menu-Labeling Usage and Its Association with Diet and Exercise: 2011 BRFSS Sugar Sweetened Beverage and Menu Labeling Module

    PubMed Central

    Bowers, Kelly M.

    2014-01-01

    Introduction The primary objective of our study was to investigate the association between menu-labeling usage and healthy behaviors pertaining to diet (consumption of fruits, vegetables, sodas, and sugar-sweetened beverages) and exercise. Methods Data from the 2011 Behavioral Risk Factor Surveillance System, Sugar Sweetened Beverage and Menu-Labeling module, were used. Logistic regression was used to determine the association between menu-labeling usage and explanatory variables that included fruit, vegetable, soda, and sugar-sweetened beverage consumption as well as exercise. Results Nearly half (52%) of the sample indicated that they used menu labeling. People who used menu labeling were more likely to be female (odds ratio [OR], 2.29; 95% confidence interval [CI], 2.04–2.58), overweight (OR, 1.13; 95% CI, 1.00–1.29) or obese (OR, 1.29; 95% CI, 1.12–1.50), obtain adequate weekly aerobic exercise (OR, 1.18; 95% CI, 1.06–1.32), eat fruits (OR, 1.20; 95% CI, 1.12–1.29) and vegetables (OR, 1.12; 95% CI, 1.05–1.20), and drink less soda (OR, 0.76; 95% CI, 0.69–0.83). Conclusion Although obese and overweight people were more likely to use menu labeling, they were also adequately exercising, eating more fruits and vegetables, and drinking less soda. Menu labeling is intended to combat the obesity epidemic; however the results indicate an association between menu-labeling usage and certain healthy behaviors. Thus, efforts may be necessary to increase menu-labeling usage among people who are not partaking in such behaviors. PMID:24384303

  6. Biological Effects of Nonionizing Electromagnetic Radiation. Volume 8, Number 1. A Digest of Current Literature.

    DTIC Science & Technology

    1983-10-01

    IIOS; 1982 ( re’ the refractive index fluctuation produced in a sam- ple when its temperature rises as a result of ab- sorption of MW radiation is...sav af ter t he f racture compared with rsie l 3*~c AM1>4 .a PFa ~i u (10(1 die Studi1es In- p or’(f ,i rab I t, to t , ttI-,rc stim .lation increaoi

  7. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1985-11-12

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label. 5 figs.

  8. Characterization of labelling and de-labelling reagents for detection and recovery of tyrosine residue in peptide.

    PubMed

    Toyo'oka, Toshimasa; Mantani, Tomomi; Kato, Masaru

    2003-01-01

    This paper characterized the labelling and de-labelling reagents for reversible labelling of tyrosine (Tyr)-containing peptide, which involves detection and recovery. The phenolic hydroxyl group (-OH) in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), and 1-fluoro-2,4-dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N-acetyl-L-cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de-labelling reactions of NBD-labelled N-acetyl-L-tyrosine (N-AcTyr) with the nucleophiles produced N-AcTyr, accompanied by NBD-nucleophile. Although DBD-F and DNFB also successfully labeled the -OH group in N-AcTyr, the efficiency of Cbond;O bond cleavage and recovery of N-AcTyr by the nucleophiles was relatively low compared with NBD-label. Among the de-labelling reagents, N-acetyl-L-cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD-F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N-terminal amine-blocked peptides, i.e. N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Copyright 2003 John Wiley & Sons, Ltd.

  9. 16 CFR Appendix L to Part 305 - Sample Labels

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Part 305—Sample Labels ER29AU07.122 PROTOTYPE LABEL 1 ER29AU07.123 PROTOTYPE LABEL 2 ER29AU07.124 PROTOTYPE LABEL 3 ER29AU07.125 PROTOTYPE LABEL 4 ER29AU07.126 SAMPLE LABEL 1 ER29AU07.127 SAMPLE LABEL 2...

  10. 9 CFR 112.2 - Final container label, carton label, and enclosure.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... carton; (10) In the case of a product which contains an antibiotic added during the production process... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Final container label, carton label, and enclosure. 112.2 Section 112.2 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

  11. Imaging, biodistribution, and dosimetry of radionuclide-labeled PD-L1 antibody in an immunocompetent mouse model of breast cancer

    PubMed Central

    Josefsson, Anders; Nedrow, Jessie R.; Park, Sunju; Banerjee, Sangeeta Ray; Rittenbach, Andrew; Jammes, Fabien; Tsui, Benjamin; Sgouros, George

    2015-01-01

    The programmed cell death ligand 1 (PD-L1) participates in an immune checkpoint system involved in preventing autoimmunity. PD-L1 is expressed on tumor cells, tumor-associated macrophages, and other cells in the tumor microenvironment. Anti-PD-L1 antibodies are active against a variety of cancers, and combined anti-PD-L1 therapy with external beam radiotherapy has been shown to increase therapeutic efficacy. PD-L1 expression status is an important indicator of prognosis and therapy responsiveness, but methods to precisely capture the dynamics of PD-L1 expression in the tumor microenvironment are still limited. In this study, we developed a murine anti-PD-L1 antibody conjugated to the radioactive isotope Indium-111 (111In) for imaging and biodistribution studies in an immune-intact mouse model of breast cancer. The distribution of 111In-DTPA-anti-PD-L1 in tumors as well as the spleen, liver, thymus, heart, and lungs peaked 72 hours after injection. Co-injection of labeled and 100-fold unlabeled antibody significantly reduced spleen uptake at 24 hours, indicating that an excess of unlabeled antibody effectively blocked PD-L1 sites in the spleen, thus shifting the concentration of 111In-DTPA-anti-PD-L1 into the blood stream and potentially increasing tumor uptake. Clearance of 111In-DTPA-anti-PD-L1 from all organs occurred at 144 hours. Moreover, dosimetry calculations revealed that radionuclide-labeled anti-PD-L1 antibody yielded tolerable projected marrow doses, further supporting its use for radiopharmaceutical therapy. Taken together, these studies demonstrate the feasibility of using anti-PD-L1 antibody for radionuclide imaging and radioimmunotherapy, and highlight a new opportunity to optimize and monitor the efficacy of immune checkpoint inhibition therapy. PMID:26554829

  12. Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

    PubMed Central

    Lerch, Michael T.; Horwitz, Joseph; McCoy, John; Hubbell, Wayne L.

    2013-01-01

    Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875–85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate. PMID:24248390

  13. Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating

    NASA Astrophysics Data System (ADS)

    Yasui, Takao; Ogawa, Kensuke; Kaji, Noritada; Nilsson, Mats; Ajiri, Taiga; Tokeshi, Manabu; Horiike, Yasuhiro; Baba, Yoshinobu

    2016-08-01

    Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications.

  14. Small-amplitude backbone motions of the spin-labeled lipopeptide trichogin GA IV in a lipid membrane as revealed by electron spin echo.

    PubMed

    Syryamina, Victoria N; Isaev, Nikolay P; Peggion, Cristina; Formaggio, Fernando; Toniolo, Claudio; Raap, Jan; Dzuba, Sergei A

    2010-09-30

    Trichogin GA IV is a lipopeptide antibiotic of fungal origin, which is known to be able to modify the membrane permeability. TOAC nitroxide spin-labeled analogues of this membrane active peptide were investigated in hydrated bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by electron spin echo (ESE) spectroscopy. Because the TOAC nitroxide spin label is rigidly attached to the peptide backbone, it may report on the backbone orientational dynamics. The ESE signal in this system is observed below ∼150 K. Previously, three-pulse stimulated ESE was found to be sensitive to two types of orientational motion of spin-labeled POPC lipid bilayers at these temperatures. The first type is fast stochastic librations, with a correlation time on the nanosecond scale (which also manifests itself in a two-pulse primary ESE experiment). The second type is slow millisecond inertial rotations. In the present work, we find that at low molar peptide to lipid ratio (1:200), where the individual peptide molecules are randomly distributed at the membrane surface, the spin labels show only a fast type of motion. At the high molar peptide to lipid ratio (1:20), a slow motion is also observed. Because at this high concentration trichogin GA IV is known to change its orientation from the in-plane topology to the transmembrane disposition, the observed onset of a slow motion may be safely attributed to the dynamics of peptides, which are elongated along the lipid molecules of the membrane. The possible interrelation between this backbone rotational motion of the peptide antibiotic and the membrane leakage is discussed.

  15. Toddlers benefit from labeling on an executive function search task.

    PubMed

    Miller, Stephanie E; Marcovitch, Stuart

    2011-03-01

    Although labeling improves executive function (EF) performance in children older than 3years, the results from studies with younger children have been equivocal. In the current study, we assessed performance in a computerized multistep multilocation search task with older 2-year-olds. The correct search location was either (a) not marked by a familiar picture or given a distinct label, (b) marked by a familiar picture but not given a distinct label, (c) marked by a familiar picture and labeled by the experimenter, or (d) marked by a familiar picture and labeled by the participant. The results revealed that accuracy improved across conditions such that children made the fewest errors when they generated the label for the hiding location. These findings support the hierarchical competing systems model, which postulates that improved performance can be explained by more powerful representations that guide search behavior. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Improved 68 Ga-labeling method using ethanol addition: Application to the α-helical peptide DOTA-FAMP.

    PubMed

    Hasegawa, Koki; Kawachi, Emi; Uehara, Yoshinari; Yoshida, Tsuyoshi; Imaizumi, Satoshi; Ogawa, Masahiro; Miura, Shin-Ichiro; Saku, Keijiro

    2017-01-01

    We examined the 68 Ga labeling of the α-helical peptide, DOTA-FAMP, and evaluated conformational changes during radiolabeling. 68 Ga-DOTA-FAMP is a positron emission tomography probe candidate for atherosclerotic plaques. The labeling yield achieved by Zhernosekov's method (using acetone for 68 Ga purification) was compared with that achieved by the original and 2 modified Mueller's methods (using NaCl solution). Modified method I involves desalting the 68 Ga prior to labeling, and modified method II involves the inclusion of ethanol in the labeling solution. The labeling yield using Zhernosekov's method was 62% ± 5.4%. In comparison, Mueller's original method gave 8.9% ± 1.7%. Modified method I gave a slight improvement of 32% ± 2.1%. Modified method II further increased the yield to 66% ± 3.4%. Conformational changes were determined by circular dichroism spectroscopy, revealing that these differences could be attributed to conformational changes. Heat treatment affects peptide conformation, which leads to aggregation and decreases the labeling yield. Mueller's method is simpler, but harsh conditions preclude its application to biomolecules. To suppress aggregation, we included a desalting process and added ethanol in the labeling solution. These changes significantly improved the labeling yield. Before use for imaging, conformational changes of biomolecules during radiolabeling should be evaluated by circular dichroism spectroscopy to ensure the homogeneity of the labeled product. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Preclinical evaluation of melanocortin-1 receptor (MC1-R) specific 68Ga- and 44Sc-labeled DOTA-NAPamide in melanoma imaging.

    PubMed

    Nagy, Gábor; Dénes, Noémi; Kis, Adrienn; Szabó, Judit P; Berényi, Ervin; Garai, Ildikó; Bai, Péter; Hajdu, István; Szikra, Dezső; Trencsényi, György

    2017-08-30

    Alpha melanocyte stimulating hormone (α-MSH) enhances melanogenesis in melanoma malignum by binding to melanocortin-1 receptors (MC1-R). Earlier studies demonstrated that alpha-MSH analog NAPamide molecule specifically binds to MC1-R receptor. Radiolabeled NAPamide is a promising radiotracer for the non-invasive detection of melanin producing melanoma tumors by Positron Emission Tomography (PET). In this present study the MC1-R selectivity of the newly developed Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using melanoma tumors. DOTA-NAPamide was labeled with Ga-68 and Sc-44 radionuclides. The MC1-R specificity of Ga-68- and Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using MC1-R positive (B16-F10) and negative (A375) melanoma cell lines. For in vivo imaging studies B16-F10 and A375 tumor-bearing mice were injected with 44 Sc/ 68 Ga-DOTA-NAPamide (in blocking studies with α-MSH) and whole body PET/MRI scans were acquired. Radiotracer uptake was expressed in terms of standardized uptake values (SUVs). 44 Sc/ 68 Ga-labeled DOTA-NAPamide were produced with high specific activity (approx. 19 GBq/μmol) and with excellent radiochemical purity (99%<). MC1-R positive B16-F10 cells showed significantly (p≤0.01) higher in vitro radiotracer accumulation than that of receptor negative A375 melanoma cells. In animal experiments, also significantly (p≤0.01) higher Ga-68-DOTA-NAPamide (SUVmean: 0.38±0.02), and Sc-44-DOTA-NAPamide (SUVmean: 0.52±0.13) uptake was observed in subcutaneously growing B16-F10 tumors, than in receptor negative A375 tumors, where the SUVmean values of Ga-68-DOTA-NAPamide and Sc-44-DOTA-NAPamide were 0.04±0.01 and 0.07±0.01, respectively. Tumor-to-muscle (T/M SUVmean) ratios were approximately 15-fold higher in B16-F10 tumor-bearing mice, than that of A375 tumors, and this difference was also significant (p≤0.01) using both radiotracers after 60 min incubation time. Our newly synthesized 44 Sc-labeled

  18. Estradiol treatment in preadolescent females enhances adolescent spatial memory and differentially modulates hippocampal region-specific phosphorylated ERK labeling.

    PubMed

    Wartman, Brianne C; Keeley, Robin J; Holahan, Matthew R

    2012-10-24

    Estrogen levels in rats are positively correlated with enhanced memory function and hippocampal dendritic spine density. There is much less work on the long-term effects of estradiol manipulation in preadolescent rats. The present work examined how injections of estradiol during postnatal days 19-22 (p19-22; preadolescence) affected water maze performance and hippocampal phosphorylated ERK labeling. To investigate this, half of the estradiol- and vehicle-treated female rats were trained on a water maze task 24h after the end of estradiol treatment (p23-27) while the other half was not trained. All female rats were tested on the water maze from p40 to p44 (adolescence) and hippocampal pERK1/2 labeling was assessed as a putative marker of neuronal plasticity. During adolescence, preadolescent-trained groups showed lower latencies than groups without preadolescent training. Retention data revealed lower latencies in both estradiol groups, whether preadolescent trained or not. Immunohistochemical detection of hippocampal pERK1/2 revealed elevations in granule cell labeling associated with the preadolescent trained groups and reductions in CA1 labeling associated with estradiol treatment. These results show a latent beneficial effect of preadolescent estradiol treatment on adolescent spatial performance and suggest an organizational effect of prepubescent exogenously applied estradiol. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  19. Expansion of CD4+CD25+FOXP3+ regulatory T cells in infants of mothers with type 1 diabetes

    PubMed Central

    Luopajärvi, Kristiina; Nieminen, Janne K; Ilonen, Jorma; Åkerblom, Hans K; Knip, Mikael; Vaarala, Outi

    2014-01-01

    Background Reduced risk for type 1 diabetes (T1D) has been reported in the offspring of mothers with T1D when compared with children of affected fathers. Objective To evaluate the hypothesis that exposure of the offspring to maternal insulin therapy induces regulatory mechanisms in utero, we compared the FOXP3 expressing regulatory T cells in cord blood (CB) of infants born to mothers with or without T1D. Subjects and Methods Cord blood mononuclear cells (CBMCs) from 20 infants with maternal T1D and from 20 infants with an unaffected mother were analyzed for the numbers of CD4+CD25+FOXP3+ cells ex vivo and after in vitro stimulation with human insulin by flow cytometry. The mRNA expression of FOXP3, NFATc2, STIM1, interleukin (IL)-10, and transforming growth factor (TGF)-β was measured by real-time reverse transcription polymerase chain reaction. Results The percentage of FOXP3+ cells in CD4+CD25high cells was higher in the CB of the infants with maternal T1D when compared with the infants of unaffected mothers (p = 0.023). After in vitro insulin stimulation an increase in the percentage of FOXP3+ cells in CD4+CD25high cells (p = 0.0002) as well as upregulation of FOXP3, NFATc2, STIM1, IL-10, and TGF-β transcripts in CBMCs (p < 0.013 for all; Wilcoxon test) was observed only in the offspring of mothers with T1D, in whom the disease-related PTPN22 allele was associated with reduced STIM1 and NFATc2 response in insulin-stimulated CBMCs (p = 0.007 and p = 0.014). Conclusions We suggest that maternal insulin treatment induces expansion of regulatory T cells in the fetus, which might contribute to the lower risk of diabetes in children with maternal vs. paternal diabetes. PMID:22332874

  20. Expansion of CD4+CD25+FOXP3+ regulatory T cells in infants of mothers with type 1 diabetes.

    PubMed

    Luopajärvi, Kristiina; Nieminen, Janne K; Ilonen, Jorma; Akerblom, Hans K; Knip, Mikael; Vaarala, Outi

    2012-08-01

    Reduced risk for type 1 diabetes (T1D) has been reported in the offspring of mothers with T1D when compared with children of affected fathers. To evaluate the hypothesis that exposure of the offspring to maternal insulin therapy induces regulatory mechanisms in utero, we compared the FOXP3 expressing regulatory T cells in cord blood (CB) of infants born to mothers with or without T1D. Cord blood mononuclear cells (CBMCs) from 20 infants with maternal T1D and from 20 infants with an unaffected mother were analyzed for the numbers of CD4+CD25+FOXP3+ cells ex vivo and after in vitro stimulation with human insulin by flow cytometry. The mRNA expression of FOXP3, NFATc2, STIM1, interleukin (IL)-10, and transforming growth factor (TGF)-β was measured by real-time reverse transcription polymerase chain reaction. The percentage of FOXP3+ cells in CD4+CD25(high) cells was higher in the CB of the infants with maternal T1D when compared with the infants of unaffected mothers (p = 0.023). After in vitro insulin stimulation an increase in the percentage of FOXP3+ cells in CD4+CD25(high) cells (p = 0.0002) as well as upregulation of FOXP3, NFATc2, STIM1, IL-10, and TGF-β transcripts in CBMCs (p < 0.013 for all; Wilcoxon test) was observed only in the offspring of mothers with T1D, in whom the disease-related PTPN22 allele was associated with reduced STIM1 and NFATc2 response in insulin-stimulated CBMCs (p = 0.007 and p = 0.014). We suggest that maternal insulin treatment induces expansion of regulatory T cells in the fetus, which might contribute to the lower risk of diabetes in children with maternal vs. paternal diabetes. © 2012 John Wiley & Sons A/S.

  1. Canonical Transient Receptor Potential Channel 2 (TRPC2) as a Major Regulator of Calcium Homeostasis in Rat Thyroid FRTL-5 Cells

    PubMed Central

    Sukumaran, Pramod; Löf, Christoffer; Kemppainen, Kati; Kankaanpää, Pasi; Pulli, Ilari; Näsman, Johnny; Viitanen, Tero; Törnquist, Kid

    2012-01-01

    Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCβ1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca2+-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca2+-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells. PMID:23144458

  2. Defective Store-Operated Calcium Entry Causes Partial Nephrogenic Diabetes Insipidus

    PubMed Central

    Mamenko, Mykola; Dhande, Isha; Tomilin, Viktor; Zaika, Oleg; Boukelmoune, Nabila; Zhu, Yaming; Gonzalez-Garay, Manuel L.

    2016-01-01

    Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca2+) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca2+ levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca2+ mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca2+ levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling. PMID:26574044

  3. Defective Store-Operated Calcium Entry Causes Partial Nephrogenic Diabetes Insipidus.

    PubMed

    Mamenko, Mykola; Dhande, Isha; Tomilin, Viktor; Zaika, Oleg; Boukelmoune, Nabila; Zhu, Yaming; Gonzalez-Garay, Manuel L; Pochynyuk, Oleh; Doris, Peter A

    2016-07-01

    Store-operated calcium entry (SOCE) is the mechanism by which extracellular signals elicit prolonged intracellular calcium elevation to drive changes in fundamental cellular processes. Here, we investigated the role of SOCE in the regulation of renal water reabsorption, using the inbred rat strain SHR-A3 as an animal model with disrupted SOCE. We found that SHR-A3, but not SHR-B2, have a novel truncating mutation in the gene encoding stromal interaction molecule 1 (STIM1), the endoplasmic reticulum calcium (Ca(2+)) sensor that triggers SOCE. Balance studies revealed increased urine volume, hypertonic plasma, polydipsia, and impaired urinary concentrating ability accompanied by elevated circulating arginine vasopressin (AVP) levels in SHR-A3 compared with SHR-B2. Isolated, split-open collecting ducts (CD) from SHR-A3 displayed decreased basal intracellular Ca(2+) levels and a major defect in SOCE. Consequently, AVP failed to induce the sustained intracellular Ca(2+) mobilization that requires SOCE in CD cells from SHR-A3. This effect decreased the abundance of aquaporin 2 and enhanced its intracellular retention, suggesting impaired sensitivity of the CD to AVP in SHR-A3. Stim1 knockdown in cultured mpkCCDc14 cells reduced SOCE and basal intracellular Ca(2+) levels and prevented AVP-induced translocation of aquaporin 2, further suggesting the effects in SHR-A3 result from the expression of truncated STIM1. Overall, these results identify a novel mechanism of nephrogenic diabetes insipidus and uncover a role of SOCE in renal water handling. Copyright © 2016 by the American Society of Nephrology.

  4. Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling.

    PubMed

    Moriya, Koko; Kimoto, Mayumi; Matsuzaki, Kanako; Kiwado, Aya; Takamitsu, Emi; Utsumi, Toshihiko

    2016-10-15

    To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [(3)H]myristic acid or [(3)H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Flow-aggregated traffic-driven label mapping in label-switching networks

    NASA Astrophysics Data System (ADS)

    Nagami, Kenichi; Katsube, Yasuhiro; Esaki, Hiroshi; Nakamura, Osamu

    1998-12-01

    Label switching technology enables high performance, flexible, layer-3 packet forwarding based on the fixed length label information mapped to the layer-3 packet stream. A Label Switching Router (LSR) forwards layer-3 packets based on their label information mapped to the layer-3 address information as well as their layer-3 address information. This paper evaluates the required number of labels under traffic-driven label mapping policy using the real backbone traffic traces. The evaluation shows that the label mapping policy requires a large number of labels. In order to reduce the required number of labels, we propose a label mapping policy which is a traffic-driven label mapping for the traffic toward the same destination network. The evaluation shows that the proposed label mapping policy requires only about one tenth as many labels compared with the traffic-driven label mapping for the host-pair packet stream,and the topology-driven label mapping for the destination network packet stream.

  6. Nutrition Label Viewing during a Food-Selection Task: Front-of-Package Labels vs Nutrition Facts Labels.

    PubMed

    Graham, Dan J; Heidrick, Charles; Hodgin, Katie

    2015-10-01

    Earlier research has identified consumer characteristics associated with viewing Nutrition Facts labels; however, little is known about those who view front-of-package nutrition labels. Front-of-package nutrition labels might appeal to more consumers than do Nutrition Facts labels, but it might be necessary to provide consumers with information about how to locate and use these labels. This study quantifies Nutrition Facts and front-of-package nutrition label viewing among American adult consumers. Attention to nutrition information was measured during a food-selection task. One hundred and twenty-three parents (mean age=38 years, mean body mass index [calculated as kg/m(2)]=28) and one of their children (aged 6 to 9 years) selected six foods from a university laboratory-turned-grocery aisle. Participants were randomized to conditions in which front-of-package nutrition labels were present or absent, and signage explaining front-of-package nutrition labels was present or absent. Adults' visual attention to Nutrition Facts labels and front-of-package nutrition labels was objectively measured via eye-tracking glasses. To examine whether there were significant differences in the percentages of participants who viewed Nutrition Facts labels vs front-of-package nutrition labels, McNemar's tests were conducted across all participants, as well as within various sociodemographic categories. To determine whether hypothesized factors, such as health literacy and education, had stronger relationships with front-of-package nutrition label vs Nutrition Facts label viewing, linear regression assessed the magnitude of relationships between theoretically and empirically derived factors and each type of label viewing. Overall, front-of-package nutrition labels were more likely to be viewed than Nutrition Facts labels; however, for all subgroups, higher rates of front-of-package nutrition label viewership occurred only when signage was present drawing attention to the presence and

  7. Co-Labeling for Multi-View Weakly Labeled Learning.

    PubMed

    Xu, Xinxing; Li, Wen; Xu, Dong; Tsang, Ivor W

    2016-06-01

    It is often expensive and time consuming to collect labeled training samples in many real-world applications. To reduce human effort on annotating training samples, many machine learning techniques (e.g., semi-supervised learning (SSL), multi-instance learning (MIL), etc.) have been studied to exploit weakly labeled training samples. Meanwhile, when the training data is represented with multiple types of features, many multi-view learning methods have shown that classifiers trained on different views can help each other to better utilize the unlabeled training samples for the SSL task. In this paper, we study a new learning problem called multi-view weakly labeled learning, in which we aim to develop a unified approach to learn robust classifiers by effectively utilizing different types of weakly labeled multi-view data from a broad range of tasks including SSL, MIL and relative outlier detection (ROD). We propose an effective approach called co-labeling to solve the multi-view weakly labeled learning problem. Specifically, we model the learning problem on each view as a weakly labeled learning problem, which aims to learn an optimal classifier from a set of pseudo-label vectors generated by using the classifiers trained from other views. Unlike traditional co-training approaches using a single pseudo-label vector for training each classifier, our co-labeling approach explores different strategies to utilize the predictions from different views, biases and iterations for generating the pseudo-label vectors, making our approach more robust for real-world applications. Moreover, to further improve the weakly labeled learning on each view, we also exploit the inherent group structure in the pseudo-label vectors generated from different strategies, which leads to a new multi-layer multiple kernel learning problem. Promising results for text-based image retrieval on the NUS-WIDE dataset as well as news classification and text categorization on several real-world multi

  8. FocusStack and StimServer: a new open source MATLAB toolchain for visual stimulation and analysis of two-photon calcium neuronal imaging data.

    PubMed

    Muir, Dylan R; Kampa, Björn M

    2014-01-01

    Two-photon calcium imaging of neuronal responses is an increasingly accessible technology for probing population responses in cortex at single cell resolution, and with reasonable and improving temporal resolution. However, analysis of two-photon data is usually performed using ad-hoc solutions. To date, no publicly available software exists for straightforward analysis of stimulus-triggered two-photon imaging experiments. In addition, the increasing data rates of two-photon acquisition systems imply increasing cost of computing hardware required for in-memory analysis. Here we present a Matlab toolbox, FocusStack, for simple and efficient analysis of two-photon calcium imaging stacks on consumer-level hardware, with minimal memory footprint. We also present a Matlab toolbox, StimServer, for generation and sequencing of visual stimuli, designed to be triggered over a network link from a two-photon acquisition system. FocusStack is compatible out of the box with several existing two-photon acquisition systems, and is simple to adapt to arbitrary binary file formats. Analysis tools such as stack alignment for movement correction, automated cell detection and peri-stimulus time histograms are already provided, and further tools can be easily incorporated. Both packages are available as publicly-accessible source-code repositories.

  9. Correlation of 125I-LSD autoradiographic labeling with serotonin voltage clamp responses in Aplysia neurons

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

    Evans, M.L.; Kadan, M.J.; Hartig, P.R.

    Autoradiographic receptor binding studies using 125I-LSD (2-(125I)lysergic acid diethyamide) revealed intense labelling on the soma of a symmetrically located pair of cells in the abdominal ganglion of Aplysia californica. This binding was blocked by micromolar concentrations of serotonin and lower concentrations of the serotonergic antagonists, cyproheptadine and mianserin. Electrophysiological investigation of responses to serotonin of neurons in the left upper quadrant, where one of the labeled neurons is located, revealed a range of serotonin responses. Cells L3 and L6 have a K+ conductance increase in response to serotonin that is not blocked by cyproheptadine or mianserin. Cells L2 and L4more » have a biphasic response to serotonin: a Na+ conductance increase, which can be blocked by cyproheptadine and mianserin, followed by a voltage dependent Ca2+ conductance which is blocked by Co2+ but not the serotonergic antagonists. Cell L1, and its symmetrical pair, R1, have in addition to the Na+ and Ca2+ responses observed in L2 and L4, a Cl- conductance increase blocked by LSD, cyproheptadine and mianserin. LSD had little effect on the other responses. The authors conclude that the symmetrically located cells L1 and R1 have a Cl- channel linked to a cyproheptadine- and mianserin-sensitive serotonin receptor that is selectively labelled by 125I-LSD. This receptor has many properties in common with the mammalian serotonin 1C receptor.« less

  10. Preparation of four 1,4-dihydropyridine derivatives (DHPs) labeled with carbon-14.

    PubMed

    Ahmadi Faghih, Mohammad Amin; Moslemin, Mohammad Hossein; Shirvani, Gholamhossein; Javaheri, Mohsen

    2018-05-23

    The importance of DHPs compounds and the need for examining the mechanism of their effect, mandated us to synthesize a number of carbon-14 labeled 1,4-dihydropyridine derivatives for pharmacological studies. Simple preparation and suitable radiochemical yield were advantages of this preparation. This article is protected by copyright. All rights reserved.

  11. Toddlers Benefit from Labeling on an Executive Function Search Task

    PubMed Central

    Miller, Stephanie E.; Marcovitch, Stuart

    2010-01-01

    Although labeling improves executive function (EF) performance in children older than 3 (e.g., Kirkham, Cruess, & Diamond, 2003), the results from studies with younger children have been equivocal (e.g., Sophian & Wellman, 1983). In the present study, we assessed performance in a computerized multistep multilocation search task with older 2-year-old children. The correct search location was either: (a) not marked by a familiar picture nor given a distinct label, (b) marked by a familiar picture but not given a distinct label (c) marked by a familiar picture and labeled by the experimenter, or (d) marked by a familiar picture and labeled by the participant. The results revealed that accuracy improved across conditions such that children made fewest errors when they generated the label for the hiding location. These findings support the hierarchical competing systems model (Marcovitch & Zelazo, 2006, 2009) that postulates that improved performance can be explained by more powerful representations that guide search behavior. PMID:21112597

  12. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Tier 1 and later locomotives. The label on the engine is replaced each time the locomotive is... 0 and Tier 1 locomotives, the label may be made up of more than one piece, as long as all pieces are... to Tier 1+ locomotives.” (4) “This locomotive conforms to U.S. EPA regulations applicable to Tier 2...

  13. Immunofluorescence reveals unusual patterns of labelling for connexin43 localized to calbindin-D28K-positive interstitial cells in the pineal gland.

    PubMed

    Tsao, D D; Wang, S G; Lynn, B D; Nagy, J I

    2017-06-01

    Gap junctions between cells in the pineal gland have been described ultrastructurally, but their connexin constituents have not been fully characterized. We used immunofluorescence in combination with markers of pineal cells to document the cellular localization of connexin43 (Cx43). Immunofluorescence labelling of Cx43 with several different antibodies was widely distributed throughout the pineal, whereas another connexin examined, connexin26, was not found in pineal but only in surrounding leptomeninges. Labelling apparently associated with plasma membranes was visualized either as fine Cx43-puncta (1-2 μm) or as unusually large pools of Cx43 ranging up to 4-7 μm in diameter or length. These puncta and pools were highly concentrated in perivascular spaces, where they were associated with numerous cells devoid of labelling for markers of pinealocytes (e.g. tryptophan hydroxylase and serotonin), and where they were minimally associated with blood vessels and lacked association with resident macrophages. Astrocytes labelled for glial fibrillary acidic protein were largely restricted to the anterior pole of the pineal gland, where they displayed only fine and sparse Cx43-puncta along their processes. Labelling for Cx43 was localized largely though not exclusively to the somata and long processes of a subpopulation of perivascular interstitial cells that were immunopositive for calbindin-D28K. These cells were often located among dense bundles or termination areas of sympathetic fibres labelled for tyrosine hydroxylase or serotonin. The results indicate that interstitial cells form abundant gap junctions composed of Cx43, and suggest that gap junction-mediated intracellular communication by these cells supports the activities of pinealocytes. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  14. Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline

    PubMed Central

    Schilling, Birgit; Rardin, Matthew J.; MacLean, Brendan X.; Zawadzka, Anna M.; Frewen, Barbara E.; Cusack, Michael P.; Sorensen, Dylan J.; Bereman, Michael S.; Jing, Enxuan; Wu, Christine C.; Verdin, Eric; Kahn, C. Ronald; MacCoss, Michael J.; Gibson, Bradford W.

    2012-01-01

    Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models. PMID:22454539

  15. The efficacy of sugar labeling formats: Implications for labeling policy.

    PubMed

    Vanderlee, Lana; White, Christine M; Bordes, Isabelle; Hobin, Erin P; Hammond, David

    2015-12-01

    To examine knowledge of sugar recommendations and test the efficacy of formats for labeling total and added sugar on pre-packaged foods. Online surveys were conducted among 2008 Canadians aged 16-24. Participants were asked to identify recommended limits for total and added sugar consumption. In Experiment 1, participants were randomized to one of six labeling conditions with varying information for total sugar for a high- or low-sugar product and were asked to identify the relative amount of total sugar in the product. In Experiment 2, participants were randomized to one of three labels with different added sugar formats and were asked if the product contained added sugar and the relative amount of added sugar. Few young people correctly identified recommendations for total sugar (5%) or added sugar (7%). In Experiment 1, those who were shown percent daily value information were more likely to correctly identify the relative amount of total sugar (P < 0.05). In Experiment 2, those shown added sugar information were more likely to correctly identify that the product contained added sugar and the relative amount of added sugar in the product (P < 0.05). Improved labeling may improve consumer understanding of the amount of sugars in food products. © 2015 The Obesity Society.

  16. Optimization and quality assessment of the post-digestion 18O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry.

    PubMed

    Wang, Hongbin; Hu, Gaofei; Zhang, Yongqian; Yuan, Zheng; Zhao, Xuan; Zhu, Yong; Cai, De; Li, Yujuan; Xiao, Shengyuan; Deng, Yulin

    2010-07-15

    The post-digestion (18)O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion (18)O labeling, the optimal (18)O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited (18)O incorporation depending on concentration. However, a urea concentration between 1 and 2M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1-2 M and pH 4.5, 98.4+/-1.9% for a standard protein mixture and 97.2+/-6.2% for a complex biological sample. For a 1:1 mixture analysis of the (16)O- and (18)O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05+/-0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion (18)O labeling method. 2010 Elsevier B.V. All rights reserved.

  17. Histochemistry of nerve fibres double labelled with anti-TRPV2 antibodies and sensory nerve marker AM1-43 in the dental pulp of rat molars.

    PubMed

    Nishikawa, Sumio

    2008-09-01

    AM1-43 can label sensory nerve fibres and sensory neurons. Permeation of non-selective cation channels of the nerve cell membrane is suggested to be the mechanism responsible for labelling. To identify these channels, two candidates, TRPV1 and TRPV2 were examined by immunocytochemistry in the dental pulp and trigeminal ganglion of rats injected with AM1-43. A part of AM1-43-labelled nerve fibres was also positive for anti-TRPV2 antibody but negative for anti-TRPV1 antibody in the dental pulp. In the trigeminal ganglion, a part of the neuron showed both bright AM1-43 labelling and anti-TRPV2 immunolabelling, but neurons double labelled with AM1-43 and TRPV1 were rare. These results suggest that TRPV2 channels, but not TRPV1 channels, contribute to the fluorescent labelling of AM1-43 in the dental pulp.

  18. A systematic review of calorie labeling and modified calorie labeling interventions: Impact on consumer and restaurant behavior

    PubMed Central

    Bleich, Sara N.; Economos, Christina D.; Spiker, Marie L.; Vercammen, Kelsey; VanEpps, Eric M.; Block, Jason P.; Elbel, Brian; Story, Mary; Roberto, Christina A.

    2017-01-01

    Background Evidence on the effects of restaurant calorie labeling on consumer and restaurant behavior is mixed. This paper examined: 1) consumer responses to calorie information alone or compared to modified calorie information, and 2) changes in restaurant offerings following or in advance of menu labeling implementation. Methods We searched PubMed, Web of Science, Policy File and PAIS International to identify restaurant calorie labeling studies through October 1, 2016, that measured calories ordered, consumed, or available for purchase on restaurant menus. We also searched reference lists of calorie labeling articles. Results Fifty-three studies were included: 18 in real-world restaurants, 9 in cafeterias, and 21 in laboratory or simulation settings. Five examined restaurant offerings. Conclusion Due to a lack of well-powered studies with strong designs, the degree to which menu labeling encourages lower calorie purchases and whether that translates to a healthier population is unclear. Although there is limited evidence that menu labeling affects calories purchased at fast-food restaurants, some evidence demonstrates that it lowers calories purchased at certain types of restaurants and in cafeteria settings. The limited data on modified calorie labels find that such labels can encourage lower-calorie purchases, but may not differ in effects relative to calorie labels alone. PMID:29045080

  19. A Systematic Review of Calorie Labeling and Modified Calorie Labeling Interventions: Impact on Consumer and Restaurant Behavior.

    PubMed

    Bleich, Sara N; Economos, Christina D; Spiker, Marie L; Vercammen, Kelsey A; VanEpps, Eric M; Block, Jason P; Elbel, Brian; Story, Mary; Roberto, Christina A

    2017-12-01

    Evidence on the effects of restaurant calorie labeling on consumer and restaurant behavior is mixed. This paper examined: (1) consumer responses to calorie information alone or compared to modified calorie information and (2) changes in restaurant offerings following or in advance of menu labeling implementation. Searches were conducted in PubMed, Web of Science, Policy File, and PAIS International to identify restaurant calorie labeling studies through October 1, 2016, that measured calories ordered, consumed, or available for purchase on restaurant menus. The reference lists of calorie labeling articles were also searched. Fifty-three studies were included: 18 in real-world restaurants, 9 in cafeterias, and 21 in laboratory or simulation settings. Five examined restaurant offerings. Because of a lack of well-powered studies with strong designs, the degree to which menu labeling encourages lower-calorie purchases and whether that translates to a healthier population are unclear. Although there is limited evidence that menu labeling affects calories purchased at fast-food restaurants, some evidence demonstrates that it lowers calories purchased at certain types of restaurants and in cafeteria settings. The limited data on modified calorie labels find that such labels can encourage lower-calorie purchases but may not differ in effects relative to calorie labels alone. © 2017 The Obesity Society.

  20. Quantitative phosphoproteomics analysis reveals a key role of insulin growth factor 1 receptor (IGF1R) tyrosine kinase in human sperm capacitation.

    PubMed

    Wang, Jing; Qi, Lin; Huang, Shaoping; Zhou, Tao; Guo, Yueshuai; Wang, Gaigai; Guo, Xuejiang; Zhou, Zuomin; Sha, Jiahao

    2015-04-01

    One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. 16 CFR 300.14 - Substitute label requirement.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Substitute label requirement. 300.14 Section 300.14 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.14 Substitute label...

  2. 16 CFR 300.3 - Required label information.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Required label information. 300.3 Section 300.3 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.3 Required label...

  3. Proximity-Directed Labeling Reveals a New Rapamycin-Induced Heterodimer of FKBP25 and FRB in Live Cells

    PubMed Central

    2016-01-01

    Mammalian target of rapamycin (mTOR) signaling is a core pathway in cellular metabolism, and control of the mTOR pathway by rapamycin shows potential for the treatment of metabolic diseases. In this study, we employed a new proximity biotin-labeling method using promiscuous biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced interactome on the FK506-rapamycin binding (FRB) domain in living cells. FKBP25 showed the strongest biotin labeling by FRB–pBirA in the presence of rapamycin. Immunoprecipitation and immunofluorescence experiments confirmed that endogenous FKBP25 has a rapamycin-induced physical interaction with the FRB domain. Furthermore, the crystal structure of the ternary complex of FRB–rapamycin–FKBP25 was determined at 1.67-Å resolution. In this crystal structure we found that the conformational changes of FRB generate a hole where there is a methionine-rich space, and covalent metalloid coordination was observed at C2085 of FRB located at the bottom of the hole. Our results imply that FKBP25 might have a unique physiological role related to metallomics in mTOR signaling. PMID:27610411

  4. Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP.

    PubMed Central

    Ostap, E. M.; Thomas, D. D.

    1991-01-01

    The most probable source of force generation in muscle fibers in the rotation of the myosin head when bound to actin. This laboratory has demonstrated that ATP induces microsecond rotational motions of spin-labeled myosin heads bound to actin (Berger, C. L. E. C. Svensson, and D. D. Thomas. 1989. Proc. Natl. Acad. Sci. USA. 86:8753-8757). Our goal is to determine whether the observed ATP-induced rotational motions of actin-bound heads are accompanied by changes in actin rotational motions. We have used saturation transfer electron paramagnetic resonance (ST-EPR) and laser-induced photolysis of caged ATP to monitor changes in the microsecond rotational dynamics of spin-labeled F-actin in the presence of myosin subfragment-1 (S1). A maleimide spin label was attached selectively to cys-374 on actin. In the absence of ATP (with or without caged ATP), the ST-EPR spectrum (corresponding to an effective rotational time of approximately 150 microseconds) was essentially the same as observed for the same spin label bound to cys-707 (SH1) on S1, indicating that S1 is rigidly bound to actin in rigor. At normal ionic strength (micro = 186 mM), a decrease in ST-EPR intensity (increase in microsecond F-actin mobility) was clearly indicated upon photolysis of 1 mM caged ATP with a 50-ms, 351-nm laser pulse. This increase in mobility is due to the complete dissociation of Si from the actin filament. At low ionic strength (micro, = 36 mM), when about half the Si heads remain bound during ATP hydrolysis, no change in the actin mobility was detected, despite much faster motions of labeled S1 bound to actin. Therefore, we conclude that the active interaction of Si, actin,and ATP induces rotation of myosin heads relative to actin, but does not affect the microsecond rotational motion of actin itself, as detected at cys-374 of actin. PMID:1651780

  5. Appalachian residents' perspectives on new U.S. cigarette warning labels.

    PubMed

    Reiter, Paul L; Broder-Oldach, Benjamin; Wewers, Mary Ellen; Klein, Elizabeth G; Paskett, Electra D; Katz, Mira L

    2012-12-01

    The U.S. Food and Drug Administration revealed new pictorial warning labels in June 2011 for cigarette packages, yet little is known about how these labels are perceived by U.S. residents. We examined the reactions to and attitudes about the new labels among residents of Appalachian Ohio, a region with a high smoking prevalence. We conducted focus groups with Appalachian Ohio residents between July and October 2011. Participants included healthcare providers (n = 30), community leaders (n = 26), parents (n = 28), and young adult men ages 18-26 (n = 18). Most participants supported the addition of the new labels to U.S. cigarette packages, though many were unaware of the labels prior to the focus groups. Participants did not think the labels would be effective in promoting smoking cessation among smokers in their communities, but they were more positive about the potential of the labels to reduce smoking initiation. Participants reported positive feedback about the more graphic labels, particularly those showing a man with a tracheal stoma or a person with severe oral disease. The labels that include a cartoon image of an ill infant and a man who quit smoking received the most negative feedback. Participants generally supported adding pictorial warning labels to U.S. cigarette packages, but only a few of labels received mostly positive feedback. Results offer early insight into how the new labels may be received if they are put into practice.

  6. 5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy.

    PubMed

    Puffer, Barbara; Kreutz, Christoph; Rieder, Ulrike; Ebert, Marc-Olivier; Konrat, Robert; Micura, Ronald

    2009-12-01

    (19)F NMR spectroscopy has proved to be a valuable tool to monitor functionally important conformational transitions of nucleic acids. Here, we present a systematic investigation on the application of 5-fluoro pyrimidines to probe DNA and RNA secondary structures. Oligonucleotides with the propensity to adapt secondary structure equilibria were chosen as model systems and analyzed by 1D (19)F and (1)H NMR spectroscopy. A comparison with the unmodified analogs revealed that the equilibrium characteristics of the bistable DNA and RNA oligonucleotides were hardly affected upon fluorine substitution at C5 of pyrimidines. This observation was in accordance with UV spectroscopic melting experiments which demonstrated that single 5-fluoro substitutions in double helices lead to comparable thermodynamic stabilities. Thus, 5-fluoro pyrimidine labeling of DNA and RNA can be reliably applied for NMR based nucleic acid secondary structure evaluation. Furthermore, we developed a facile synthetic route towards 5-fluoro cytidine phosphoramidites that enables their convenient site-specific incorporation into oligonucleotides by solid-phase synthesis.

  7. The Development of Ethnic/Racial Self-Labeling: Individual Differences in Context.

    PubMed

    Cheon, Yuen Mi; Bayless, Sara Douglass; Wang, Yijie; Yip, Tiffany

    2018-03-15

    Ethnic/racial self-labeling represents one's knowledge of and preference for ethnic/racial group membership, which is related to, but distinguishable from, ethnic/racial identity. This study examined the development of ethnic/racial self-labeling over time by including the concept of elaboration among a diverse sample of 297 adolescents (Time 1 mean age 14.75, 67% female, 37.4% Asian or Asian American, 10.4% Black, African American, or West Indian, 23.2% Hispanic or Latinx, 24.2% White, 4.4% other). Growth mixture modeling revealed two distinct patterns-low and high self-labeling elaboration from freshman to sophomore year of high school. Based on logistic regression analyses, the level of self-labeling elaboration was generally low among the adolescents who were foreign-born, reported low levels of ethnic/racial identity exploration, or attended highly diverse schools. We also found a person-by-context interaction where the impact of school diversity varied for foreign-born and native-born adolescents (b = 12.81, SE = 6.30, p < 0.05) and by the level of ethnic/racial identity commitment (b = 14.32, SE = 6.65, p < 0.05). These findings suggest varying patterns in ethnic/racial self-labeling elaboration among adolescents from diverse backgrounds and their linkage to individual and contextual factors.

  8. 27 CFR 7.22 - Mandatory label information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ..., DEPARTMENT OF THE TREASURY LIQUORS LABELING AND ADVERTISING OF MALT BEVERAGES Labeling Requirements for Malt Beverages § 7.22 Mandatory label information. There shall be stated: (a) On the brand label: (1) Brand name....27. (5) Alcohol content in accordance with § 7.71, for malt beverages that contain any alcohol...

  9. Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

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

    Kito, Hiroaki; Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto; Yamamura, Hisao

    2015-04-10

    Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cyclemore » progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.« less

  10. Associations between Public Library Use and Reading Aloud among Families with Young Children.

    PubMed

    Chen, Pamela; Rea, Corinna; Shaw, Rebecca; Bottino, Clement J

    2016-06-01

    To measure public library use in a sample of families with young children and examine associations with reading aloud. We interviewed 200 parents of 6- to 18-month-old children visiting a hospital-based pediatric clinic. We assessed public library card ownership, public library visitation, and awareness of public library programming. We assessed reading aloud using the StimQ READ questionnaire. We used multivariable logistic and linear regression to examine associations while adjusting for sociodemographic characteristics. In multivariable analysis, parents who owned a public library card had greater odds of reading aloud daily to their 6- to 18-month-old child (aOR, 2.0; 95% CI, 1.0-3.8) and higher StimQ READ scores (β = 0.9; 95% CI, 0.2-1.6). Parents who visited a public library once a month or more often had greater odds of reading aloud daily (aOR, 3.4; 95% CI, 1.8-6.7) and higher StimQ READ scores (β = 1.3; 95% CI, 0.6-2.0). Parents whose 6- to 18-month-old child had ever visited a public library did not have greater odds of reading aloud daily (aOR, 1.4; 95% CI, 0.7-2.9), but did have higher StimQ read scores (β = 1.2; 95% CI, 0.4-2.0). Parents who felt informed about available public library programs for children had greater odds of reading aloud daily (aOR, 2.5; 95% CI, 1.3-5.1) and higher StimQ READ scores (β = 1.1; 95% CI, 0.4-1.9). In this sample of families with young children, we found positive associations between public library use and reading aloud. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Carbon transitions from either Calvin cycle or transitory starch to heteroglycans as revealed by (14) C-labeling experiments using protoplasts from Arabidopsis.

    PubMed

    Malinova, Irina; Steup, Martin; Fettke, Joerg

    2013-09-01

    Plants metabolize transitory starch by precisely coordinated plastidial and cytosolic processes. The latter appear to include the action of water-soluble heteroglycans (SHGin ) whose monosaccharide pattern is similar to that of apoplastic glycans (SHGex ) but, unlike SHGex , SHGin strongly interacts with glucosyl transferases. In this study, we analyzed starch metabolism using mesophyll protoplasts from wild-type plants and two knock-out mutants [deficient in the cytosolic transglucosidase, disproportionating isoenzyme 2 (DPE2) or the plastidial phosphoglucomutase (PGM1)] from Arabidopsis thaliana. Protoplasts prelabeled by photosynthetic (14) CO2 fixation were transferred to an unlabeled medium and were darkened or illuminated. Carbon transitions from the Calvin cycle or from starch to both SHGin and SHGex were analyzed. In illuminated protoplasts, starch turn-over was undetectable but darkened protoplasts continuously degraded starch. During illumination, neither the total (14) C content nor the labeling patterns of the sugar residues of SHGin were significantly altered but both the total amount and the labeling of the constituents of SHGex increased with time. In darkened protoplasts, the (14) C-content of most of the sugar residues of SHGin transiently and strongly increased and then declined. This effect was not observed in any SHGex constituent. In darkened DPE2-deficient protoplasts, none of the SHGin constituents exhibited an essential transient increase in labeling. In contrast, some residues of SHGin from the PGM1 mutant exhibited a transient increase in label but this effect significantly differed from that of the wild type. Two conclusions are reached: first, SHGin and SHGex exert different metabolic functions and second, SHGin is directly involved in starch degradation. © 2013 Scandinavian Plant Physiology Society.

  12. Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation.

    PubMed

    Azimzadeh, Omid; Scherthan, Harry; Yentrapalli, Ramesh; Barjaktarovic, Zarko; Ueffing, Marius; Conrad, Marcus; Neff, Frauke; Calzada-Wack, Julia; Aubele, Michaela; Buske, Christian; Atkinson, Michael J; Hauck, Stefanie M; Tapio, Soile

    2012-04-18

    Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p≤0.05) in irradiated hearts 24h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Detection of CdSe quantum dot photoluminescence for security label on paper

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

    Isnaeni,, E-mail: isnaeni@lipi.go.id; Sugiarto, Iyon Titok; Bilqis, Ratu

    CdSe quantum dot has great potential in various applications especially for emitting devices. One example potential application of CdSe quantum dot is security label for anti-counterfeiting. In this work, we present a practical approach of security label on paper using one and two colors of colloidal CdSe quantum dot, which is used as stamping ink on various types of paper. Under ambient condition, quantum dot is almost invisible. The quantum dot security label can be revealed by detecting emission of quantum dot using photoluminescence and cnc machine. The recorded quantum dot emission intensity is then analyzed using home-made program tomore » reveal quantum dot pattern stamp having the word ’RAHASIA’. We found that security label using quantum dot works well on several types of paper. The quantum dot patterns can survive several days and further treatment is required to protect the quantum dot. Oxidation of quantum dot that occurred during this experiment reduced the emission intensity of quantum dot patterns.« less

  14. A combined EPR and MD simulation study of a nitroxyl spin label with restricted internal mobility sensitive to protein dynamics.

    PubMed

    Oganesyan, Vasily S; Chami, Fatima; White, Gaye F; Thomson, Andrew J

    2017-01-01

    EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Making food labels social: The impact of colour of nutritional labels and injunctive norms on perceptions and choice of snack foods☆

    PubMed Central

    Vasiljevic, Milica; Pechey, Rachel; Marteau, Theresa M.

    2015-01-01

    Recent studies report that using green labels to denote healthier foods, and red to denote less healthy foods increases consumption of green- and decreases consumption of red-labelled foods. Other symbols (e.g. emoticons conveying normative approval and disapproval) could also be used to signal the healthiness and/or acceptability of consuming such products. The present study tested the combined effects of using emoticons and colours on labels amongst a nationally representative sample of the UK population (n = 955). In a 3 (emoticon expression: smiling vs. frowning vs. no emoticon) × 3 (colour label: green vs. red vs. white) ×2 (food option: chocolate bar vs. cereal bar) between-subjects experiment, participants rated the level of desirability, healthiness, tastiness, and calorific content of a snack bar they had been randomised to view. At the end they were further randomised to view one of nine possible combinations of colour and emoticon labels and asked to choose between a chocolate and a cereal bar. Regardless of label, participants rated the chocolate as tastier and more desirable when compared to the cereal bar, and the cereal bar as healthier than the chocolate bar. A series of interactions revealed that a frowning emoticon on a white background decreased perceptions of healthiness and tastiness of the cereal bar, but not the chocolate bar. In the explicit choice task selection was unaffected by label. Overall nutritional labels had limited effects on perceptions and no effects on choice of snack foods. Emoticon labels yielded stronger effects on perceptions of taste and healthiness of snacks than colour labels. Frowning emoticons may be more potent than smiling emoticons at influencing the perceived healthiness and tastiness of foods carrying health halos. PMID:25841647

  16. Making food labels social: The impact of colour of nutritional labels and injunctive norms on perceptions and choice of snack foods.

    PubMed

    Vasiljevic, Milica; Pechey, Rachel; Marteau, Theresa M

    2015-08-01

    Recent studies report that using green labels to denote healthier foods, and red to denote less healthy foods increases consumption of green- and decreases consumption of red-labelled foods. Other symbols (e.g. emoticons conveying normative approval and disapproval) could also be used to signal the healthiness and/or acceptability of consuming such products. The present study tested the combined effects of using emoticons and colours on labels amongst a nationally representative sample of the UK population (n = 955). In a 3 (emoticon expression: smiling vs. frowning vs. no emoticon) × 3 (colour label: green vs. red vs. white) ×2 (food option: chocolate bar vs. cereal bar) between-subjects experiment, participants rated the level of desirability, healthiness, tastiness, and calorific content of a snack bar they had been randomised to view. At the end they were further randomised to view one of nine possible combinations of colour and emoticon labels and asked to choose between a chocolate and a cereal bar. Regardless of label, participants rated the chocolate as tastier and more desirable when compared to the cereal bar, and the cereal bar as healthier than the chocolate bar. A series of interactions revealed that a frowning emoticon on a white background decreased perceptions of healthiness and tastiness of the cereal bar, but not the chocolate bar. In the explicit choice task selection was unaffected by label. Overall nutritional labels had limited effects on perceptions and no effects on choice of snack foods. Emoticon labels yielded stronger effects on perceptions of taste and healthiness of snacks than colour labels. Frowning emoticons may be more potent than smiling emoticons at influencing the perceived healthiness and tastiness of foods carrying health halos. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. 27 CFR 4.32 - Mandatory label information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Mandatory label information. (a) There shall be stated on the brand label: (1) Brand name, in accordance... the bottle. (c) There shall be stated on the brand label or on a back label a statement that the... the Office of Management and Budget under Control Number 1512-0469) [T.D. 6521, 25 FR 13835, Dec. 29...

  18. Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

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

    Bandhuvula, Padmavathi; Li Zaiguo; Bittman, Robert

    2009-03-06

    Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an {omega}-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K{sub m} ofmore » 35 {mu}M for BODIPY-sphingosine 1-phosphate.« less

  19. Performance limitations of label-free sensors in molecular diagnosis using complex samples

    NASA Astrophysics Data System (ADS)

    Varma, Manoj

    2016-03-01

    Label-free biosensors promised a paradigm involving direct detection of biomarkers from complex samples such as serum without requiring multistep sample processing typical of labelled methods such as ELISA or immunofluorescence assays. Label-free sensors have witnessed decades of development with a veritable zoo of techniques available today exploiting a multitude of physical effects. It is appropriate now to critically assess whether label-free technologies have succeeded in delivering their promise with respect to diagnostic applications, particularly, ambitious goals such as early cancer detection using serum biomarkers, which require low limits of detection (LoD). Comparison of nearly 120 limits of detection (LoD) values reported by labelled and label-free sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Data from experiments where labelled and label-free assays were performed simultaneously using the same assay parameters also confirm that the LoD achieved by labelled techniques is 2 to 3 orders of magnitude better than that by label-free techniques. Furthermore, label-free techniques required significant signal amplification, for e.g. using nanoparticle conjugated secondary antibodies, to achieve LoDs comparable to labelled methods substantially deviating from the original "direct detection" paradigm. This finding has important implications on the practical limits of applying label-free detection methods for molecular diagnosis.

  20. The influence of calorie labeling on food orders and consumption: A review of the literature1

    PubMed Central

    Kiszko, Kamila M.; Martinez, Olivia D.; Abrams, Courtney; Elbel, Brian

    2014-01-01

    Obesity is a challenging public health problem that affects millions of Americans. Increasingly policy makers are seeking environmental and policy-based solutions to combat and prevent its serious health effects. Calorie labeling mandates, including the provision in the 2010 Patient Protection and Affordable Care Act that is set to begin in 2014, have been one of the most popular and most studied approaches. This review examines 31 studies published from January 1, 2007 through July 19, 2013. It builds on Harnack and French's 2008 review and assesses the evidence on the effectiveness of calorie labeling at the point of purchase. We find that, while there are some positive results reported from studies examining the effects of calorie labeling, overall the best designed studies (real world studies, with a comparison group) show that calorie labels do not have the desired effect in reducing total calories ordered at the population level. Moving forward, researchers should consider novel, more effective ways of presenting nutrition information, while keeping a focus on particular subgroups that may be differentially affected by nutrition policies. PMID:24760208

  1. Audit of manufactured products: use of allergen advisory labels and identification of labeling ambiguities.

    PubMed

    Pieretti, Mariah M; Chung, Danna; Pacenza, Robert; Slotkin, Todd; Sicherer, Scott H

    2009-08-01

    The Food Allergy Labeling and Consumer Protection Act became effective January 1, 2006, and mandates disclosure of the 8 major allergens in plain English and as a source of ingredients in the ingredient statement. It does not regulate advisory labels. We sought to determine the frequency and language used in voluntary advisory labels among commercially available products and to identify labeling ambiguities affecting consumers with allergy. Trained surveyors performed a supermarket survey of 20,241 unique manufactured food products (from an original assessment of 49,604 products) for use of advisory labels. A second detailed survey of 744 unique products evaluated additional labeling practices. Overall, 17% of 20,241 products surveyed contain advisory labels. Chocolate candy, cookies, and baking mixes were the 3 categories of 24 with the greatest frequency (> or = 40%). Categorically, advisory warnings included "may contain" (38%), "shared equipment" (33%), and "within plant" (29%). The subsurvey disclosed 25 different types of advisory terminology. Nonspecific terms, such as "natural flavors" and "spices," were found on 65% of products and were not linked to a specific ingredient for 83% of them. Additional ambiguities included unclear sources of soy (lecithin vs protein), nondisclosure of sources of gelatin and lecithin, and simultaneous disclosure of "contains" and "may contain" for the same allergen, among others. Numerous products have advisory labeling and ambiguities that present challenges to consumers with food allergy. Additional allergen labeling regulation could improve safety and quality of life for individuals with food allergy.

  2. 9 CFR 381.500 - Exemption from nutrition labeling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Exemption from nutrition labeling. 381... INSPECTION AND CERTIFICATION POULTRY PRODUCTS INSPECTION REGULATIONS Nutrition Labeling § 381.500 Exemption from nutrition labeling. (a) The following poultry products are exempt from nutrition labeling: (1...

  3. 9 CFR 381.500 - Exemption from nutrition labeling.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Exemption from nutrition labeling. 381... INSPECTION AND CERTIFICATION POULTRY PRODUCTS INSPECTION REGULATIONS Nutrition Labeling § 381.500 Exemption from nutrition labeling. (a) The following poultry products are exempt from nutrition labeling: (1...

  4. 9 CFR 381.500 - Exemption from nutrition labeling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Exemption from nutrition labeling. 381... INSPECTION AND CERTIFICATION POULTRY PRODUCTS INSPECTION REGULATIONS Nutrition Labeling § 381.500 Exemption from nutrition labeling. (a) The following poultry products are exempt from nutrition labeling: (1...

  5. Analysis of the Effects of Fixed Costs on Learning Curve Calculations

    DTIC Science & Technology

    1994-09-01

    Gansler, Jacques S . The Defense Industry. Cambridge MA: MIT Press, 1980. 11. Horngren , Charles T. and George Foster. Cost Accounting : A Managerial...Incorrect Total Cost Estimates and Comparison to Correct/Correct Total C o st E stim a te s ...7 1 12. Incorrect/Correct Total Cost Estimates and Comparison to Correct/Correct Total C o st E stim a te s

  6. Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

    NASA Astrophysics Data System (ADS)

    Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

    2015-05-01

    Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1

  7. Treatment of post-stroke dysphagia by vitalstim therapy coupled with conventional swallowing training.

    PubMed

    Xia, Wenguang; Zheng, Chanjuan; Lei, Qingtao; Tang, Zhouping; Hua, Qiang; Zhang, Yangpu; Zhu, Suiqiang

    2011-02-01

    To investigate the effects of VitalStim therapy coupled with conventional swallowing training on recovery of post-stroke dysphagia, a total of 120 patients with post-stroke dysphagia were randomly and evenly divided into three groups: conventional swallowing therapy group, VitalStim therapy group, and VitalStim therapy plus conventional swallowing therapy group. Prior to and after the treatment, signals of surface electromyography (sEMG) of swallowing muscles were detected, swallowing function was evaluated by using the Standardized Swallowing Assessment (SSA) and Videofluoroscopic Swallowing Study (VFSS) tests, and swallowing-related quality of life (SWAL-QOL) was evaluated using the SWAL-QOL questionnaire. There were significant differences in sEMG value, SSA, VFSS, and SWAL-QOL scores in each group between prior to and after treatment. After 4-week treatment, sEMG value, SSA, VFSS and SWAL-QOL scores were significantly greater in the VitalStim therapy plus conventional swallowing training group than in the conventional swallowing training group and VitalStim therapy group, but no significant difference existed between conventional swallowing therapy group and VitalStim therapy group. It was concluded that VitalStim therapy coupled with conventional swallowing training was conducive to recovery of post-stroke dysphagia.

  8. Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling1[C][W

    PubMed Central

    Gaupels, Frank; Sarioglu, Hakan; Beckmann, Manfred; Hause, Bettina; Spannagl, Manuel; Draper, John; Lindermayr, Christian; Durner, Jörg

    2012-01-01

    In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling. PMID:23085839

  9. Facilitated Visual Interpretation of Scores in Principal Component Analysis by Bioactivity-Labeling of 1H-NMR Spectra-Metabolomics Investigation and Identification of a New α-Glucosidase Inhibitor in Radix Astragali.

    PubMed

    Liu, Yueqiu; Nyberg, Nils T; Jäger, Anna K; Staerk, Dan

    2017-03-06

    Radix Astragali is a component of several traditional medicines used for the treatment of type 2 diabetes in China. Radix Astragali is known to contain isoflavones, which inhibit α-glucosidase in the small intestines, and thus lowers the blood glucose levels. In this study, 21 samples obtained from different regions of China were extracted with ethyl acetate, then the IC50-values were determined, and the crude extracts were analyzed by 1H-NMR spectroscopy. A principal component analysis of the 1H-NMR spectra labeled with their IC50-values, that is, bioactivity-labeled 1H-NMR spectra, showed a clear correlation between spectral profiles and the α-glucosidase inhibitory activity. The loading plot and LC-HRMS/NMR of microfractions indicated that previously unknown long chain ferulates could be partly responsible for the observed antidiabetic activity of Radix Astragali. Subsequent preparative scale isolation revealed a compound not previously reported, linoleyl ferulate (1), showing α-glucosidase inhibitory activity (IC50 0.5 mM) at a level comparable to the previously studied isoflavones. A closely related analogue, hexadecyl ferulate (2), did not show significant inhibitory activity, and the double bonds in the alcohol part of 1 seem to be important structural features for the α-glucosidase inhibitory activity. This proof of concept study demonstrates that bioactivity-labeling of the 1H-NMR spectral data of crude extracts allows global and nonselective identification of individual constituents contributing to the crude extract's bioactivity.

  10. 16 CFR 300.30 - Deceptive labeling in general.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Deceptive labeling in general. 300.30 Section 300.30 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.30 Deceptive labeling...

  11. Consumer purchasing patterns in response to calorie labeling legislation in New York City.

    PubMed

    Vadiveloo, Maya K; Dixon, L Beth; Elbel, Brian

    2011-05-27

    Obesity is a major public health threat and policies aimed at curbing this epidemic are emerging. National calorie labeling legislation is forthcoming and requires rigorous evaluation to examine its impact on consumers. The purpose of this study was to examine whether point-of-purchase calorie labels in New York City (NYC) chain restaurants affected food purchasing patterns in a sample of lower income adults in NYC and Newark, NJ. This study utilized a difference-in-difference design to survey 1,170 adult patrons of four popular chain restaurants in NYC and Newark, NJ (which did not introduce labeling) before and after calorie labeling was implemented in NYC. Receipt data were collected and analyzed to examine food and beverage purchases and frequency of fast food consumption. Descriptive statistics were generated, and linear and logistic regression, difference-in-difference analysis, and predicted probabilities were used to analyze the data. A difference-in-difference analysis revealed no significant favorable differences and some unfavorable differences in food purchasing patterns and frequency of fast food consumption between adult patrons of fast food restaurants in NYC and Newark, NJ. Adults in NYC who reported noticing and using the calorie labels consumed fast food less frequently compared to adults who did not notice the labels (4.9 vs. 6.6 meals per week, p <0.05). While no favorable differences in purchasing as a result of labeling were noted, self-reported use of calorie labels was associated with some favorable behavioral patterns in a subset of adults in NYC. However, overall impact of the legislation may be limited. More research is needed to understand the most effective way to deliver calorie information to consumers.

  12. Influence of nutritional knowledge on the use and interpretation of Spanish nutritional food labels.

    PubMed

    Carrillo, E; Varela, P; Fiszman, S

    2012-01-01

    The present study analyzed the nutritional knowledge of Spanish consumers and its relationship with the correct use of food labels. Consumers were asked about their nutritional knowledge and some functional foods and about their understanding of food labeling and their use of it to select healthy food. A 2-part questionnaire was employed. The 1st part concerned their knowledge of nutritional facts, including their knowledge about macronutrients and perception of certain functional foods, while the 2nd part addressed some questions regarding food labels. The results revealed no statistically significant differences in nutritional knowledge by either age or gender, but a direct relationship with educational level. The association between nutritional knowledge and the perception and understanding of food labeling showed that the nutritional label rarely influenced the food purchases of the group with low nutritional knowledge, who considered that this information was too technical. More than half of the consumers did not consider the calorie or sugar content important for selecting food. In addition, the group with low nutritional knowledge stated that they never or rarely looked at the food labels to check whether it was low-fat food that they were buying. Knowing the status of the consumer's nutritional knowledge allows health campaigns to be designed; considering the influence of cultural factors and the perception of food labeling is very useful for promoting better nutritional information. © 2011 Institute of Food Technologists®

  13. Food Labels

    MedlinePlus

    ... Staying Safe Videos for Educators Search English Español Food Labels KidsHealth / For Teens / Food Labels What's in ... to have at least 95% organic ingredients. Making Food Labels Work for You The first step in ...

  14. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... auxiliary, variable-speed propulsion engines used with fixed-pitch propellers, recreational, etc.) (c) The... EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General requirements. (1) Each new engine covered by a certificate of conformity under § 94.208 shall be labeled by the...

  15. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... auxiliary, variable-speed propulsion engines used with fixed-pitch propellers, recreational, etc.) (c) The... EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General requirements. (1) Each new engine covered by a certificate of conformity under § 94.208 shall be labeled by the...

  16. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... auxiliary, variable-speed propulsion engines used with fixed-pitch propellers, recreational, etc.) (c) The... EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General requirements. (1) Each new engine covered by a certificate of conformity under § 94.208 shall be labeled by the...

  17. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... auxiliary, variable-speed propulsion engines used with fixed-pitch propellers, recreational, etc.) (c) The... EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General requirements. (1) Each new engine covered by a certificate of conformity under § 94.208 shall be labeled by the...

  18. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... auxiliary, variable-speed propulsion engines used with fixed-pitch propellers, recreational, etc.) (c) The... EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General requirements. (1) Each new engine covered by a certificate of conformity under § 94.208 shall be labeled by the...

  19. 16 CFR 300.6 - Labels to be avoided.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Labels to be avoided. 300.6 Section 300.6 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.6 Labels to be avoided. Stamps, tags...

  20. 27 CFR 5.32 - Mandatory label information.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Mandatory label information. 5.32 Section 5.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF DISTILLED SPIRITS Labeling Requirements for...

  1. Localization of insulinomas to regions of the pancreas by intraarterial calcium stimulation: the NIH experience.

    PubMed

    Guettier, Jean-Marc; Kam, Anthony; Chang, Richard; Skarulis, Monica C; Cochran, Craig; Alexander, H Richard; Libutti, Steven K; Pingpank, James F; Gorden, Phillip

    2009-04-01

    Selective intraarterial calcium injection of the major pancreatic arteries with hepatic venous sampling [calcium arterial stimulation (CaStim)] has been used as a localizing tool for insulinomas at the National Institutes of Health (NIH) since 1989. The accuracy of this technique for localizing insulinomas was reported for all cases until 1996. The aim of the study was to assess the accuracy and track record of the CaStim over time and in the context of evolving technology and to review issues related to result interpretation and procedure complications. CaStim was the only invasive preoperative localization modality used at our center. Endoscopic ultrasound (US) was not studied. We conducted a retrospective case review at a referral center. Twenty-nine women and 16 men (mean age, 47 yr; range, 13-78) were diagnosed with an insulinoma from 1996-2008. A supervised fast was conducted to confirm the diagnosis of insulinoma. US, computed tomography (CT), magnetic resonance imaging (MRI), and CaStim were used as preoperative localization studies. Localization predicted by each preoperative test was compared to surgical localization for accuracy. We measured the accuracy of US, CT, MRI, and CaStim for localization of insulinomas preoperatively. All 45 patients had surgically proven insulinomas. Thirty-eight of 45 (84%) localized to the correct anatomical region by CaStim. In five of 45 (11%) patients, the CaStim was falsely negative. Two of 45 (4%) had false-positive localizations. The CaStim has remained vastly superior to abdominal US, CT, or MRI over time as a preoperative localizing tool for insulinomas. The utility of the CaStim for this purpose and in this setting is thus validated.

  2. In vivo kinetic approach reveals slow SOD1 turnover in the CNS

    PubMed Central

    Crisp, Matthew J.; Mawuenyega, Kwasi G.; Patterson, Bruce W.; Reddy, Naveen C.; Chott, Robert; Self, Wade K.; Weihl, Conrad C.; Jockel-Balsarotti, Jennifer; Varadhachary, Arun S.; Bucelli, Robert C.; Yarasheski, Kevin E.; Bateman, Randall J.; Miller, Timothy M.

    2015-01-01

    Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins. PMID:26075819

  3. In vivo kinetic approach reveals slow SOD1 turnover in the CNS.

    PubMed

    Crisp, Matthew J; Mawuenyega, Kwasi G; Patterson, Bruce W; Reddy, Naveen C; Chott, Robert; Self, Wade K; Weihl, Conrad C; Jockel-Balsarotti, Jennifer; Varadhachary, Arun S; Bucelli, Robert C; Yarasheski, Kevin E; Bateman, Randall J; Miller, Timothy M

    2015-07-01

    Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins.

  4. Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate. Part I. Chemistry and labeling technique.

    PubMed

    Schuhmacher, J; Matys, R; Hauser, H; Maier-Borst, W; Matzku, S

    1986-01-01

    As a chelating agent for labeling antibodies (Abs) with metallic radionuclides, a propionic acid substituted ethylenediamine N,N'-di-[(o-hydroxyphenyl) acetic acid] (P-EDDHA), which tightly complexes 67Ga, was synthesized. The 67Ga-P-EDDHA chelate was coupled in aqueous solution to IgG at a molar ratio of 1:1 via carbodiimide. The average coupling yield was 15%. A specific activity of 4 mCi/mg IgG could be obtained with commercially supplied 67Ga. In vitro stability was evaluated in human serum at 37 degrees C and showed a half-life of about 120 h for the release of 67Ga from the labeled Ab during the initial phase of incubation. This in vitro halflife is similar to that measured for 111In-DTPA labeled Abs. Because of the high stability of the 67Ga-P-EDDHA chelate, the in vivo formation of radioactive labeled transferrin by transchelation, as described for 111In-DTPA labeled Abs, should, however, be reduced by this labeling technique.

  5. 5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy

    PubMed Central

    Puffer, Barbara; Kreutz, Christoph; Rieder, Ulrike; Ebert, Marc-Olivier; Konrat, Robert; Micura, Ronald

    2009-01-01

    19F NMR spectroscopy has proved to be a valuable tool to monitor functionally important conformational transitions of nucleic acids. Here, we present a systematic investigation on the application of 5-fluoro pyrimidines to probe DNA and RNA secondary structures. Oligonucleotides with the propensity to adapt secondary structure equilibria were chosen as model systems and analyzed by 1D 19F and 1H NMR spectroscopy. A comparison with the unmodified analogs revealed that the equilibrium characteristics of the bistable DNA and RNA oligonucleotides were hardly affected upon fluorine substitution at C5 of pyrimidines. This observation was in accordance with UV spectroscopic melting experiments which demonstrated that single 5-fluoro substitutions in double helices lead to comparable thermodynamic stabilities. Thus, 5-fluoro pyrimidine labeling of DNA and RNA can be reliably applied for NMR based nucleic acid secondary structure evaluation. Furthermore, we developed a facile synthetic route towards 5-fluoro cytidine phosphoramidites that enables their convenient site-specific incorporation into oligonucleotides by solid-phase synthesis. PMID:19843610

  6. Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

    PubMed Central

    2015-01-01

    The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

  7. Label-free sensing of the binding state of MUC1 peptide and anti-MUC1 aptamer solution in fluidic chip by terahertz spectroscopy.

    PubMed

    Zhao, Xiang; Zhang, Mingkun; Wei, Dongshan; Wang, Yunxia; Yan, Shihan; Liu, Mengwan; Yang, Xiang; Yang, Ke; Cui, Hong-Liang; Fu, Weiling

    2017-10-01

    The aptamer and target molecule binding reaction has been widely applied for construction of aptasensors, most of which are labeled methods. In contrast, terahertz technology proves to be a label-free sensing tool for biomedical applications. We utilize terahertz absorption spectroscopy and molecular dynamics simulation to investigate the variation of binding-induced collective vibration of hydrogen bond network in a mixed solution of MUC1 peptide and anti-MUC1 aptamer. The results show that binding-induced alterations of hydrogen bond numbers could be sensitively reflected by the variation of terahertz absorption coefficients of the mixed solution in a customized fluidic chip. The minimal detectable concentration is determined as 1 pmol/μL, which is approximately equal to the optimal immobilized concentration of aptasensors.

  8. Label-free, real-time interaction and adsorption analysis 1: surface plasmon resonance.

    PubMed

    Fee, Conan J

    2013-01-01

    A key requirement for the development of proteins for use in nanotechnology is an understanding of how individual proteins bind to other molecules as they assemble into larger structures. The introduction of labels to enable the detection of biomolecules brings the inherent risk that the labels themselves will influence the nature of biomolecular interactions. Thus, there is a need for label-free interaction and adsorption analysis. In this and the following chapter, two biosensor techniques are reviewed: surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM). Both allow real-time analysis of biomolecular interactions and both are label-free. The first of these, SPR, is an optical technique that is highly sensitive to the changes in refractive index that occur with protein (or other molecule) accumulation near an illuminated gold surface. Unlike QCM ( Chapter 18 ) SPR is not affected by the water that may be associated with the adsorbed layer nor by conformational changes in the adsorbed species. SPR thus provides unique information about the interaction of a protein with its binding partners.

  9. Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.

    PubMed

    Gómez-Cortés, Pilar; Brenna, J Thomas; Sacks, Gavin L

    2012-06-19

    Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve

  10. 30 CFR 47.41 - Requirement for container labels.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Requirement for container labels. 47.41 Section... TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.41 Requirement for container labels. (a) The operator must ensure that each container of a hazardous chemical has a label. If a...

  11. Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

    PubMed

    Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

    2017-08-01

    We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

  12. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Label. 18.55 Section 18.55 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container...

  13. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Label. 18.55 Section 18.55 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container...

  14. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Label. 18.55 Section 18.55 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container...

  15. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Label. 18.55 Section 18.55 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container...

  16. 27 CFR 4.32 - Mandatory label information.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Mandatory label information. 4.32 Section 4.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF WINE Labeling Requirements for Wine § 4.32...

  17. 27 CFR 4.32 - Mandatory label information.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Mandatory label information. 4.32 Section 4.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF WINE Labeling Requirements for Wine § 4.32...

  18. Selective dye-labeling of newly synthesized proteins in bacterial cells.

    PubMed

    Beatty, Kimberly E; Xie, Fang; Wang, Qian; Tirrell, David A

    2005-10-19

    We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell.

  19. Food nutrition labelling practice in China.

    PubMed

    Tao, Yexuan; Li, Ji; Lo, Y Martin; Tang, Qingya; Wang, Youfa

    2011-03-01

    The present study aimed to scrutinize the food nutrition labelling practice in China before the Chinese Food Nutrition Labeling Regulation (CFNLR) era. Nutrition information of pre-packaged foods collected from a supermarket between December 2007 and January 2008 was analysed and compared with findings from a survey conducted in Beijing. Information collected from a supermarket in Shanghai. A total of 850 pre-packaged foods. In the Shanghai survey, the overall labelling rate was 30·9 %, similar to that found in the Beijing study (29·7 %). While only 20·5 % of the snacks in Shanghai had nutrition labelling, the percentage of food items labelled with SFA (8·6 %), trans fatty acid (4·7 %) or fibre (12·1 %) was very low. Of those food items with nutrition labels, a considerable proportion (7-15 %) did not label energy, fat, carbohydrate or protein. Food products manufactured by Taiwan and Hong Kong companies had a lower labelling rate (13·6 %) than those manufactured by domestic (31·6 %) or international manufacturers (33·8 %). The very low food nutrition labelling rate among products sold in large chain supermarkets in major cities of China before CFNLR emphasizes the need for such critical regulations to be implemented in order to reinforce industrial compliance with accurate nutrition labelling.

  20. A label distance maximum-based classifier for multi-label learning.

    PubMed

    Liu, Xiaoli; Bao, Hang; Zhao, Dazhe; Cao, Peng

    2015-01-01

    Multi-label classification is useful in many bioinformatics tasks such as gene function prediction and protein site localization. This paper presents an improved neural network algorithm, Max Label Distance Back Propagation Algorithm for Multi-Label Classification. The method was formulated by modifying the total error function of the standard BP by adding a penalty term, which was realized by maximizing the distance between the positive and negative labels. Extensive experiments were conducted to compare this method against state-of-the-art multi-label methods on three popular bioinformatic benchmark datasets. The results illustrated that this proposed method is more effective for bioinformatic multi-label classification compared to commonly used techniques.

  1. Less label, more free: approaches in label-free quantitative mass spectrometry.

    PubMed

    Neilson, Karlie A; Ali, Naveid A; Muralidharan, Sridevi; Mirzaei, Mehdi; Mariani, Michael; Assadourian, Gariné; Lee, Albert; van Sluyter, Steven C; Haynes, Paul A

    2011-02-01

    In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. 21 CFR 1.382 - What labeling or marking requirements apply to a detained article of food?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 false What labeling or marking requirements apply to a detained article of food? 1.382 Section 1.382 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL GENERAL ENFORCEMENT REGULATIONS Administrative Detention of Food for...

  3. Nutritional information and health warnings on wine labels: Exploring consumer interest and preferences.

    PubMed

    Annunziata, A; Pomarici, E; Vecchio, R; Mariani, A

    2016-11-01

    This paper aims to contribute to the current debate on the inclusion of nutritional information and health warnings on wine labels, exploring consumers' interest and preferences. The results of a survey conducted on a sample of Italian wine consumers (N = 300) show the strong interest of respondents in the inclusion of such information on the label. Conjoint analysis reveals that consumers assign greater utility to health warnings, followed by nutritional information. Cluster analysis shows the existence of three different consumer segments. The first cluster, which included mainly female consumers (over 55) and those with high wine involvement, revealed greater awareness of the links between wine and health and better knowledge of wine nutritional properties, preferring a more detailed nutritional label, such as a panel with GDA%. By contrast, the other two clusters, consisting of individuals who generally find it more difficult to understand nutritional labels, preferred the less detailed label of a glass showing calories. The second and largest cluster comprising mainly younger men (under 44), showed the highest interest in health warnings while the third cluster - with a relatively low level of education - preferred the specification of the number of glasses not to exceed. Our results support the idea that the policy maker should consider introducing a mandatory nutritional label in the easier-to-implement and not-too-costly form of a glass with calories, rotating health warnings and the maximum number of glasses not to exceed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Allergen labelling in meat, dairy and cereal products from the Serbian market

    NASA Astrophysics Data System (ADS)

    Spirić, D.; Nikolić, D.; Ćirić, J.; Janković, S.; Stefanović, S.; Janković, V.; Teodorović, V.

    2017-09-01

    Allergens in food are a great health risk, because of the ratio of severity of problems compared to small amounts of ingested allergen. Since 2014, Serbian producers and importers of food have been obliged to declare allergens from the list of Codex Alimentarius on the product packaging. Surveillance of different meat, diary, and cereal product took place in 2016, with aim of checking if the Serbian regulatory requirements for labelling of allergens in food are being fulfilled. Out of 68 different meat products, 20 were not labelled for allergens. Thirty-six labels of various dairy products were examined revealing that allergen information was included on 27 of them. Only one of eight examined cereal products did not have allergen labelling.

  5. 16 CFR 1633.12 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Labeling. 1633.12 Section 1633.12 Commercial... (OPEN FLAME) OF MATTRESS SETS Rules and Regulations § 1633.12 Labeling. (a) Each mattress set subject to... information (and no other information) in English: (1) Name of the manufacturer, or for imported mattress sets...

  6. 21 CFR 1271.370 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) You must label each HCT/P made available for distribution clearly and accurately. (b) The following information must appear on the HCT/P label: (1) Distinct identification code affixed to the HCT/P container, and assigned in accordance with § 1271.290(c); (2) Description of the type of HCT/P; (3) Expiration...

  7. 21 CFR 1271.370 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) You must label each HCT/P made available for distribution clearly and accurately. (b) The following information must appear on the HCT/P label: (1) Distinct identification code affixed to the HCT/P container, and assigned in accordance with § 1271.290(c); (2) Description of the type of HCT/P; (3) Expiration...

  8. The C(2)1pi(u) state of Na2 molecule studied by polarization labelling spectroscopy method.

    PubMed

    Jastrzebski, W; Kowalczyk, P; Camacho, J J; Pardo, A; Poyato, J M

    2001-08-01

    The C1pi(u) <-- X1sigma(g)+ system of Na2 is studied by the polarization labelling spectroscopy technique. Accurate molecular constants are derived for the observed levels nu = 0-12, J = 12-100 in the C1pi(u) state.

  9. Consumer purchasing patterns in response to calorie labeling legislation in New York City

    PubMed Central

    2011-01-01

    Background Obesity is a major public health threat and policies aimed at curbing this epidemic are emerging. National calorie labeling legislation is forthcoming and requires rigorous evaluation to examine its impact on consumers. The purpose of this study was to examine whether point-of-purchase calorie labels in New York City (NYC) chain restaurants affected food purchasing patterns in a sample of lower income adults in NYC and Newark, NJ. Methods This study utilized a difference-in-difference design to survey 1,170 adult patrons of four popular chain restaurants in NYC and Newark, NJ (which did not introduce labeling) before and after calorie labeling was implemented in NYC. Receipt data were collected and analyzed to examine food and beverage purchases and frequency of fast food consumption. Descriptive statistics were generated, and linear and logistic regression, difference-in-difference analysis, and predicted probabilities were used to analyze the data. Results A difference-in-difference analysis revealed no significant favorable differences and some unfavorable differences in food purchasing patterns and frequency of fast food consumption between adult patrons of fast food restaurants in NYC and Newark, NJ. Adults in NYC who reported noticing and using the calorie labels consumed fast food less frequently compared to adults who did not notice the labels (4.9 vs. 6.6 meals per week, p <0.05). Conclusion While no favorable differences in purchasing as a result of labeling were noted, self-reported use of calorie labels was associated with some favorable behavioral patterns in a subset of adults in NYC. However, overall impact of the legislation may be limited. More research is needed to understand the most effective way to deliver calorie information to consumers. PMID:21619632

  10. Off-label use of vaccines.

    PubMed

    Neels, Pieter; Southern, James; Abramson, Jon; Duclos, Philippe; Hombach, Joachim; Marti, Melanie; Fitzgerald-Husek, Alanna; Fournier-Caruana, Jacqueline; Hanquet, Germaine

    2017-04-25

    This article reviews the off-label recommendations and use of vaccines, and focuses on the differences between the labelled instructions on how to use the vaccine as approved by the regulatory authorities (or "label" 1 ), and the recommendations for use issued by public health advisory bodies at national and international levels. Differences between public health recommendations and the product label regarding the vaccine use can lead to confusion at the level of vaccinators and vaccinees and possibly result in lower compliance with national vaccination schedules. In particular, in many countries, the label may contain regulatory restrictions and warnings against vaccination of specific population groups (e.g. pregnant women) due to a lack of evidence of safety from controlled trials at the time of initial licensure of the vaccine, while public health authorities may recommend the same vaccine for that group, based on additional post-marketing data and benefit risk analyses. We provide an overview of the different responsibilities between regulatory authorities and public health advisory bodies, and the rationale for off-label use 2 of vaccines, the challenges involved based on the impact of off-label use in real-life. We propose to reduce off-label use of vaccines by requiring the manufacturer to regularly adapt the label as much as possible to the public health needs as supported by new evidence. This would require manufacturers to collect and report post-marketing data, communicate them to all stakeholders and regulators to extrapolate existing evidence (when acceptable) to other groups or to other brands of a vaccine (class effect 3 ). Regulatory authorities have a key role to play by requesting additional post-marketing data, e.g. in specific target groups. When public health recommendations for vaccine use that are outside labelled indications are considered necessary, good communication between regulatory bodies, public health authorities, companies and

  11. What good is labeling what's good? A field experimental investigation of parental labeled praise and child compliance.

    PubMed

    Leijten, Patty; Thomaes, Sander; Orobio de Castro, Bram; Dishion, Thomas J; Matthys, Walter

    2016-12-01

    There is a need to identify the "effective ingredients" of evidence-based behavior therapies. We tested the effects of one of the most common ingredients in parenting interventions for preventing disruptive child behavior, referred to as labeled praise (e.g., "well done picking up your toys"), which is typically recommended in preference to unlabeled praise (e.g., "well done"). We compared the effects of labeled praise, unlabeled praise, and no praise on child compliance in two experiments. Experiment 1 included 161 4 to 8 year-old community sample children and tested immediate effects of praise. Experiment 2 included 132 3 to 9 year-old children with varying levels of disruptive behavior and tested immediate and two-week effects of praise. In Experiment 1, teaching parents to use labeled praise did not increase immediate child compliance, whereas teaching them to use unlabeled praise did. In Experiment 2, teaching parents to use labeled praise for two weeks reduced disruptive child behavior, but this effect was of a similar magnitude to that for unlabeled praise. Parents preferred the use of unlabeled over labeled praise. These findings suggest that parental praise promotes child compliance, but the addition of labeling the specific positive behavior may not be of incremental value. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Carbon-13 and carbon-14 labeled dabigatran etexilate and tritium labeled dabigatran.

    PubMed

    Latli, Bachir; Kiesling, Ralf; Aßfalg, Stefan; Chevliakov, Max; Hrapchak, Matt; Campbell, Scot; Gonnella, Nina; Busacca, Carl A; Senanayake, Chris H

    2016-12-01

    Dabigatran etexilate or pradaxa, a novel oral anticoagulant, is a reversible, competitive, direct thrombin inhibitor. It is used to prevent strokes in patients with atrial fibrillation and the formation of blood clots in the veins (deep venous thrombosis) in adults who have had an operation to replace a hip or a knee. Pradaxa is the only novel oral anticoagulant available with both proven superiority to warfarin and a specific reversal agent for use in rare emergency situations. The detailed description of the synthesis of carbon-13 and carbon-14 labeled dabigatran etexilate, and tritium labeled dabigatran is described. The synthesis of carbon-13 dabigatran etexilate was accomplished in eight steps and in 6% overall yield starting from aniline- 13 C 6 . Ethyl bromoacetate-1- 14 C was the reagent of choice in the synthesis of carbon-14 labeled dabigatran etexilate in six steps and 17% overall yield. Tritium labeled dabigatran was prepared using either direct tritium incorporation under Crabtree's catalytic conditions or tritium-dehalogenation of a diiodo-precursor of dabigatran. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Humans make near-optimal adjustments of control to initial body configuration in vertical squat jumping.

    PubMed

    Bobbert, Maarten F; Richard Casius, L J; Kistemaker, Dinant A

    2013-05-01

    We investigated adjustments of control to initial posture in squat jumping. Eleven male subjects jumped from three initial postures: preferred initial posture (PP), a posture in which the trunk was rotated 18° more backward (BP) and a posture in which it was rotated 15° more forward (FP) than in PP. Kinematics, ground reaction forces and electromyograms (EMG) were collected. EMG was rectified and smoothed to obtain smoothed rectified EMG (srEMG). Subjects showed adjustments in srEMG histories, most conspicuously a shift in srEMG-onset of rectus femoris (REC): from early in BP to late in FP. Jumps from the subjects' initial postures were simulated with a musculoskeletal model comprising four segments and six Hill-type muscles, which had muscle stimulation (STIM) over time as input. STIM of each muscle changed from initial to maximal at STIM-onset, and STIM-onsets were optimized using jump height as criterion. Optimal simulated jumps from BP, PP and FP were similar to jumps of the subjects. Optimal solutions primarily differed in STIM-onset of REC: from early in BP to late in FP. Because the subjects' adjustments in srEMG-onsets were similar to adjustments of the model's optimal STIM-onsets, it was concluded that the former were near-optimal. With the model we also showed that near-maximum jumps from BP, PP and FP could be achieved when STIM-onset of REC depended on initial hip joint angle and STIM-onsets of the other muscles were posture-independent. A control theory that relies on a mapping from initial posture to STIM-onsets seems a parsimonious alternative to theories relying on internal optimal control models. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Label-free detection of HIV-1 infected cells via integration of optical tweezers and photoluminescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Lugongolo, Masixole Yvonne; Ombinda-Lemboumba, Saturnin; Noto, Luyanda Lunga; Maaza, Malik; Mthunzi-Kufa, Patience

    2018-02-01

    The human immunodeficiency virus-1 (HIV-1) is currently detected using conventional qualitative and quantitative tests to determine the presence or absence of HIV in blood samples. However, the approach of these tests detects the presence of either viral antibodies or viral RNA that require labelling which may be costly, sophisticated and time consuming. A label-free approach of detecting the presence of HIV is therefore desirable. Of note optical tweezers can be coupled with other technologies including spectroscopy, which also investigates light-matter interactions. For example, coupling of optical tweezers with luminescence spectroscopy techniques has emerged as a powerful tool in biology for micro-manipulation, detection and analysis of individual cells. Integration of optical techniques has enabled studying biological particles in a label-free manner, whilst detecting functional groups and other essential molecules within mixed populations of cells. In the current study, an optical trapping system coupled to luminescence spectroscopy was utilised to detect the presence of HIV infection in TZM-bl cells in vitro. This was performed by infecting TZM-bl cells with the ZM53 HIV-1 pseudovirus, and incubating them for 48 hours prior analysis. The differences between infected and uninfected cells were thereafter displayed as shown by the spectrographs obtained. Combination of these two techniques has a potential in the field of infectious disease diagnostics.

  15. 101 Labeled Brain Images and a Consistent Human Cortical Labeling Protocol

    PubMed Central

    Klein, Arno; Tourville, Jason

    2012-01-01

    We introduce the Mindboggle-101 dataset, the largest and most complete set of free, publicly accessible, manually labeled human brain images. To manually label the macroscopic anatomy in magnetic resonance images of 101 healthy participants, we created a new cortical labeling protocol that relies on robust anatomical landmarks and minimal manual edits after initialization with automated labels. The “Desikan–Killiany–Tourville” (DKT) protocol is intended to improve the ease, consistency, and accuracy of labeling human cortical areas. Given how difficult it is to label brains, the Mindboggle-101 dataset is intended to serve as brain atlases for use in labeling other brains, as a normative dataset to establish morphometric variation in a healthy population for comparison against clinical populations, and contribute to the development, training, testing, and evaluation of automated registration and labeling algorithms. To this end, we also introduce benchmarks for the evaluation of such algorithms by comparing our manual labels with labels automatically generated by probabilistic and multi-atlas registration-based approaches. All data and related software and updated information are available on the http://mindboggle.info/data website. PMID:23227001

  16. Diagnostic labels assigned to patients with orthopedic conditions and the influence of the label on selection of interventions: a qualitative study of orthopaedic clinical specialists.

    PubMed

    Miller-Spoto, Marcia; Gombatto, Sara P

    2014-06-01

    A variety of diagnostic classification systems are used by physical therapists, but little information about how therapists assign diagnostic labels and how the labels are used to direct intervention is available. The purposes of this study were: (1) to examine the diagnostic labels assigned to patient problems by physical therapists who are board-certified Orthopaedic Clinical Specialists (OCSs) and (2) to determine whether the label influences selection of interventions. A cross-sectional survey was conducted. Two written cases were developed for patients with low back and shoulder pain. A survey was used to evaluate the diagnostic label assigned and the interventions considered important for each case. The cases and survey were sent to therapists who are board-certified OCSs. Respondents assigned a diagnostic label and rated the importance of intervention categories for each case. Each diagnostic label was coded based on the construct it represented. Percentage responses for each diagnostic label code and intervention category were calculated. Relative importance of intervention category based on diagnostic label was examined. For the low back pain and shoulder pain cases, respectively, "Combination" (48.5%, 34.9%) and "Pathology/Pathophysiology" (32.7%, 57.3%) diagnostic labels were most common. Strengthening (85.9%, 98.1%), stretching (86.8%, 84.9%), neuromuscular re-education (87.6%, 93.4%), functional training (91.4%, 88.6%), and mobilization/manipulation (85.1%, 86.8%) were considered the most important interventions. Relative importance of interventions did not differ based on diagnostic label (χ2=0.050-1.263, P=.261-.824). The low response rate may limit the generalizability of the findings. Also, examples provided for labels may have influenced responses, and some of the label codes may have represented overlapping constructs. There is little consistency with which OCS therapists assign diagnostic labels, and the label does not seem to influence

  17. Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

    PubMed

    Rash, John E; Kamasawa, Naomi; Davidson, Kimberly G V; Yasumura, Thomas; Pereda, Alberto E; Nagy, James I

    2012-06-01

    Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

  18. Label-free Raman observation of cytochrome c dynamics during apoptosis

    PubMed Central

    Okada, Masaya; Smith, Nicholas Isaac; Palonpon, Almar Flotildes; Endo, Hiromi; Kawata, Satoshi; Sodeoka, Mikiko; Fujita, Katsumasa

    2012-01-01

    We performed label-free observation of molecular dynamics in apoptotic cells by Raman microscopy. Dynamic changes in cytochrome c distribution at the Raman band of 750 cm-1 were observed after adding an apoptosis inducer to the cells. The comparison of mitochondria fluorescence images and Raman images of cytochrome c confirmed that changes in cytochrome c distribution can be distinguished as release of cytochrome c from mitochondria. Our observation also revealed that the redox state of cytochrome c was maintained during the release from the mitochondria. Monitoring mitochondrial membrane potential with JC-1 dye confirmed that the observed cytochrome c release was associated with apoptosis. PMID:22184220

  19. In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

    PubMed

    Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

    2018-04-19

    Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Fluorine-19 Labeling of Stromal Vascular Fraction Cells for Clinical Imaging Applications

    PubMed Central

    Rose, Laura C.; Kadayakkara, Deepak K.; Wang, Guan; Bar-Shir, Amnon; Helfer, Brooke M.; O’Hanlon, Charles F.; Kraitchman, Dara L.; Rodriguez, Ricardo L.

    2015-01-01

    Stromal vascular fraction (SVF) cells are used clinically for various therapeutic targets. The location and persistence of engrafted SVF cells are important parameters for determining treatment failure versus success. We used the GID SVF-1 platform and a clinical protocol to harvest and label SVF cells with the fluorinated (19F) agent CS-1000 as part of a first-in-human phase I trial (clinicaltrials.gov identifier NCT02035085) to track SVF cells with magnetic resonance imaging during treatment of radiation-induced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0% ± 15.8% CD45+, 24.6% ± 12.5% CD34+, and 7.5% ± 3.3% CD31+ cells, with 2.1 ± 0.7 × 105 cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% ± 13.5% of CD34+ progenitor cells compared with 47.8% ± 18.5% of hematopoietic CD45+ cells, with an average of 2.8 ± 2.0 × 1012 19F atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7% ± 5.0%) of CD31+ cells were also labeled, although most coexpressed CD34. Only 16% ± 22.3% of CD45−/CD31−/CD34− (triple-negative) cells were labeled with CS-ATM DM Green. After induction of cell death by either apoptosis or necrosis, >95% of 19F was released from the cells, indicating that fluorine retention can be used as a surrogate marker for cell survival. Labeled-SVF cells engrafted in a silicone breast phantom could be visualized with a clinical 3-Tesla magnetic resonance imaging scanner at a sensitivity of approximately 2 × 106 cells at a depth of 5 mm. The current protocol can be used to image transplanted SVF cells at clinically relevant cell concentrations in patients. Significance Stromal vascular fraction (SVF) cells harvested from adipose tissue offer great promise in regenerative medicine, but methods to track such cell therapies are needed to ensure correct administration and monitor survival. A clinical protocol

  1. 21 CFR 640.70 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Source Plasma § 640.70 Labeling. Link to an amendment published... information shall appear on the label affixed to each container of Source Plasma: (1) The proper name of the... shall follow the proper name in the same size and type of print as the proper name. If the Source Plasma...

  2. Labeling and tracking exosomes within the brain using gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Betzer, Oshra; Perets, Nisim; Barnoy, Eran; Offen, Daniel; Popovtzer, Rachela

    2018-02-01

    Cell-to-cell communication system involves Exosomes, small, membrane-enveloped nanovesicles. Exosomes are evolving as effective therapeutic tools for different pathologies. These extracellular vesicles can bypass biological barriers such as the blood-brain barrier, and can function as powerful nanocarriers for drugs, proteins and gene therapeutics. However, to promote exosomes' therapy development, especially for brain pathologies, a better understanding of their mechanism of action, trafficking, pharmacokinetics and bio-distribution is needed. In this research, we established a new method for non-invasive in-vivo neuroimaging of mesenchymal stem cell (MSC)-derived exosomes, based on computed tomography (CT) imaging with glucose-coated gold nanoparticle (GNP) labeling. We demonstrated that the exosomes were efficiently and directly labeled with GNPs, via an energy-dependent mechanism. Additionally, we found the optimal parameters for exosome labeling and neuroimaging, wherein 5 nm GNPs enhanced labeling, and intranasal administration produced superior brain accumulation. We applied our technique in a mouse model of focal ischemia. Imaging and tracking of intranasally-administered GNP-labeled exosomes revealed specific accumulation and prolonged presence at the lesion area, up to 24 hrs. We propose that this novel exosome labeling and in-vivo neuroimaging technique can serve as a general platform for brain theranostics.

  3. Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection.

    PubMed

    Yan, Weiying; Sloat, Amy L; Yagi, Shigeyuki; Nakazumi, Hiroyuki; Colyer, Christa L

    2006-04-01

    Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.

  4. Optimal design of isotope labeling experiments.

    PubMed

    Yang, Hong; Mandy, Dominic E; Libourel, Igor G L

    2014-01-01

    Stable isotope labeling experiments (ILE) constitute a powerful methodology for estimating metabolic fluxes. An optimal label design for such an experiment is necessary to maximize the precision with which fluxes can be determined. But often, precision gained in the determination of one flux comes at the expense of the precision of other fluxes, and an appropriate label design therefore foremost depends on the question the investigator wants to address. One could liken ILE to shadows that metabolism casts on products. Optimal label design is the placement of the lamp; creating clear shadows for some parts of metabolism and obscuring others.An optimal isotope label design is influenced by: (1) the network structure; (2) the true flux values; (3) the available label measurements; and, (4) commercially available substrates. The first two aspects are dictated by nature and constrain any optimal design. The second two aspects are suitable design parameters. To create an optimal label design, an explicit optimization criterion needs to be formulated. This usually is a property of the flux covariance matrix, which can be augmented by weighting label substrate cost. An optimal design is found by using such a criterion as an objective function for an optimizer. This chapter uses a simple elementary metabolite units (EMU) representation of the TCA cycle to illustrate the process of experimental design of isotope labeled substrates.

  5. [(64) Cu]-labelled trastuzumab: optimisation of labelling by DOTA and NODAGA conjugation and initial evaluation in mice.

    PubMed

    Schjoeth-Eskesen, Christina; Nielsen, Carsten Haagen; Heissel, Søren; Højrup, Peter; Hansen, Paul Robert; Gillings, Nic; Kjaer, Andreas

    2015-05-30

    The human epidermal growth factor receptor-2 (HER2) is overexpressed in 20-30% of all breast cancer cases, leading to increased cell proliferation, growth and migration. The monoclonal antibody, trastuzumab, binds to HER2 and is used for treatment of HER2-positive breast cancer. Trastuzumab has previously been labelled with copper-64 by conjugation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. The aim of this study was to optimise the (64) Cu-labelling of DOTA-trastuzumab and as the first to produce and compare with its 1,4,7-triazacyclononane, 1-glutaric acid-5,7 acetic acid (NODAGA) analogue in a preliminary HER2 tumour mouse model. The chelators were conjugated to trastuzumab using the activated esters DOTA mono-N-hydroxysuccinimide (NHS) and NODAGA-NHS. (64) Cu-labelling of DOTA-trastuzumab was studied by varying the amount of DOTA-trastuzumab used, reaction temperature and time. Full (64) Cu incorporation could be achieved using a minimum of 10-µg DOTA-trastuzumab, but the fastest labelling was obtained after 15 min at room temperature using 25 µg of DOTA-trastuzumab. In comparison, 80% incorporation was achieved for (64) Cu-labelling of NODAGA-trastuzumab. Both [(64) Cu]DOTA-trastuzumab and [(64) Cu]NODAGA-trastuzumab were produced after purification with radiochemical purities of >97%. The tracers were injected into mice with HER2 expressing tumours. The mice were imaged by positron emission tomography and showed high tumour uptake of 3-9% ID/g for both tracers. © 2015 The Authors Journal of Labelled Compounds and Radiopharmaceuticals published by John Wiley & Sons Ltd.

  6. Dynamics of Dengue Virus (DENV)–Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions

    PubMed Central

    Woda, Marcia; Friberg, Heather; Currier, Jeffrey R.; Srikiatkhachorn, Anon; Macareo, Louis R.; Green, Sharone; Jarman, Richard G.; Rothman, Alan L.; Mathew, Anuja

    2016-01-01

    Background. The development of reagents to identify and characterize antigen-specific B cells has been challenging. Methods. We recently developed Alexa Fluor–labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. Results. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV+ class-switched memory B cells (IgD−CD27+ CD19+ cells) reached up to 8% during acute infection and early convalescence. AF DENV–labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38−CD27+) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. Conclusions. AF DENVs reveal changes in the phenotype of DENV serotype–specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination. PMID:27443614

  7. Effects of subthalamic nucleus stimulation on motor cortex plasticity in Parkinson disease

    PubMed Central

    Kim, Sang Jin; Udupa, Kaviraja; Ni, Zhen; Moro, Elena; Gunraj, Carolyn; Mazzella, Filomena; Lozano, Andres M.; Hodaie, Mojgan; Lang, Anthony E.

    2015-01-01

    Objective: We hypothesized that subthalamic nucleus (STN) deep brain stimulation (DBS) will improve long-term potentiation (LTP)-like plasticity in motor cortex in Parkinson disease (PD). Methods: We studied 8 patients with PD treated with STN-DBS and 9 age-matched healthy controls. Patients with PD were studied in 4 sessions in medication (Med) OFF/stimulator (Stim) OFF, Med-OFF/Stim-ON, Med-ON/Stim-OFF, and Med-ON/Stim-ON states in random order. Motor evoked potential amplitude and cortical silent period duration were measured at baseline before paired associated stimulation (PAS) and at 3 different time intervals (T0, T30, T60) up to 60 minutes after PAS in the abductor pollicis brevis and abductor digiti minimi muscles. Results: Motor evoked potential size significantly increased after PAS in controls (+67.7% of baseline at T30) and in patients in the Med-ON/Stim-ON condition (+55.8% of baseline at T30), but not in patients in the Med-OFF/Stim-OFF (−0.4% of baseline at T30), Med-OFF/Stim-ON (+10.3% of baseline at T30), and Med-ON/Stim-OFF conditions (+17.3% of baseline at T30). Cortical silent period duration increased after PAS in controls but not in patients in all test conditions. Conclusions: Our findings suggest that STN-DBS together with dopaminergic medications restore LTP-like plasticity in motor cortex in PD. Restoration of cortical plasticity may be one of the mechanisms of how STN-DBS produces clinical benefit. PMID:26156511

  8. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash

    2004-06-01

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  9. Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

    PubMed Central

    Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

    2009-01-01

    OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

  10. On cordial labeling of double duplication for some families of graph

    NASA Astrophysics Data System (ADS)

    Shobana, L.; Remigius Perpetua Mary, F.

    2018-04-01

    Let G (V, E) be a simple undirected graph where V,E are its vertex set and edge set respectively. Consider a labeling where f bea function from the vertices of G to {0, 1} and for each edge xy assign the label|f(x)-f(y)|. Then f is called cordial of G if the number of vertices labeled 0 and the number of vertices labeled 1 differs by at most 1 and the number of edges labeled 0 and the number of edges labeled 1 differs by at most 1. In this paper we proved the existence of cordial labeling for double duplication of path graph Pn: n≥2, cycle graph Cn: n≥3 except for n≡2 (mod 4), wheel graph Wn:n≥5 except for n≥3 (mod 4), flower graph Fn: n≥5 and bistar graph Bm,n: m,n≥2.

  11. Potent and selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 labeled with carbon-13 and carbon-14.

    PubMed

    Latli, Bachir; Hrapchak, Matt; Savoie, Jolaine; Zhan, Yongda; Busacca, Carl A; Senanayake, Chris H

    2017-07-01

    (S)-6-(2-Hydroxy-2-methylpropyl)-3-((S)-1-(4-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)phenyl)ethyl)-6-phenyl-1,3-oxazinan-2-one (1) and (4aR,9aS)-1-(1H-benzo[d]midazole-5-carbonyl)-2,3,4,4a,9,9a-hexahydro-1-H-indeno[2,1-b]pyridine-6-carbonitrile hydrochloride (2) are potent and selective inhibitor of 11β-hydroxysteroid dehydrogenase type 1 enzyme. These 2 drug candidates developed for the treatment of type-2 diabetes were prepared labeled with carbon-13 and carbon-14 to enable drug metabolism, pharmacokinetics, bioanalytical, and other studies. In the carbon-13 synthesis, benzoic- 13 C 6 acid was converted in 7 steps and in 16% overall yield to [ 13 C 6 ]-(1). Aniline- 13 C 6 was converted in 7 steps to 1H-benzimidazole-1-2,3,4,5,6- 13 C 6 -5-carboxylic acid and then coupled to a tricyclic chiral indenopiperidine to afford [ 13 C 6 ]-(2) in 19% overall yield. The carbon-14 labeled (1) was prepared efficiently in 2 radioactive steps in 41% overall yield from an advanced intermediate using carbon-14 labeled methyl magnesium iodide and Suzuki-Miyaura cross coupling via in situ boronate formation. As for the synthesis of [ 14 C]-(2), 1H-benzimidazole-5-carboxylic- 14 C acid was first prepared in 4 steps using potassium cyanide- 14 C, then coupled to the chiral indenopiperidine using amide bond formation conditions in 26% overall yield. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Anhedonia and amotivation in psychiatric outpatients with fully remitted stimulant use disorder.

    PubMed

    Leventhal, Adam M; Kahler, Christopher W; Ray, Lara A; Stone, Kristen; Young, Diane; Chelminski, Iwona; Zimmerman, Mark

    2008-01-01

    This study evaluated whether psychiatric outpatients with a past stimulant use disorder in full remission for >/= 2 months (STIM+, n = 204) and those with no history of stimulant use disorder (STIM-, n = 2070) differed in the prevalence of current anhedonia and amotivation. Results showed that a significantly greater proportion of STIM+ participants reported anhedonia and amotivation than STIM- participants. The relation between stimulant use disorder history and anhedonia remained robust after controlling for other relevant clinical and demographic factors. These findings suggest that anhedonia may be a preexisting risk factor or protracted effect of stimulant misuse.

  13. (3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

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

    Norman, A.B.; Battaglia, G.; Creese, I.

    1985-12-01

    In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5more » mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.« less

  14. Spin-Label Oximetry at Q- and W-Band

    PubMed Central

    Subczynski, W.K.; Mainali, L.; Camenisch, T.G.; Froncisz, W.; Hyde, J.S.

    2011-01-01

    Spin-lattice relaxation times (T1s) of both small water-soluble spin labels in the aqueous phase as well as lipid-type spin labels in membranes increase when the microwave frequency increases from 2 to 35 GHz (Hyde et al., J. Phys. Chem. B 108 [2004] 9524–9529). The T1 measured at W-band (94 GHz) for the water-soluble spin labels CTPO and TEMPONE (Froncisz et al., J. Magn. Reson. 193 [2008] 297–304) is, however, shorter than when measured at Q-band (35 GHz). In this paper, the decreasing trends at W-band have been confirmed for commonly used lipid-type spin labels in model membranes. It is concluded that the longest values of T1 will generally be found at Q-band, noting that long values are advantageous for measurement of bimolecular collisions with oxygen. The contribution of dissolved molecular oxygen to the relaxation rate was found to be independent of microwave frequency up to 94 GHz for lipid-type spin labels in membranes. This contribution is expressed in terms of the oxygen transport parameter W = T11(Air) − T11(N2), which is a function of both concentration and translational diffusion of oxygen in the local environment of a spin label. The new capabilities in measurement of the oxygen transport parameter using saturation-recovery (SR) EPR at Q- and W-band have been demonstrated in saturated (DMPC) and unsaturated (POPC) lipid bilayer membranes with the use of stearic acid (n-SASL) and phosphatidylcholine (n-PC) spin labels, and compared with results obtained earlier at X-band. SR EPR spin-label oximetry at Q- and W-band has the potential to be a powerful tool for studying samples of small volume, ~30 nL. These benefits, together with other factors such as a higher resonator efficiency parameter and a new technique for canceling free induction decay signals, are discussed. PMID:21277814

  15. Consumer knowledge and attitudes toward nutritional labels.

    PubMed

    Cannoosamy, Komeela; Pugo-Gunsam, Prity; Jeewon, Rajesh

    2014-01-01

    To determine Mauritian consumers' attitudes toward nutritional labels based on the Kano model and to identify determinants of the use and understanding of nutrition labels. The researchers also used a Kano model questionnaire to determine consumers' attitudes toward nutrition labeling. Four hundred consumers residing in Mauritius. Information was elicited via a questionnaire that assessed nutritional knowledge and information about the use and understanding of nutritional labels and demographic factors. Nutritional label use and understanding, nutrition knowledge, and association of demographic factors with label use. Statistical tests performed included 1-way ANOVA and independent samples t tests. Statistically significant relationships (P < .05) were found for nutritional knowledge and nutritional label usage with demographic factors. All demographic factors with the exception of gender were significantly associated (P < .05) with nutritional label understanding. Based on the outcome of the Kano survey, calorie content, trans fat content, protein content, and cholesterol content were found to be must-be attributes: that is, attributes that, when not present, result in consumer dissatisfaction. Age, education, income, household size, and nutrition knowledge had an impact on nutritional label use. Health promoters should aim to increase the use of nutritional labels. Copyright © 2014 Society for Nutrition Education and Behavior. Published by Elsevier Inc. All rights reserved.

  16. Labeling of lectin receptors during the cell cycle.

    PubMed

    Garrido, J

    1976-12-01

    Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.

  17. Carbon-11 labelling of 8[[3-[4-(2-[(11)C]methoxyphenyl)piperazin-1-yl]-2-hydroxypropyl]oxy]thiochroman, a presynaptic 5-HT(1A) receptor agonist, and its in vivo evaluation in anaesthetised rat and in awake cat.

    PubMed

    Zimmer, Luc; Fournet, Guy; Benoît, Joseph; Guillaumet, Gérald; Le Bars, Didier

    2003-07-01

    A new compound, 8[[3-[4-(2-[(11)C]methoxyphenyl)piperazin-1-yl]-2-hydroxypropyl]oxy]thiochroman was labeled via O-methylation with [(11)C]methyl iodide in good yield and specific activity. Original biological evaluations included (i) the study in anesthetized rat with a beta-sensitive intracerebral probe (beta-Microprobe), allowing to measure locally the kinetic of the new PET ligand, and (ii) a PET-scan on a conditioned cat maintained awake during the acquisition. In both in vivo techniques, the new ligand did not reveal any specific binding in hippocampus indicating that this radiotracer is not suitable for mapping 5HT(1A) receptors using positron emission tomography.

  18. Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.

    PubMed

    Woda, Marcia; Friberg, Heather; Currier, Jeffrey R; Srikiatkhachorn, Anon; Macareo, Louis R; Green, Sharone; Jarman, Richard G; Rothman, Alan L; Mathew, Anuja

    2016-10-01

    The development of reagents to identify and characterize antigen-specific B cells has been challenging. We recently developed Alexa Fluor-labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV(+) class-switched memory B cells (IgD(-)CD27(+) CD19(+) cells) reached up to 8% during acute infection and early convalescence. AF DENV-labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38(-)CD27(+)) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. AF DENVs reveal changes in the phenotype of DENV serotype-specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  19. Single Upconversion Nanoparticle-Bacterium Cotrapping for Single-Bacterium Labeling and Analysis.

    PubMed

    Xin, Hongbao; Li, Yuchao; Xu, Dekang; Zhang, Yueli; Chen, Chia-Hung; Li, Baojun

    2017-04-01

    Detecting and analyzing pathogenic bacteria in an effective and reliable manner is crucial for the diagnosis of acute bacterial infection and initial antibiotic therapy. However, the precise labeling and analysis of bacteria at the single-bacterium level are a technical challenge but very important to reveal important details about the heterogeneity of cells and responds to environment. This study demonstrates an optical strategy for single-bacterium labeling and analysis by the cotrapping of single upconversion nanoparticles (UCNPs) and bacteria together. A single UCNP with an average size of ≈120 nm is first optically trapped. Both ends of a single bacterium are then trapped and labeled with single UCNPs emitting green light. The labeled bacterium can be flexibly moved to designated locations for further analysis. Signals from bacteria of different sizes are detected in real time for single-bacterium analysis. This cotrapping method provides a new approach for single-pathogenic-bacterium labeling, detection, and real-time analysis at the single-particle and single-bacterium level. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. The Impact of Standard Nutrition Labels on Alcoholic Beverages

    ERIC Educational Resources Information Center

    Martinez, Julia A.; Dale, Chelsea F.; Fontana, Victoria C.; Collier, Suzanne L.

    2015-01-01

    Whether or not to mandate nutrition labels on alcoholic beverages is a topic of debate. We examined the effect of nutrition labels on (1) plans for drinking and (2) alcohol expectancies. Study 1, n = 80 underage college drinkers responded to an image of a beer with or without a nutrition label. Study 2, n = 98 community drinkers responded to…

  1. Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel Gating Motions

    PubMed Central

    Dellisanti, Cosma D.; Ghosh, Borna; Hanson, Susan M.; Raspanti, James M.; Grant, Valerie A.; Diarra, Gaoussou M.; Schuh, Abby M.; Satyshur, Kenneth; Klug, Candice S.; Czajkowski, Cynthia

    2013-01-01

    Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2–M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2–M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our

  2. 16 CFR 300.11 - Improper methods of labeling.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Improper methods of labeling. 300.11 Section 300.11 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.11 Improper methods of...

  3. 9 CFR 112.10 - Special packaging and labeling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Special packaging and labeling. 112.10... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING § 112.10 Special packaging and labeling. A biological product, which requires special packaging and/or...

  4. GEO Label: User and Producer Perspectives on a Label for Geospatial Data

    NASA Astrophysics Data System (ADS)

    Lush, V.; Lumsden, J.; Masó, J.; Díaz, P.; McCallum, I.

    2012-04-01

    One of the aims of the Science and Technology Committee (STC) of the Group on Earth Observations (GEO) was to establish a GEO Label- a label to certify geospatial datasets and their quality. As proposed, the GEO Label will be used as a value indicator for geospatial data and datasets accessible through the Global Earth Observation System of Systems (GEOSS). It is suggested that the development of such a label will significantly improve user recognition of the quality of geospatial datasets and that its use will help promote trust in datasets that carry the established GEO Label. Furthermore, the GEO Label is seen as an incentive to data providers. At the moment GEOSS contains a large amount of data and is constantly growing. Taking this into account, a GEO Label could assist in searching by providing users with visual cues of dataset quality and possibly relevance; a GEO Label could effectively stand as a decision support mechanism for dataset selection. Currently our project - GeoViQua, - together with EGIDA and ID-03 is undertaking research to define and evaluate the concept of a GEO Label. The development and evaluation process will be carried out in three phases. In phase I we have conducted an online survey (GEO Label Questionnaire) to identify the initial user and producer views on a GEO Label or its potential role. In phase II we will conduct a further study presenting some GEO Label examples that will be based on Phase I. We will elicit feedback on these examples under controlled conditions. In phase III we will create physical prototypes which will be used in a human subject study. The most successful prototypes will then be put forward as potential GEO Label options. At the moment we are in phase I, where we developed an online questionnaire to collect the initial GEO Label requirements and to identify the role that a GEO Label should serve from the user and producer standpoint. The GEO Label Questionnaire consists of generic questions to identify whether

  5. Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

    PubMed

    Swaminathan Iyer, K; Gaikwad, R M; Woodworth, C D; Volkov, D O; Sokolov, Igor

    2012-06-01

    A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p < 0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, premalignant cells.

  6. Physical Labeling of Papillomavirus-Infected, Immortal, and Cancerous Cervical Epithelial Cells Reveal Surface Changes at Immortal Stage

    PubMed Central

    Iyer, K. Swaminathan; Gaikwad, R. M.; Woodworth, C. D.; Volkov, D. O.

    2013-01-01

    A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p <0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, pre-malignant cells. PMID:22351422

  7. 27 CFR 5.32 - Mandatory label information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Distilled Spirits § 5.32 Mandatory label information. There shall be stated: (a) On the brand label: (1) Brand name. (2) Class and type, in accordance with § 5.35. (3) Alcoholic content, in accordance with § 5... prescribed in § 5.47, net contents in accordance with § 5.38(b) or § 5.38a(b)(2). (b) On the brand label or...

  8. Formaldehyde metabolism by Escherichia coli. Carbon and solvent deuterium incorporation into glycerol, 1,2-propanediol, and 1,3-propanediol

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

    Hunter, B.K.; Nicholls, K.M.; Sanders, J.K.

    1985-07-16

    Escherichia coli were grown on 14.3% uniformly TC-labeled glucose as the sole carbon source and challenged anaerobically with 90% TC-labeled formaldehyde. The major multiply labeled metabolites were identified by TC NMR spectroscopy to be glycerol and 1,2-propanediol, and a minor metabolite was shown to be 1,3-propanediol. In each case, formaldehyde is incorporated only into the C1 position. A novel form of TC NMR isotope dilution analysis of the major products reveals that all the 1,2-diol C1 is formaldehyde derived but that about 40% of the glycerol C1 is derived from bacterial sources. Glycerokinase converted the metabolite (1- TC)glycerol to equalmore » amounts of (3- TC)glycerol 3-phosphate and (1- TC)glycerol 3-phosphate, demonstrating that the metabolite is racemic. When ( TC)formaldehyde incubation was carried out in H2O/D2O mixtures, deuterium incorporation was detected by beta- and gamma-isotope shifts. The 1,3-diol is deuterium labeled only at C2 and only once, while the 1,2-diol and glycerol are each labeled independently at both C2 and C3; C3 is multiply labeled. Deuterium incorporation levels are different for each metabolite, indicating that the biosynthetic pathways probably diverge early.« less

  9. 49 CFR 172.402 - Additional labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 30, 2001, such as, a label without the hazard class or division number displayed in the lower corner... this section); and (2)For other than Class 1 or Class 2 materials (for subsidiary labeling requirements for Class 1 or Class 2 materials see paragraph (e) or paragraphs (f) and (g), respectively, of this...

  10. Dynamic map labeling.

    PubMed

    Been, Ken; Daiches, Eli; Yap, Chee

    2006-01-01

    We address the problem of filtering, selecting and placing labels on a dynamic map, which is characterized by continuous zooming and panning capabilities. This consists of two interrelated issues. The first is to avoid label popping and other artifacts that cause confusion and interrupt navigation, and the second is to label at interactive speed. In most formulations the static map labeling problem is NP-hard, and a fast approximation might have O(nlogn) complexity. Even this is too slow during interaction, when the number of labels shown can be several orders of magnitude less than the number in the map. In this paper we introduce a set of desiderata for "consistent" dynamic map labeling, which has qualities desirable for navigation. We develop a new framework for dynamic labeling that achieves the desiderata and allows for fast interactive display by moving all of the selection and placement decisions into the preprocessing phase. This framework is general enough to accommodate a variety of selection and placement algorithms. It does not appear possible to achieve our desiderata using previous frameworks. Prior to this paper, there were no formal models of dynamic maps or of dynamic labels; our paper introduces both. We formulate a general optimization problem for dynamic map labeling and give a solution to a simple version of the problem. The simple version is based on label priorities and a versatile and intuitive class of dynamic label placements we call "invariant point placements". Despite these restrictions, our approach gives a useful and practical solution. Our implementation is incorporated into the G-Vis system which is a full-detail dynamic map of the continental USA. This demo is available through any browser.

  11. Nuclear and Fluorescent Labeled PD-1-Liposome-DOX-64Cu/IRDye800CW Allows Improved Breast Tumor Targeted Imaging and Therapy.

    PubMed

    Du, Yang; Liang, Xiaolong; Li, Yuan; Sun, Ting; Jin, Zhengyu; Xue, Huadan; Tian, Jie

    2017-11-06

    The overexpression of programmed cell death-1 (PD-1) in tumors as breast cancer makes it a possible target for cancer imaging and therapy. Advances in molecular imaging, including radionuclide imaging and near-infrared fluorescence (NIRF) imaging, enable the detection of tumors with high sensitivity. In this study, we aim to develop a novel PD-1 antibody targeted positron emission tomography (PET) and NIRF labeled liposome loaded with doxorubicin (DOX) and evaluate its application for in vivo cancer imaging and therapy. IRDye800CW and 64 Cu were conjugated to liposomes with PD-1 antibody labeling, and DOX was inside the liposomes to form theranostic nanoparticles. The 4T1 tumors were successfully visualized with PD-1-Liposome-DOX- 64 Cu/IRDye800CW using NIRF/PET imaging. The bioluminescent imaging (BLI) results showed that tumor growth was significantly inhibited in the PD-1-Liposome-DOX-treated group than the IgG control. Our results highlight the potential of using dual-labeled theranostic PD-1 mAb-targeted Liposome-DOX- 64 Cu/IRDye800CW for the management of breast tumor.

  12. 10 CFR 34.35 - Labeling, storage, and transportation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 1 2011-01-01 2011-01-01 false Labeling, storage, and transportation. 34.35 Section 34.35... REQUIREMENTS FOR INDUSTRIAL RADIOGRAPHIC OPERATIONS Equipment § 34.35 Labeling, storage, and transportation. (a... changer or the storage container has securely attached to it a durable, legible, and clearly visible label...

  13. 10 CFR 34.35 - Labeling, storage, and transportation.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 1 2012-01-01 2012-01-01 false Labeling, storage, and transportation. 34.35 Section 34.35... REQUIREMENTS FOR INDUSTRIAL RADIOGRAPHIC OPERATIONS Equipment § 34.35 Labeling, storage, and transportation. (a... changer or the storage container has securely attached to it a durable, legible, and clearly visible label...

  14. 42 CFR 84.257 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Labeling requirements. (a) A warning shall be placed on the label of each gas mask, chemical-cartridge... performance of any gas mask, chemical-cartridge respirator, or powered air-purifying respirator approved under... this subpart shall be specified as follows: Chemical-cartridge respirator 1 hour. Gas mask 4 hours...

  15. 47 CFR 15.19 - Labelling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Labelling requirements. 15.19 Section 15.19 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL RADIO FREQUENCY DEVICES General § 15.19 Labelling requirements. (a) In addition to the requirements in part 2 of this chapter, a device subject to certification...

  16. Positron Emission Tomography and Near-Infrared Fluorescence Imaging of Vascular Endothelial Growth Factor with Dual-Labeled Bevacizumab.

    PubMed

    Zhang, Yin; Hong, Hao; Engle, Jonathan W; Yang, Yunan; Barnhart, Todd E; Cai, Weibo

    2012-01-01

    The pivotal role of vascular endothelial growth factor (VEGF) in cancer is underscored by the approval of bevacizumab (Bev, a humanized anti-VEGF monoclonal antibody) for first line treatment of cancer patients. The aim of this study was to develop a dual-labeled Bev for both positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of VEGF. Bev was conjugated to a NIRF dye (i.e. 800CW) and 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) before (64)Cu-labeling. Flow cytometry analysis of U87MG human glioblastoma cells revealed no difference in VEGF binding affinity/specificity between Bev and NOTA-Bev-800CW. (64)Cu-labeling of NOTA-Bev-800CW was achieved with high yield. Serial PET imaging of U87MG tumor-bearing female nude mice revealed that tumor uptake of (64)Cu-NOTA-Bev-800CW was 4.6 ± 0.7, 16.3 ± 1.6, 18.1 ± 1.4 and 20.7 ± 3.7 %ID/g at 4, 24, 48 and 72 h post-injection respectively (n = 4), corroborated by in vivo/ex vivo NIRF imaging and biodistribution studies. Tumor uptake as measured by ex vivo NIRF imaging had a good linear correlation with the %ID/g values obtained from PET (R(2) = 0.93). Blocking experiments and histology both confirmed the VEGF specificity of (64)Cu-NOTA-Bev-800CW. The persistent, prominent, and VEGF-specific uptake of (64)Cu-NOTA-Bev-800CW in the tumor, observed by both PET and NIRF imaging, warrants further investigation and future clinical translation of such Bev-based imaging agents.

  17. PRN 2001-1: First Aid Statements on Pesticide Product Labels

    EPA Pesticide Factsheets

    This PR notice is intended to provide guidance for what the Agency believes is the most updated appropriate first aid language for pesticide product labels to ensure that they continue to adequately protect the public.

  18. Drug Labeling and Exposure in Neonates

    PubMed Central

    Laughon, Matthew M.; Avant, Debbie; Tripathi, Nidhi; Hornik, Christoph P.; Cohen-Wolkowiez, Michael; Clark, Reese H.; Smith, P. Brian; Rodriguez, William

    2014-01-01

    Importance Federal legislation has led to a notable increase in pediatric studies submitted to the Food and Drug Administration (FDA), resulting in new pediatric information in product labeling. However, approximately 50% of drug labels still have insufficient information on safety, efficacy, or dosing in children. Neonatal information in labeling is even scarcer because neonates comprise a vulnerable subpopulation for which end point development is lagging and studies are more challenging. Objective To quantify progress made in neonatal studies and neonatal information in product labeling as result of recent legislation. Design 1. Cohort of neonatal drug studies; and 2. Cohort of infants exposed to these drugs.. Setting 1. Neonatal drug studies: FDA website; 2. National review: infants admitted to a neonatal intensive care unit (NICU) Participants 1) We identified drug studies between 1997 and 2010 that included neonates as a result of pediatric legislation using information available on the FDA website. We determined what studies were published in the medical literature, the legislation responsible for the studies, and the resulting neonatal labeling changes. 2) We then examined the use of these drugs in neonates admitted to 290 NICUs (the Pediatrix Data Warehouse) in the United States from 2005–2010. Exposures Infants exposed to a drug studied in neonates as identified by the FDA website Main outcome measures Number of drug studies with neonates and rate of exposure per 1000 admission among infants admitted to a NICU Results In a review of the FDA databases, we identified 28 drugs studied in neonates and 24 related labeling changes. Forty-one studies encompassed the 28 drugs, and 31 (76%) of these were published. Eleven (46%) of the 24 neonatal labeling changes established safety and effectiveness. In a review of a cohort of 446,335 hospitalized infants, we identified 399 drugs used and 1,525,739 drug exposures in the first 28 postnatal days. Thirteen (46%) of

  19. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or (2...

  20. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or (2...

  1. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or (2...

  2. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) COSMETICS COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in...) Such statement of identity shall be in terms of: (1) The common or usual name of the cosmetic; or (2...

  3. Scanning transmission ion microscopy mass measurements for quantitative trace element analysis within biological samples and validation using atomic force microscopy thickness measurements

    NASA Astrophysics Data System (ADS)

    Devès, Guillaume; Cohen-Bouhacina, Touria; Ortega, Richard

    2004-10-01

    We used the nuclear microprobe techniques, micro-PIXE (particle-induced X-ray emission), micro-RBS (Rutherford backscattering spectrometry) and scanning transmission ion microscopy (STIM) in order to perform the characterization of trace element content and spatial distribution within biological samples (dehydrated cultured cells, tissues). The normalization of PIXE results was usually expressed in terms of sample dry mass as determined by micro-RBS recorded simultaneously to micro-PIXE. However, the main limit of RBS mass measurement is the sample mass loss occurring during irradiation and which could be up to 30% of the initial sample mass. We present here a new methodology for PIXE normalization and quantitative analysis of trace element within biological samples based on dry mass measurement performed by mean of STIM. The validation of STIM cell mass measurements was obtained in comparison with AFM sample thickness measurements. Results indicated the reliability of STIM mass measurement performed on biological samples and suggested that STIM should be performed for PIXE normalization. Further information deriving from direct confrontation of AFM and STIM analysis could as well be obtained, like in situ measurements of cell specific gravity within cells compartment (nucleolus and cytoplasm).

  4. Understanding Food Labels

    MedlinePlus

    ... Healthy eating for girls Understanding food labels Understanding food labels There is lots of info on food ... need to avoid because of food allergies. Other food label terms top In addition to the Nutrition ...

  5. Inter-labeler and intra-labeler variability of condition severity classification models using active and passive learning methods.

    PubMed

    Nissim, Nir; Shahar, Yuval; Elovici, Yuval; Hripcsak, George; Moskovitch, Robert

    2017-09-01

    Labeling instances by domain experts for classification is often time consuming and expensive. To reduce such labeling efforts, we had proposed the application of active learning (AL) methods, introduced our CAESAR-ALE framework for classifying the severity of clinical conditions, and shown its significant reduction of labeling efforts. The use of any of three AL methods (one well known [SVM-Margin], and two that we introduced [Exploitation and Combination_XA]) significantly reduced (by 48% to 64%) condition labeling efforts, compared to standard passive (random instance-selection) SVM learning. Furthermore, our new AL methods achieved maximal accuracy using 12% fewer labeled cases than the SVM-Margin AL method. However, because labelers have varying levels of expertise, a major issue associated with learning methods, and AL methods in particular, is how to best to use the labeling provided by a committee of labelers. First, we wanted to know, based on the labelers' learning curves, whether using AL methods (versus standard passive learning methods) has an effect on the Intra-labeler variability (within the learning curve of each labeler) and inter-labeler variability (among the learning curves of different labelers). Then, we wanted to examine the effect of learning (either passively or actively) from the labels created by the majority consensus of a group of labelers. We used our CAESAR-ALE framework for classifying the severity of clinical conditions, the three AL methods and the passive learning method, as mentioned above, to induce the classifications models. We used a dataset of 516 clinical conditions and their severity labeling, represented by features aggregated from the medical records of 1.9 million patients treated at Columbia University Medical Center. We analyzed the variance of the classification performance within (intra-labeler), and especially among (inter-labeler) the classification models that were induced by using the labels provided by seven

  6. Abandoning a label doesn’t make it disappear: The perseverance of labeling effects

    PubMed Central

    Foroni, Francesco; Rothbart, Myron

    2012-01-01

    Labels exert strong influence on perception and judgment. The present experiment examines the possibility that such effects may persist even when labels are abandoned. Participants judged the similarity of pairs of silhouette drawings of female body types, ordered on a continuum from very thin to very heavy, under conditions where category labels were, and were not, superimposed on the ordered stimuli. Consistent with earlier research, labels had strong effects on perceived similarity, with silhouettes sharing the same label judged as more similar than those having different labels. Moreover, when the labels were removed and no longer present, the effect of the labels, although diminished, persisted. It did not make any difference whether the labels were simply abandoned or, in addition, had their validity challenged. The results are important for our understanding of categorization and labeling processes. The potential theoretical and practical implications of these results for social processes are discussed. PMID:23105148

  7. 7 CFR 201.31a - Labeling treated seed.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... been treated shall be labeled in type no smaller than 8 point to indicate that the seed has been... labeling all types of mercurials. Examples of commonly accepted abbreviated chemical names are: BHC (1, 2... the size of the type used for information required to be on the label under paragraph (a) and shall...

  8. 47 CFR 15.19 - Labelling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false Labelling requirements. 15.19 Section 15.19 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL RADIO FREQUENCY DEVICES General § 15.19 Labelling requirements. (a) In addition to the requirements in part 2 of this chapter, a device subject to certification, or verification shall be...

  9. Isotope-Labeled Composition B for Tracing Detonation Signatures

    NASA Astrophysics Data System (ADS)

    Manner, Virginia; Podlesak, David; Huber, Rachel; Amato, Ronald; Giambra, Anna; Bowden, Patrick; Hartline, Ernest; Dattelbaum, Dana

    2017-06-01

    To better understand how solid carbon forms and evolves during detonation, we have prepared Composition B with 13 C and 15 N-labeled 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) and 2,4,6-trinitrotoluene (TNT) in order to trace the formation of soot from the carbon and nitrogen atoms in these explosives. Isotope-labeling of explosives has been performed in the recent past for a variety of reasons, including environmental remediation and reaction mechanism studies. Because it is expensive and time consuming to prepare these materials, and our detection equipment only requires trace amounts of isotopes, we have prepared fully-labeled materials and substituted them into unlabeled RDX and TNT at less than the 1% level. We will discuss the preparation and full characterization of this labeled Composition B, the detonation tests performed, along with the results of the post-detonation soot analysis. Various detonation models predict differing amounts and forms of carbon and nitrogen; these isotopically-labeled precursors have allowed these models to be tested.

  10. Noncharged and Charged Monodendronised Perylene Bisimides as Highly Fluorescent Labels and their Bioconjugates.

    PubMed

    Huth, Katharina; Heek, Timm; Achazi, Katharina; Kühne, Christian; Urner, Leonhard H; Pagel, Kevin; Dernedde, Jens; Haag, Rainer

    2017-04-06

    A series of water-soluble, hydroxylated and sulphated, polyglycerol (PG) dendronised, monofunctional perylene bisimides (PBIs) were synthesised in three generations. Their photophysical properties were determined by absorption and emission spectroscopy and their suitability as potential biolabels examined by biological in vitro studies after bioconjugation. It could be shown that the photophysical properties of the PBI labels can be improved by increasing the sterical demand and ionic charge of the attached dendron. Thereby, charged labels show superior suppression of aggregation over charge neutral labels owing to electrostatic repulsion forces on the PG-dendron. The ionic charges also enabled a reduction in dendron generation while retaining the labels' outstanding fluorescence quantum yields (FQYs) up to 100 %. These core-unsubstituted perylene derivatives were successfully applied as fluorescent labels upon bioconjugation to the therapeutic antibody cetuximab. The dye-antibody conjugates showed a strongly enhanced aggregation tendency compared to the corresponding free dyes. Biological evaluation by receptor-binding, cellular uptake, and cytotoxicity studies revealed that labelling did not affect the antibody's function, which renders the noncharged and charged dendronised PBIs suitable candidates as fluorescent labels in biological imaging. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A novel broad specificity fucosidase capable of core α1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes.

    PubMed

    Vainauskas, Saulius; Kirk, Charlotte H; Petralia, Laudine; Guthrie, Ellen P; McLeod, Elizabeth; Bielik, Alicia; Luebbers, Alex; Foster, Jeremy M; Hokke, Cornelis H; Rudd, Pauline M; Shi, Xiaofeng; Taron, Christopher H

    2018-06-22

    Exoglycosidases are often used for detailed characterization of glycan structures. Bovine kidney α-fucosidase is commonly used to determine the presence of core α1-6 fucose on N-glycans, an important modification of glycoproteins. Recently, several studies have reported that removal of core α1-6-linked fucose from N-glycans labeled with the reactive N-hydroxysuccinimide carbamate fluorescent labels 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) and RapiFluor-MS is severely impeded. We report here the cloning, expression and biochemical characterization of an α-fucosidase from Omnitrophica bacterium (termed fucosidase O). We show that fucosidase O can efficiently remove α1-6- and α1-3-linked core fucose from N-glycans. Additionally, we demonstrate that fucosidase O is able to efficiently hydrolyze core α1-6-linked fucose from N-glycans labeled with any of the existing NHS-carbamate activated fluorescent dyes.

  12. Inter-Labeler and Intra-Labeler Variability of Condition Severity Classification Models Using Active and Passive Learning Methods

    PubMed Central

    Nissim, Nir; Shahar, Yuval; Boland, Mary Regina; Tatonetti, Nicholas P; Elovici, Yuval; Hripcsak, George; Moskovitch, Robert

    2018-01-01

    Background and Objectives Labeling instances by domain experts for classification is often time consuming and expensive. To reduce such labeling efforts, we had proposed the application of active learning (AL) methods, introduced our CAESAR-ALE framework for classifying the severity of clinical conditions, and shown its significant reduction of labeling efforts. The use of any of three AL methods (one well known [SVM-Margin], and two that we introduced [Exploitation and Combination_XA]) significantly reduced (by 48% to 64%) condition labeling efforts, compared to standard passive (random instance-selection) SVM learning. Furthermore, our new AL methods achieved maximal accuracy using 12% fewer labeled cases than the SVM-Margin AL method. However, because labelers have varying levels of expertise, a major issue associated with learning methods, and AL methods in particular, is how to best to use the labeling provided by a committee of labelers. First, we wanted to know, based on the labelers’ learning curves, whether using AL methods (versus standard passive learning methods) has an effect on the Intra-labeler variability (within the learning curve of each labeler) and inter-labeler variability (among the learning curves of different labelers). Then, we wanted to examine the effect of learning (either passively or actively) from the labels created by the majority consensus of a group of labelers. Methods We used our CAESAR-ALE framework for classifying the severity of clinical conditions, the three AL methods and the passive learning method, as mentioned above, to induce the classifications models. We used a dataset of 516 clinical conditions and their severity labeling, represented by features aggregated from the medical records of 1.9 million patients treated at Columbia University Medical Center. We analyzed the variance of the classification performance within (intra-labeler), and especially among (inter-labeler) the classification models that were induced by

  13. RNA synthesis in the pancreatic acinar cells of aging mice as revealed by electron microscopic radioautography.

    PubMed

    Nagata, Tetsuji

    2012-01-01

    For the purpose of studying the aging changes of macromolecular synthesis in the pancreatic acinar cells of experimental animals, we studied 10 groups of aging mice during development and aging from fetal day 19 to postnatal month 24. They were injected with 3H-uridine, a precursor for RNA synthesis, sacrificed and the pancreatic tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating RNA synthesis in pancreatic acinar cells in respective aging groups were analyzed qualitatively. The number of mitochondria per cell, the number of labeled mitochondria with silver grains and the number of silver grains in each cell in respective aging groups were analyzed quantitatively in relation to the aging of animals. The results revealed that the RNA synthetic activity as expressed by the incorporations of RNA precursor, i.e., the number of silver grains in cell nuclei, cell organelles, changed due to the aging of animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each pancreatic acinar cell. It was demonstrated that the number of mitochondria, the number of labeled mitochondria and the labeling indices showing RNA synthesis at various ages increased from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile stage at postnatal year 1 to 2, indicating the aging changes. Based upon our findings, available literature on macromolecular synthesis in mitochondria of various cells are reviewed.

  14. Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.

    PubMed

    Basu, Sankha S; Mesaros, Clementina; Gelhaus, Stacy L; Blair, Ian A

    2011-02-15

    Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

  15. Dynamic Labeling Reveals Temporal Changes in Carbon Re-Allocation within the Central Metabolism of Developing Apple Fruit

    PubMed Central

    Beshir, Wasiye F.; Mbong, Victor B. M.; Hertog, Maarten L. A. T. M.; Geeraerd, Annemie H.; Van den Ende, Wim; Nicolaï, Bart M.

    2017-01-01

    In recent years, the application of isotopically labeled substrates has received extensive attention in plant physiology. Measuring the propagation of the label through metabolic networks may provide information on carbon allocation in sink fruit during fruit development. In this research, gas chromatography coupled to mass spectrometry based metabolite profiling was used to characterize the changing metabolic pool sizes in developing apple fruit at five growth stages (30, 58, 93, 121, and 149 days after full bloom) using 13C-isotope feeding experiments on hypanthium tissue discs. Following the feeding of [U-13C]glucose, the 13C-label was incorporated into the various metabolites to different degrees depending on incubation time, metabolic pathway activity, and growth stage. Evidence is presented that early in fruit development the utilization of the imported sugars was faster than in later developmental stages, likely to supply the energy and carbon skeletons required for cell division and fruit growth. The declined 13C-incorporation into various metabolites during growth and maturation can be associated with the reduced metabolic activity, as mirrored by the respiratory rate. Moreover, the concentration of fructose and sucrose increased during fruit development, whereas concentrations of most amino and organic acids and polyphenols declined. In general, this study showed that the imported compounds play a central role not only in carbohydrate metabolism, but also in the biosynthesis of amino acid and related protein synthesis and secondary metabolites at the early stage of fruit development. PMID:29093725

  16. Blood specimen labelling errors: Implications for nephrology nursing practice.

    PubMed

    Duteau, Jennifer

    2014-01-01

    Patient safety is the foundation of high-quality health care, as recognized both nationally and worldwide. Patient blood specimen identification is critical in ensuring the delivery of safe and appropriate care. The practice of nephrology nursing involves frequent patient blood specimen withdrawals to treat and monitor kidney disease. A critical review of the literature reveals that incorrect patient identification is one of the major causes of blood specimen labelling errors. Misidentified samples create a serious risk to patient safety leading to multiple specimen withdrawals, delay in diagnosis, misdiagnosis, incorrect treatment, transfusion reactions, increased length of stay and other negative patient outcomes. Barcode technology has been identified as a preferred method for positive patient identification leading to a definitive decrease in blood specimen labelling errors by as much as 83% (Askeland, et al., 2008). The use of a root cause analysis followed by an action plan is one approach to decreasing the occurrence of blood specimen labelling errors. This article will present a review of the evidence-based literature surrounding blood specimen labelling errors, followed by author recommendations for completing a root cause analysis and action plan. A failure modes and effects analysis (FMEA) will be presented as one method to determine root cause, followed by the Ottawa Model of Research Use (OMRU) as a framework for implementation of strategies to reduce blood specimen labelling errors.

  17. Multimodal Molecular Imaging Reveals High Target Uptake and Specificity of 111In- and 68Ga-Labeled Fibrin-Binding Probes for Thrombus Detection in Rats.

    PubMed

    Oliveira, Bruno L; Blasi, Francesco; Rietz, Tyson A; Rotile, Nicholas J; Day, Helen; Caravan, Peter

    2015-10-01

    We recently showed the high target specificity and favorable imaging properties of 64Cu and Al18F PET probes for noninvasive imaging of thrombosis. Here, our aim was to evaluate new derivatives labeled with either with 68Ga, 111In, or 99mTc as thrombus imaging agents for PET and SPECT. In this study, the feasibility and potential of these probes for thrombus imaging was assessed in detail in 2 animal models of arterial thrombosis. The specificity of the probes was further evaluated using a triple-isotope approach with multimodal SPECT/PET/CT imaging. Radiotracers were synthesized using a known fibrin-binding peptide conjugated to 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid monoamide (DOTA-MA), or a diethylenetriamine ligand (DETA-propanoic acid [PA]), followed by labeling with 68Ga (FBP14, 68Ga-NODAGA), 111In (FBP15, 111In-DOTA-MA), or 99mTc (FBP16, 99mTc(CO)3-DETA-PA), respectively. PET or SPECT imaging, biodistribution, pharmacokinetics, and metabolic stability were evaluated in rat models of mural and occlusive carotid artery thrombosis. In vivo target specificity was evaluated by comparing the distribution of the SPECT and PET probes with preformed 125I-labeled thrombi and with a nonbinding control probe using SPECT/PET/CT imaging. All 3 radiotracers showed affinity similar to soluble fibrin fragment DD(E) (inhibition constant=0.53-0.83 μM). After the kidneys, the highest uptake of 68Ga-FBP14 and 111In-FBP15 was in the thrombus (1.0±0.2 percentage injected dose per gram), with low off-target accumulation. Both radiotracers underwent fast systemic elimination (half-life, 8-15 min) through the kidneys, which led to highly conspicuous thrombi on PET and SPECT images. 99mTc-FBP16 displayed low target uptake and distribution consistent with aggregation or degradation. Triple-isotope imaging experiments showed that both 68Ga-FBP14 and 111In-FBP15, but not the nonbinding derivative 64Cu

  18. Label-free and live cell imaging by interferometric scattering microscopy.

    PubMed

    Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

    2018-03-14

    Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

  19. Store-operated Ca{sup 2+} entry in rhabdomyosarcoma cells

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

    Schmid, Evi, E-mail: Evi.Schmid@med.uni-tuebingen.de; Stagno, Matias Julian; Yan, Jing

    Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an intrinsic or early-acquisition of resistance to chemo- and radiation therapy. Molecular determinants pivotal for RMS migration, metastatic invasion, cell proliferation, and survival are incompletely identified. Migration and cell proliferation were shown to correlate with cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}). Store-operated Ca{sup 2+}-entry (SOCE) that increases intracellular [Ca{sup 2+}] is accomplished by Orai1, a pore-forming ion channel unit, the expression of which is stimulated by the transcription factor NFκB. The present study explored the expression of Orai1 and its regulators STIM1 and NFκB in human rhabdomyosarcoma cell linesmore » and analyzed their impact on cell proliferation and migration. For the study human rhabdomyosarcoma cell lines RD (embryonal) and RH30 (alveolar) were analyzed for Orai1, STIM1, and NFκB transcription by RT-PCR and their corresponding proteins in Western blot. [Ca{sup 2+}]{sub i} was detected via Fura-2 fluorescence and SOCE – resulting from [Ca{sup 2+}]{sub i} increase following store depletion with extracellular Ca{sup 2+} removal and inhibition of the sarcoendoplasmatic reticular Ca{sup 2+} ATPase – detected with thapsigargin. Cell migration was analyzed in transwell and mitotic cell death with the clonogenic assay. In summary, Orai1, STIM1, and NFκB are expressed in embryonal (RD) and alveolar (RH30) rhabdomyosarcoma. SOCE inhibitor BTP2, Orai1 inhibitor 2-APB, or NFκB inhibitor wogonin virtually abrogated (BTP2, 2-APB) or significantly reduced (wogonin) SOCE. Moreover, SOCE inhibitors 2-APB and BTP2 and wogonin significantly inhibited migration and proliferation of both, RD and RH30 cells. These results suggest that Orai1 signaling is involved in SOCE into rhabdomyosarcoma cells thus contributing to migration, invasion and proliferation. - Highlights: • Orai1, STIM1, and NFκB are expressed in RD and RH30

  20. Introduction to Pesticide Labels

    EPA Pesticide Factsheets

    Pesticide product labels provide critical information about how to safely and legally handle and use pesticide products. Unlike most other types of product labels, pesticide labels are legally enforceable. Learn about pesticide product labels.

  1. Restaurant menu labelling: Is it worth adding sodium to the label?

    PubMed

    Scourboutakos, Mary J; Corey, Paul N; Mendoza, Julio; Henson, Spencer J; L'Abbe, Mary R

    2014-07-31

    Several provincial and federal bills have recommended various forms of menu labelling that would require information beyond just calories; however, the additional benefit of including sodium information is unknown. The objective of this study was to determine whether sodium information on menus helps consumers make lower-sodium choices and to understand what other factors influence the effect of menu labelling on consumers' meal choices. A total of 3,080 Canadian consumers completed an online survey that included a repeated measures experiment in which consumers were asked to select what they would typically order from four mock-restaurant menus. Subsequently, consumers were randomly allocated to see one of three menu-labelling treatments (calories; calories and sodium; or calories, sodium and serving size) and were given the option to change their order. There was a significant difference in the proportion of consumers who changed their order, varying from 17% to 30%, depending on the restaurant type. After participants had seen menu labelling, sodium levels decreased in all treatments (p<0.0001). However, in three of the four restaurant types, consumers who saw calorie and sodium information ordered meals with significantly less sodium than consumers who saw only calorie information (p<0.01). Consumers who saw sodium labelling decreased the sodium level of their meal by an average of 171-384 mg, depending on the restaurant. In the subset of consumers who saw sodium information and chose to change their order, sodium levels decreased by an average of 681-1,360 mg, depending on the restaurant. Sex, intent to lose weight and the amount of calories ordered at baseline were the most important predictors of who used menu labelling. Eighty percent of survey panelists wanted to see nutrition information when dining out. Including sodium information alongside calorie information may result in a larger decrease in the amount of sodium ordered by restaurant-goers.

  2. Advancing understanding of affect labeling with dynamic causal modeling

    PubMed Central

    Torrisi, Salvatore J.; Lieberman, Matthew D.; Bookheimer, Susan Y.; Altshuler, Lori L.

    2013-01-01

    Mechanistic understandings of forms of incidental emotion regulation have implications for basic and translational research in the affective sciences. In this study we applied Dynamic Causal Modeling (DCM) for fMRI to a common paradigm of labeling facial affect to elucidate prefrontal to subcortical influences. Four brain regions were used to model affect labeling, including right ventrolateral prefrontal cortex (vlPFC), amygdala and Broca’s area. 64 models were compared, for each of 45 healthy subjects. Family level inference split the model space to a likely driving input and Bayesian Model Selection within the winning family of 32 models revealed a strong pattern of endogenous network connectivity. Modulatory effects of labeling were most prominently observed following Bayesian Model Averaging, with the dampening influence on amygdala originating from Broca’s area but much more strongly from right vlPFC. These results solidify and extend previous correlation and regression-based estimations of negative corticolimbic coupling. PMID:23774393

  3. 16 CFR 300.5 - Required label and method of affixing.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Required label and method of affixing. 300.5 Section 300.5 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.5 Required label and...

  4. 40 CFR 85.530 - Vehicle/engine labels and packaging labels.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... PROGRAMS (CONTINUED) CONTROL OF AIR POLLUTION FROM MOBILE SOURCES Exemption of Clean Alternative Fuel... from the tampering prohibition: (1) You must make a supplemental emission control information label for..., consistent with the requirements of this subpart. You may do this by identifying the OEM test group/engine...

  5. 40 CFR 85.530 - Vehicle/engine labels and packaging labels.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... PROGRAMS (CONTINUED) CONTROL OF AIR POLLUTION FROM MOBILE SOURCES Exemption of Clean Alternative Fuel... from the tampering prohibition: (1) You must make a supplemental emission control information label for..., consistent with the requirements of this subpart. You may do this by identifying the OEM test group/engine...

  6. Evaluation of 99mTc labeled diadenosine tetraphosphate as an atherosclerotic plaque imaging agent in experimental models.

    PubMed

    Cao, Wei; Zhang, Yongxue; An, Rui

    2006-01-01

    The potential of 99mTc labeled P1, P4-di (adenosine-5')-tetraphosphate (Ap4A) for imaging experimental atherosclerotic plaques was evaluated in New Zealand white (NZW) rabbits. To label the 99mTc to Ap4A, stannous tartrate solution was used. 99mTc-Ap4A was purified on a Sephadex G-25 column. The radiochemistry purities of 99mTc-Ap4A were 85% to 91%. Biodistribution study revealed 99mTc-Ap4A cleared from blood rapidly. Thirty min after 99mTc-Ap4A administrated on NZW atherosclerotic rabbits, lesion to blood (target/blood, T/B) ratio was 3.17 +/- 1.27, and lesions to normal (target/non-target, T/NT) ratio was 5.23 +/- 1.87. Shadows of atherosclerotic plaques were clearly visible on radioautographic film. Aortas with atherosclerotic plaques also could be seen on ex vivo gamma camera images. Atherosclerotic abdominal aortas were clearly visible on in vivo images 15 min to 3 h after 99mTc-Ap4A administration. 99mTc-labeled Ap4A can be used for rapid noninvasive detection of experimental atherosclerotic plaque.

  7. 49 CFR 172.405 - Authorized label modifications.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... appear on the label. (c) For a package containing a Division 6.1, Packing Group III material, the POISON label specified in § 172.430 may be modified to display the text “PG III” instead of “POISON” or “TOXIC...

  8. 49 CFR 172.405 - Authorized label modifications.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... appear on the label. (c) For a package containing a Division 6.1, Packing Group III material, the POISON label specified in § 172.430 may be modified to display the text “PG III” instead of “POISON” or “TOXIC...

  9. 49 CFR 172.405 - Authorized label modifications.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... appear on the label. (c) For a package containing a Division 6.1, Packing Group III material, the POISON label specified in § 172.430 may be modified to display the text “PG III” instead of “POISON” or “TOXIC...

  10. 49 CFR 172.405 - Authorized label modifications.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... appear on the label. (c) For a package containing a Division 6.1, Packing Group III material, the POISON label specified in § 172.430 may be modified to display the text “PG III” instead of “POISON” or “TOXIC...

  11. Predicament of Chinese legislation on genetically modified food (GMF) labeling management and solutions - from the perspective of the new food safety law.

    PubMed

    Li, Wei; Li, Han

    2017-11-01

    This paper considers the background of Article 69 of the newly revised Food Safety Law in China in combination with the current situation of Chinese legislation on GMF labeling management, compared with a foreign genetically modified food labeling management system, revealing deficiencies in the Chinese legislation with respect to GMF labeling management, and noting that institutions should properly consider the GMF labeling management system in China. China adheres to the principle of mandatory labeling based on both product and processes in relation to GMFs and implements a system of process-centered mandatory labeling under a negotiation-construction form. However, China has not finally defined the supervision mode of mandatory labeling of GMFs through laws, and this remains a challenge for GMF labeling management when two mandatory labeling modes coexist. Since April 2015 and October 1, 2015 when the Food Safety Law was revised and formally implemented respectively, the applicable judicial interpretations and enforcement regulations have not made applicable revisions and only principle-based terms have been included in the Food Safety Law, it is still theoretically and practically difficult for mandatory labeling of GMFs in juridical practices and conflicts between the principle of GMF labeling and the purpose that safeguards consumers' right to know remain. The GMF labeling system should be legislatively and practically improved to an extent that protects consumers' right to know. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  12. Increased cholecystokinin labeling in the hippocampus of a mouse model of epilepsy maps to spines and glutamatergic terminals

    PubMed Central

    Wyeth, Megan S.; Zhang, Nianhui; Houser, Carolyn R.

    2011-01-01

    The neuropeptide cholecystokinin (CCK) is abundant in the central nervous system and expressed in a subset of inhibitory interneurons, particularly in their axon terminals. The expression profile of CCK undergoes numerous changes in several models of temporal lobe epilepsy. Previous studies in the pilocarpine model of epilepsy have shown that CCK immunohistochemical labeling is substantially reduced in several regions of the hippocampal formation, consistent with decreased CCK expression as well as selective neuronal degeneration. However, in a mouse pilocarpine model of recurrent seizures, increases in CCK-labeling also occur and are especially striking in the hippocampal dendritic layers of strata oriens and radiatum. Characterizing these changes and determining the cellular basis of the increased labeling were the major goals of the current study. One possibility was that the enhanced CCK labeling could be associated with an increase in GABAergic terminals within these regions. However, in contrast to the marked increase in CCK-labeled structures, labeling of GABAergic axon terminals was decreased in the dendritic layers. Likewise, cannabinoid receptor 1-labeled axon terminals, many of which are CCK-containing GABAergic terminals, were also decreased. These findings suggested that the enhanced CCK labeling was not due to an increase in GABAergic axon terminals. The subcellular localization of CCK immunoreactivity was then examined using electron microscopy, and the identities of the structures that formed synaptic contacts were determined. In pilocarpine-treated mice, CCK was observed in dendritic spines and these were proportionally increased relative to controls, whereas the proportion of CCK-labeled terminals forming symmetric synapses was decreased. In addition, CCK-positive axon terminals forming asymmetric synapses were readily observed in these mice. Double labeling with vesicular glutamate transporter 1 and CCK revealed co-localization in numerous

  13. Specific lysine labeling by 18OH- during alkaline cleavage of the alpha-1-antitrypsin-trypsin complex.

    PubMed Central

    Cohen, A B; Gruenke, L D; Craig, J C; Geczy, D

    1977-01-01

    alpha-1-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the alpha-1-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the alpha-1-antitrypsin-trypsin complex can be split at high pH, releasing a component of alpha-1-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the alpha-1-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 18OH-. When the new carboxy-terminal lysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the alpha-1-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on alpha-1-antitrypsin bind covalently to each enzyme. PMID:303770

  14. Person Perception and Verbal Labeling: The Development of Social Labels.

    ERIC Educational Resources Information Center

    Brooks-Gunn, Jeanne; Lewis, Michael

    This study examined the social labels which are first used by infants, social differentiation on the basis of labeling behavior, and overgeneralization of social labels. Subjects were 81 infants from 9 to 36 months of age. The 9- to 24-month-olds were shown slides of themselves, their mothers, their fathers, and unfamiliar children, babies, and…

  15. Photoaffinity labeling of the human erythrocyte glucose transporter with /sup 4/H-labelled forskolin

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

    Shanahan, M.F.; Edwards, B.M.; Morris, D.P.

    1986-05-01

    Forskolin, a potent activator of adenylate cyclase, is also known to inhibit glucose transport in a number of cells. The authors have investigated photoincorporation of (/sup 3/H)forskolin into erythrocyte membrane proteins using a technique they previously developed for photolabeling the erythrocyte glucose transporter with cytochalasin B (CB). A 30-40s irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into all of the major membrane protein bands. However, most of the incorporation occurred in only three regions of the gel. Peak 1 was a sharp peak near the top of the gelmore » in the region corresponding to spectrin, peak 2 appeared to be associated with band 3 (approx. 90kDa), and the third region labeled was between 41-60 kDa which corresponds to the region of the glucose transporter. This region appeared to contain several overlapping peaks with the largest incorporation of label occurring around 45 kDa in the area of red cell actin. When photolabeling was performed in the presence of 400 ..mu..M cytochalasin B (8.0 ..mu..M forskolin) the labeling in the 41-60 kDa region was totally inhibited while labeling of the 90 kDa peak was partially blocked. CB had no effect on the photolabeling of peak 1 by forskolin.« less

  16. Specific coupling between the 13-keto carbonyl and chlorin skeletal vibrational modes of synthetic 13 1- 18O-(un)labelled metallochlorophyll derivatives

    NASA Astrophysics Data System (ADS)

    Morishita, Hidetada; Tamiaki, Hitoshi

    2009-03-01

    Metal complexes of methyl 13 1- 18O-labelled pyropheophorbide- a1-M- 18O (M = Zn, Cu and Ni) were prepared by metallation of the 18O-labelled free base ( 1- 18O) and 18O-labelling of unlabelled nickel complex ( 1-Ni). The FT-IR spectra of 1-Zn and 1-Zn- 18O in CH 2Cl 2 showed that the 13-keto carbonyl stretching vibration mode moved to about a 30-cm -1 lower wavenumber by 18O-labelling of the 13 1-oxo moiety. In 1-Cu- 18O and 1-Ni- 18O, the 13-C dbnd 18O stretching modes were close to the highest-energy wavenumber mode of chlorin skeletal C-C/C-N vibrations at around 1650 cm -1 and they were coupled in CH 2Cl 2 to give two split IR bands (Fermi resonance). A similar coupling was observed in the resonance Raman scattering of 1-Ni- 18O in the solid state. The hydrogen-bonded 13-C dbnd 16O vibration mode of 1-Ni similarly coupled with the skeletal C-C/C-N mode in CCl 4 containing 1% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, while such a coupling was not observed in a neat CCl 4 solution of 1-Ni possessing the 13-C dbnd 16O free from any interaction. The skeletal C-C/C-N band selectively coupled with the 13-C dbnd O, not with the 3-C dbnd O, when the difference in their peak maxima was less than 20 cm -1.

  17. Effectiveness of cigarette warning labels: examining the impact of graphics, message framing, and temporal framing.

    PubMed

    Nan, Xiaoli; Zhao, Xiaoquan; Yang, Bo; Iles, Irina

    2015-01-01

    This study examines the effectiveness of cigarette warning labels, with a specific focus on the impact of graphics, message framing (gain vs. loss), and temporal framing (present-oriented vs. future-oriented) among nonsmokers in the United States. A controlled experiment (N = 253) revealed that graphic warning labels were perceived as more effective, stronger in argument strength, and were generally liked more compared to text-only labels. In addition, loss-framed labels, compared to their gain-framed counterparts, were rated higher in perceived effectiveness, argument strength, and liking. No significant difference was observed between the present- and future-oriented frames on any of the dependent variables. Implications of the findings for antismoking communication efforts are discussed.

  18. Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs.

    PubMed

    Chitneni, Satish K; Reitman, Zachary J; Gooden, David M; Yan, Hai; Zalutsky, Michael R

    2016-08-25

    Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). A small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled with radioiodine ((125)I) and/or (18)F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [(125)I]1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. Enzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values of 1.7 μM and 2.3 μM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [(125)I]1 (n = 4) and 67.5 ± 6.6% for [(18)F]4 (n = 3). [(125)I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary

  19. Technological advances in site-directed spin labeling of proteins.

    PubMed

    Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

    2013-10-01

    Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Label-Free Raman Imaging to Monitor Breast Tumor Signatures

    NASA Astrophysics Data System (ADS)

    Ciubuc, John

    Methods built on Raman spectroscopy have shown major potential in describing and discriminating between malignant and benign specimens. Accurate, real-time medical diagnosis benefits in substantial improvements through this vibrational optical method. Not only is acquisition of data possible in milliseconds and analysis in minutes, Raman allows concurrent detection and monitoring of all biological components. Besides validating a significant Raman signature distinction between non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, this study reveals a label-free method to assess overexpression of epidermal growth factor receptors (EGFR) in tumor cells. EGFR overexpression sires Raman features associated with phosphorylated threonine and serine, and modifications of DNA/RNA characteristics. Investigations by gel electrophoresis reveal EGF induction of phosphorylated Akt, agreeing with the Raman results. The analysis presented is a vital step toward Raman-based evaluation of EGF receptors in breast cancer cells. With the goal of clinically applying Raman-guided methods for diagnosis of breast tumors, the current results lay the basis for proving label-free optical alternatives in making prognosis of the disease.

  1. Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel.

    PubMed

    Zhang, Hai-zhen; Xu, Xiao-lan; Chen, Hua-yan; Ali, Sher; Wang, Dan; Yu, Jun-wei; Xu, Tao; Nan, Fa-jun

    2015-09-01

    Ca(2+)-release-activated Ca(2+) (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors. HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca(2+) influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP. A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl)urea inhibited Ca(2+) influx with IC50 of 3.25±0.17 μmol/L. SAR study on its derivatives showed that the alkyl substituent on the α-position of the left-side benzylic amine (R1) was essential for Ca(2+) influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N-H of urea was not necessary to maintain the high potency of Ca(2+) influx inhibition. The N,N'-disubstituted or N'-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity. 1-Phenyl-3-(1-phenylethyl)urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a

  2. Comparison of 68Ga- and fluorescence-labeled microspheres for measurement of relative pulmonary perfusion in anesthetized pigs.

    PubMed

    Braune, Anja; Scharffenberg, Martin; Naumann, Anne; Bluth, Thomas; de Abreu, Marcelo Gama; Kotzerke, Jörg

    2018-06-01

    We compared 68 Gallium ( 68 Ga)- and fluorescence-labeled microspheres for measurement of pulmonary perfusion distribution in anesthetized pigs without lung injury. In two mechanically ventilated pigs, the distribution of pulmonary perfusion was marked in vivo with 68 Ga- and fluorescence-labeled microspheres in supine and prone position. After each injection, the distribution of 68 Ga-labeled microspheres was measured in vivo with positron emission tomography/ computed tomography (PET/CT) in the position in which microspheres were injected and vice versa. The distribution of fluorescence-labeled microspheres was measured ex vivo . Perfusion distributions were compared between methods and postures within four lung regions and along the ventro-dorsal gradient. After each injection of 68 Ga-labeled microspheres, changes in ventro-dorsal perfusion gradients induced by repositioning were compared for volume- and mass-normalized PET/CT measurements. Regional and gradient analyses of in vivo and ex vivo measurements, respectively, consistently revealed higher pulmonary perfusion in dorsal than ventral regions in supine positioned animals. Both methods showed more pronounced perfusion gradients in supine compared to prone position. Changes in animal position were associated with alterations in the ventro-dorsal perfusion gradient when volume-, but not mass-normalization was conducted for PET/CT data. Ex vivo fluorescence- and in vivo 68 Ga-labeled microspheres measurements revealed similar perfusion distributions. Mass-normalized perfusion measurements by 68 Ga-labeled microspheres and PET/CT were not affected by positioning artifacts. Schattauer GmbH.

  3. Cortical and subcortical connections of V1 and V2 in early postnatal macaque monkeys.

    PubMed

    Baldwin, Mary K L; Kaskan, Peter M; Zhang, Bin; Chino, Yuzo M; Kaas, Jon H

    2012-02-15

    Connections of primary (V1) and secondary (V2) visual areas were revealed in macaque monkeys ranging in age from 2 to 16 weeks by injecting small amounts of cholera toxin subunit B (CTB). Cortex was flattened and cut parallel to the surface to reveal injection sites, patterns of labeled cells, and patterns of cytochrome oxidase (CO) staining. Projections from the lateral geniculate nucleus and pulvinar to V1 were present at 4 weeks of age, as were pulvinar projections to thin and thick CO stripes in V2. Injections into V1 in 4- and 8-week-old monkeys labeled neurons in V2, V3, middle temporal area (MT), and dorsolateral area (DL)/V4. Within V1 and V2, labeled neurons were densely distributed around the injection sites, but formed patches at distances away from injection sites. Injections into V2 labeled neurons in V1, V3, DL/V4, and MT of monkeys 2-, 4-, and 8-weeks of age. Injections in thin stripes of V2 preferentially labeled neurons in other V2 thin stripes and neurons in the CO blob regions of V1. A likely thick stripe injection in V2 at 4 weeks of age labeled neurons around blobs. Most labeled neurons in V1 were in superficial cortical layers after V2 injections, and in deep layers of other areas. Although these features of adult V1 and V2 connectivity were in place as early as 2 postnatal weeks, labeled cells in V1 and V2 became more restricted to preferred CO compartments after 2 weeks of age. Copyright © 2011 Wiley-Liss, Inc.

  4. Cysteine shotgun–mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells

    PubMed Central

    Krieger, Christine C.; An, Xiuli; Tang, Hsin-Yao; Mohandas, Narla; Speicher, David W.; Discher, Dennis E.

    2011-01-01

    Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells—particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin–actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 °C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography–tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin–actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of α- and β-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin’s triple-helical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption. PMID:21527722

  5. 16 CFR 300.10 - Disclosure of information on labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Disclosure of information on labels. 300.10 Section 300.10 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.10 Disclosure of...

  6. Some constructions on total labelling of m triangles

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

    Voon, Chen Huey, E-mail: chenhv@utar.edu.my; Hui, Liew How, E-mail: liewhh@utar.edu.my; How, Yim Kheng, E-mail: tidusyimhome@hotmail.com

    2016-06-02

    Let mK{sub 3} = (V{sub m}, E{sub m}) be a finite disconnected graph consisting of m disjoint triangles K{sub 3}, where V{sub m} is the set of vertices, E{sub m} is the set of edges and both V{sub m} and E{sub m} are of the same size 3m. A total labelling of mK{sub 3} is a function f which maps the elements in V{sub m} and E{sub m} to positive integer values, i.e. f : V{sub m} ∪ E{sub m} → {1, 2, 3,···}. Let c be a positive integer. A triangle is said have a c-Erdősian triangle labelling ifmore » it is a total labelling f : V{sub m} ∪ E{sub m} → {c, c + 1, ···, c + 6m − 1} such that f (x) + f (y) = f (xy) for any x, y ∈ V{sub m} and an edge xy ∈ E{sub m} joining them. In order to find all the c-Erdősian triangle labelling, a straightforward is to use the exhaustive search. However, the exhaustive search is only able to find c-Erdősian triangle labelling for m ≤ 5 due to combinatorial explosion. By studying the constant sum of vertex labels, we propose a strong permutation approach, which allows us to generate a certain classes of c-Erdősian triangle labelling up until m = 8.« less

  7. Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

    NASA Astrophysics Data System (ADS)

    Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

    2011-03-01

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

  8. SimLabel: a graphical user interface to simulate continuous wave EPR spectra from site-directed spin labeling experiments.

    PubMed

    Etienne, E; Le Breton, N; Martinho, M; Mileo, E; Belle, V

    2017-08-01

    Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the residue level, structural transitions in proteins. SDSL-EPR is based on the selective grafting of a paramagnetic label on the protein under study, followed by cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give reliable interpretation on biological system dynamics, numerical simulations of the spectra are required. Such spectral simulations can be carried out by coding in MATLAB using functions from the EasySpin toolbox. For non-expert users of MATLAB, this could be a complex task or even impede the use of such simulation tool. We developed a graphical user interface called SimLabel dedicated to run cw EPR spectra simulations particularly coming from SDSL-EPR experiments. Simlabel provides an intuitive way to visualize, simulate, and fit such cw EPR spectra. An example of SDSL-EPR spectra simulation concerning the study of an intrinsically disordered region undergoing a local induced folding is described and discussed. We believe that this new tool will help the users to rapidly obtain reliable simulated spectra and hence facilitate the interpretation of their results. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  9. Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

    PubMed Central

    Snyder, Nathaniel W.; Tombline, Gregory; Worth, Andrew J.; Parry, Robert C.; Silvers, Jacob A.; Gillespie, Kevin P.; Basu, Sankha S.; Millen, Jonathan; Goldfarb, David S.; Blair, Ian A.

    2015-01-01

    Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the gold standard for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope labeled metabolites such as acyl-coenzyme A thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope labeled CoA and acyl-CoAs from [13C3 15N1]-pantothenate using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC) in Pan6 deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof-of-concept for generating other labeled metabolites in yeast mutants. PMID:25572876

  10. Csf1r-mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System.

    PubMed

    Hawley, Catherine A; Rojo, Rocio; Raper, Anna; Sauter, Kristin A; Lisowski, Zofia M; Grabert, Kathleen; Bain, Calum C; Davis, Gemma M; Louwe, Pieter A; Ostrowski, Michael C; Hume, David A; Pridans, Clare; Jenkins, Stephen J

    2018-03-15

    CSF1 is the primary growth factor controlling macrophage numbers, but whether expression of the CSF1 receptor differs between discrete populations of mononuclear phagocytes remains unclear. We have generated a Csf1r -mApple transgenic fluorescent reporter mouse that, in combination with lineage tracing, Alexa Fluor 647-labeled CSF1-Fc and CSF1, and a modified Δ Csf1- enhanced cyan fluorescent protein (ECFP) transgene that lacks a 150 bp segment of the distal promoter, we have used to dissect the differentiation and CSF1 responsiveness of mononuclear phagocyte populations in situ. Consistent with previous Csf1r- driven reporter lines, Csf1r -mApple was expressed in blood monocytes and at higher levels in tissue macrophages, and was readily detectable in whole mounts or with multiphoton microscopy. In the liver and peritoneal cavity, uptake of labeled CSF1 largely reflected transgene expression, with greater receptor activity in mature macrophages than monocytes and tissue-specific expression in conventional dendritic cells. However, CSF1 uptake also differed between subsets of monocytes and discrete populations of tissue macrophages, which in macrophages correlated with their level of dependence on CSF1 receptor signaling for survival rather than degree of transgene expression. A double Δ Csf1r -ECFP- Csf1r -mApple transgenic mouse distinguished subpopulations of microglia in the brain, and permitted imaging of interstitial macrophages distinct from alveolar macrophages, and pulmonary monocytes and conventional dendritic cells. The Csf1r- mApple mice and fluorescently labeled CSF1 will be valuable resources for the study of macrophage and CSF1 biology, which are compatible with existing EGFP-based reporter lines. Copyright © 2018 The Authors.

  11. Efficient Thread Labeling for Monitoring Programs with Nested Parallelism

    NASA Astrophysics Data System (ADS)

    Ha, Ok-Kyoon; Kim, Sun-Sook; Jun, Yong-Kee

    It is difficult and cumbersome to detect data races occurred in an execution of parallel programs. Any on-the-fly race detection techniques using Lamport's happened-before relation needs a thread labeling scheme for generating unique identifiers which maintain logical concurrency information for the parallel threads. NR labeling is an efficient thread labeling scheme for the fork-join program model with nested parallelism, because its efficiency depends only on the nesting depth for every fork and join operation. This paper presents an improved NR labeling, called e-NR labeling, in which every thread generates its label by inheriting the pointer to its ancestor list from the parent threads or by updating the pointer in a constant amount of time and space. This labeling is more efficient than the NR labeling, because its efficiency does not depend on the nesting depth for every fork and join operation. Some experiments were performed with OpenMP programs having nesting depths of three or four and maximum parallelisms varying from 10,000 to 1,000,000. The results show that e-NR is 5 times faster than NR labeling and 4.3 times faster than OS labeling in the average time for creating and maintaining the thread labels. In average space required for labeling, it is 3.5 times smaller than NR labeling and 3 times smaller than OS labeling.

  12. 76 FR 79057 - Appliance Labeling Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-21

    ...) television labels (76 FR 1038 (Jan. 6, 2011)). The effective dates of these two final rules differ. The television label amendments, published on January 6, 2011, became effective on May 10, 2011 while the earlier... ANSI C78.20-2003 and C79.1-2002) and that uses not more than 40 watts or has a length of more than 10...

  13. 9 CFR 381.400 - Nutrition labeling of poultry products.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Nutrition labeling of poultry products... INSPECTION AND CERTIFICATION POULTRY PRODUCTS INSPECTION REGULATIONS Nutrition Labeling § 381.400 Nutrition... was delayed until Mar. 1, 2012, at 76 FR 76890, Dec. 9, 2011. (a) Nutrition labeling shall be provided...

  14. 21 CFR 501.17 - Animal food labeling warning statements.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Animal food labeling warning statements. 501.17... food labeling warning statements. (a) Self-pressurized containers. (1) The label of a food packaged in... the following warning: Warning Avoid spraying in eyes. Contents under pressure. Do not puncture or...

  15. The Influence of Sugar-Sweetened Beverage Health Warning Labels on Parents' Choices.

    PubMed

    Roberto, Christina A; Wong, Diandra; Musicus, Aviva; Hammond, David

    2016-02-01

    US states have introduced bills requiring sugar-sweetened beverages (SSBs) to display health warning labels. This study examined how such labels may influence parents and which labels are most impactful. In this study, 2381 demographically and educationally diverse parents participated in an online survey. Parents were randomly assigned to 1 of 6 conditions: (1) no warning label (control); (2) calorie label; or (3-6) 1 of 4 text versions of a warning label (eg, Safety Warning: Drinking beverages with added sugar[s] contributes to obesity, diabetes, and tooth decay). Parents chose a beverage for their child in a vending machine choice task, rated perceptions of different beverages, and indicated interest in receiving beverage coupons. Regression analyses controlling for frequency of beverage purchases were used to compare the no warning label group, calorie label group, and all warning label groups combined. Significantly fewer parents chose an SSB for their child in the warning label condition (40%) versus the no label (60%) and calorie label conditions (53%). Parents in the warning label condition also chose significantly fewer SSB coupons, believed that SSBs were less healthy for their child, and were less likely to intend to purchase SSBs. All P values <.05 after correcting for multiple comparisons. There were no consistent differences among different versions of the warning labels. Health warning labels on SSBs improved parents' understanding of health harms associated with overconsumption of such beverages and may reduce parents' purchase of SSBs for their children. Copyright © 2016 by the American Academy of Pediatrics.

  16. PRN 2012-1: Material Safety Data Sheets as Pesticide Labeling

    EPA Pesticide Factsheets

    This Notice updates and clarifies EPA’s determination in PRN 92-4 that a Material Safety Data Sheet that accompanies a pesticide product is considered part of the pesticide’s labeling but does not need EPA approval if it is consistent with 40CFR Part 156.

  17. Densities of Serotonergic Projections as Revealed by In Situ Synthesised Labelled α-Methyl-Serotonin: an Autoradiographic Evaluation

    PubMed Central

    Nishi, Kyoko; Takahashi, Sho

    2013-01-01

    An estimate of serotonergic innervation density and regional serotonin (5-HT) concentration was performed from the distribution of in situ produced labelled α-methyl-serotonin. Rats were injected with (3H) labelled α-methyl-L-tryptophan and the tracer distribution was measured using the autoradiographic method 14 days following the injection. In a separate experiment, the total brain concentration of 5-HT in the rat brain was found to be 2.4 ± 0.2 nmol/g. Based on this, and the assumption that the specific activity of in situ produced α-methyl-serotonin is the same as that of the injected tracer, it was possible to estimate the regional concentrations of 5-HT and the relative concentration of regional serotonergic innervations. It was found, and reported for the first time here, that the highest concentration of serotonergic innervation is present in the solitary nucleus. Regionally measured 5-HT concentrations accord well with previously reported concentrations of 5-HT. PMID:21472458

  18. Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    sugar 13C analysis showed that oxidizing catabolic pathways and anabolic pathways, i.e. building-up new cellular compounds, occurred in soils simultaneously. This involved an intensive C recycling within the microorganisms that was observed not only for cytosolic compounds but also for cell wall polymers. Fungal metabolism and fluxes were slower than bacterial intracellular C recycling and turnover. Furthermore, position-specific labeling of glutamate and subsequent 13C analysis of microbial phospholipid fatty acids (PLFA) revealed starvation pathways, which were only active in specific microbial groups in soils. These studies revealed that position-specific labeling enables the reconstruction of metabolic pathways of LMWOS within diverse microbial communities in complex media such as soil. Processes occurring simultaneously in soil i.e. 1) within individual, reversible metabolic pathways and 2) in various microbial groups could be traced by position-specific labeling in soils in situ. Tracing these pathways and understanding their regulating factors are crucial for soil C fluxomics, the extremely complex network of transformations towards mineralization versus the formation of microbial biomass compounds. Quantitative models to assess microbial group specific metabolic networks can be generated and parameterized by this approach. The submolecular knowledge of transformation steps and biochemical pathways in soils and their regulating factors is essential for understanding C cycling and long-term C storage in soils.

  19. Tandem mass spectrometry of isomeric aniline-labeled N-glycans separated on porous graphitic carbon: Revealing the attachment position of terminal sialic acids and structures of neutral glycans.

    PubMed

    Michael, Claudia; Rizzi, Andreas M

    2015-07-15

    Quantitative monitoring of changes in the N-glycome upon disease has gained significance in the context of biomarker discovery. Separation and quantification of isobaric glycan isomers can be attained by using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Collision-induced dissociation (CID)-based fragmentation of separated isobaric glycans is evaluated in respect to its potential of providing fragment ions specific for the linkage positions of terminal sialic acids and the presence of intersecting GlcNAc moieties, respectively. N-Glycans were labeled via reductive amination using (12)C6-aniline and (13)C6-aniline as isotope-coded labeling reagents. The differently labeled glycans were merged and separated into various species using a porous graphitic carbon (PGC) stationary phase. Identification of structural features of separated isobaric isomers was performed by CID-based tandem mass spectrometry (MS/MS) carried out in a quadrupole time-of-flight (QqTOF) or a quadrupole ion-trap (IT) mass spectrometer. Working in the negative ion mode, new diagnostic CID fragment ions could be found that are indicative for the α2,6-type linkage of sialic acids. Other diagnostic ions, identified before as being indicative for the substitution of the 6-antenna, could be confirmed as being of relevance also in the case of aniline labeling. In the positive ion mode, CID fragment ions indicative for the structure of short neutral N-glycans were identified. One new diagnostic ion specific for the linkage position of the terminal sialic acids and one for the presence of bisecting GlcNAc in N-glycans were identified. The aniline label introduced for improved relative quantitation in MS(1) was found not to significantly alter the CID fragmentation patterns that were reported previously by other authors for unlabeled/reduced glycans or for glycans with more polar labels. Copyright © 2015 John Wiley & Sons, Ltd.

  20. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans*

    PubMed Central

    Rhoads, Timothy W.; Prasad, Aman; Kwiecien, Nicholas W.; Merrill, Anna E.; Zawack, Kelson; Westphall, Michael S.; Schroeder, Frank C.; Kimble, Judith; Coon, Joshua J.

    2015-01-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress. PMID:26392051

  1. 99mTc: Labeling Chemistry and Labeled Compounds

    NASA Astrophysics Data System (ADS)

    Alberto, R.; Abram, U.

    This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section

  2. Fluorescently-labeled RNA packaging into HIV-1 particles: Direct examination of infectivity across central nervous system cell types.

    PubMed

    Xu, Ruqiang; El-Hage, Nazira; Dever, Seth M

    2015-11-01

    HIV penetrates the central nervous system (CNS), and although it is clear that microglia and to a lesser extent astrocytes are infected, whether certain other cell types such as neurons are infected remains unclear. Here, we confirmed the finding that RNAs of both cellular and viral origins are present in native HIV-1 particles and exploited this phenomenon to directly examine HIV-1 infectivity of CNS cell types. Using in vitro transcribed mRNAs that were labeled with a fluorescent dye, we showed that these fluorescent mRNAs were packaged into HIV-1 particles by directly examining infected cells using fluorescence microscopy. Cells in culture infected with these labeled virions showed the fluorescent signals of mRNA labels by a distinct pattern of punctate, focal signals within the cells which was used to demonstrate that the CXCR4-tropic NL4-3 strain was able to enter microglia and to a lesser extent astrocytes, but not neurons. The strategy used in the present study may represent a novel approach of simplicity, robustness and reliability for versatile applications in HIV studies, such as the determination of infectivity across a broad range of cell types and within sub-populations of an individual cell type by direct visualization of viral entry into cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. The power of putting a label on it: green labels weigh heavier than contradicting product information for consumers' purchase decisions and post-purchase behavior.

    PubMed

    Hahnel, Ulf J J; Arnold, Oliver; Waschto, Michael; Korcaj, Liridon; Hillmann, Karen; Roser, Damaris; Spada, Hans

    2015-01-01

    Green products are appealing. Thus, labeling products as environmentally friendly is an effective strategy to increase sales. However, the labels often promise more than the products can actually deliver. In the present research, we examined the expectation that consumers with high ecological motivation have strong preferences for green-labeled products - even when presented product information contradicts the label's image. This unsettling hypothesis is grounded in the labels' potential to create a cognitive match between the labeled product and consumers' motives. For labels indicating environmental friendliness (green product labels), this link should be strongest when consumers' ecological motivation is high. Findings in a series of three experiments support our assumption, showing that consumers with high ecological motivation had strong preferences (i.e., product evaluations, purchase intentions, and simulated purchase decisions) for green-labeled products as compared to consumers with low ecological motivation (Studies 1-3). Crucially, these preferences were robust, despite contradicting environmental product information (Studies 1 and 2). We extended our findings by additionally examining the impact of product labels and motivation on moral self-regulation processes. This was established by assessing participants' pro-social behavior after the purchase task: participants with high ecological motivation acted, consistent with their motives, more pro-socially in post-decision occasions. In accordance with moral cleansing effects, pro-social behavior was intensified after purchasing conventional products (Studies 2 and 3). Green labels protected participants with high ecological motivation from moral threats due to the purchase, thus making pro-social behavior less likely. Findings suggest that highly ecologically motivated consumers are most susceptible to green labels, which may override detailed product information.

  4. PolSK Label Over VSB-CSRZ Payload Scheme in AOLS Network

    NASA Astrophysics Data System (ADS)

    Chen, Hongwei; Chen, Minghua; Xie, Shizhong

    2007-06-01

    A novel orthogonal modulation scheme of polarization-shift-keying label over vestigial sideband payload is proposed and experimentally demonstrated over a 43-Gb/s all-optical-label-switching transmission. With this scheme, a high extinction ratio of the label is reached, while a spectral efficiency of 0.8 b/s/Hz is achieved by weakening the imperfection of the high-speed pulse sequence in polarization modulation. Furthermore, this modulation scheme is relatively irrelevant with payload bit rate. The payload and label penalties after 80-km NZDSF fiber transmission and label erasure are 1.5 and 1 dB, respectively. The influence of filter center frequency offset is also investigated. Besides, a label rewriting method is introduced to simplify label processing in intermedia nodes. All these results show that this scheme can be a candidate technique for the next-generation optical network.

  5. Proximity labeling of cis-ligands of CD22/Siglec-2 reveals stepwise α2,6 sialic acid-dependent and -independent interactions.

    PubMed

    Alborzian Deh Sheikh, Amin; Akatsu, Chizuru; Imamura, Akihiro; Abdu-Allah, Hajjaj H M; Takematsu, Hiromu; Ando, Hiromune; Ishida, Hideharu; Tsubata, Takeshi

    2018-01-01

    Lectins expressed on the cell surface are often bound and regulated by the membrane molecules containing the glycan ligands on the same cell (cis-ligands). However, molecular nature and function of cis-ligands are generally poorly understood partly because of weak interaction between lectins and glycan ligands. Cis-ligands are most extensively studied in CD22 (also known as Siglec-2), an inhibitory B lymphocyte receptor specifically recognizing α2,6 sialic acids. CD22, CD45 and IgM are suggested to be ligands of CD22. Here we labeled molecules in the proximity of CD22 in situ on B cell surface using biotin-tyramide. Molecules including CD22, CD45 and IgM were labeled in wild-type but not ST6GalI -/- B cells that lack α2,6 sialic acids, indicating that these molecules associate with CD22 by lectin-glycan interaction, and are therefore cis-ligands. In ST6GalI -/- B cells, these cis-ligands are located in a slightly more distance from CD22. Thus, the lectin-glycan interaction recruits cis-ligands already located in the relative proximity of CD22 through non-lectin-glycan interaction to the close proximity. Moreover, cis-ligands are labeled in Cmah -/- B cells that lack Neu5Gc preferred by mouse CD22 as efficiently as in wild-type B cells, indicating that very low affinity lectin-glycan interaction is sufficient for recruiting cis-ligands, and can be detected by proximity labeling. Thus, proximity labeling with tyramide appears to be a useful method to identify cis-ligands and to analyze their interaction with the lectins. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Labelling fashion magazine advertisements: Effectiveness of different label formats on social comparison and body dissatisfaction.

    PubMed

    Tiggemann, Marika; Brown, Zoe

    2018-06-01

    The experiment investigated the impact on women's body dissatisfaction of different forms of label added to fashion magazine advertisements. Participants were 340 female undergraduate students who viewed 15 fashion advertisements containing a thin and attractive model. They were randomly allocated to one of five label conditions: no label, generic disclaimer label (indicating image had been digitally altered), consequence label (indicating that viewing images might make women feel bad about themselves), informational label (indicating the model in the advertisement was underweight), or a graphic label (picture of a paint brush). Although exposure to the fashion advertisements resulted in increased body dissatisfaction, there was no significant effect of label type on body dissatisfaction; no form of label demonstrated any ameliorating effect. In addition, the consequence and informational labels resulted in increased perceived realism and state appearance comparison. Yet more extensive research is required before the effective implementation of any form of label. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Bar Code Labels

    NASA Technical Reports Server (NTRS)

    1988-01-01

    American Bar Codes, Inc. developed special bar code labels for inventory control of space shuttle parts and other space system components. ABC labels are made in a company-developed anodizing aluminum process and consecutively marketed with bar code symbology and human readable numbers. They offer extreme abrasion resistance and indefinite resistance to ultraviolet radiation, capable of withstanding 700 degree temperatures without deterioration and up to 1400 degrees with special designs. They offer high resistance to salt spray, cleaning fluids and mild acids. ABC is now producing these bar code labels commercially or industrial customers who also need labels to resist harsh environments.

  8. Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein.

    PubMed

    Jennings, M L; Anderson, M P

    1987-02-05

    A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.

  9. Maternal literacy and associations between education and the cognitive home environment in low-income families.

    PubMed

    Green, Cori M; Berkule, Samantha B; Dreyer, Benard P; Fierman, Arthur H; Huberman, Harris S; Klass, Perri E; Tomopoulos, Suzy; Yin, Hsiang Shonna; Morrow, Lesley M; Mendelsohn, Alan L

    2009-09-01

    To determine whether maternal literacy level accounts for associations between educational level and the cognitive home environment in low-income families. Analysis of 369 mother-infant dyads participating in a long-term study related to early child development. Urban public hospital. Low-income mothers of 6-month-old infants. Maternal literacy level was assessed using the Woodcock-Johnson III/Bateria III Woodcock-Munoz Tests of Achievement, Letter-Word Identification Test. Maternal educational level was assessed by determining the last grade that had been completed by the mother. The cognitive home environment (provision of learning materials, verbal responsivity, teaching, and shared reading) was assessed using StimQ, an office-based interview measure. In unadjusted analyses, a maternal literacy level of ninth grade or higher was associated with increases in scores for the overall StimQ and each of 4 subscales, whereas a maternal educational level of ninth grade or higher was associated with increases in scores for the overall StimQ and 3 of 4 subscales. In simultaneous multiple linear regression models including both literacy and educational levels, literacy continued to be associated with scores for the overall StimQ (adjusted mean difference, 3.7; 95% confidence interval, 1.7-5.7) and all subscales except teaching, whereas maternal educational level was no longer significantly associated with scores for the StimQ (1.8; 0.5-4.0) or any of its subscales. Literacy level may be a more specific indicator of risk than educational level in low-income families. Studies of low-income families should include direct measures of literacy. Pediatricians should develop strategies to identify mothers with low literacy levels and promote parenting behaviors to foster cognitive development in these at-risk families.

  10. Maternal Literacy and Associations Between Education and the Cognitive Home Environment in Low-Income Families

    PubMed Central

    Green, Cori M.; Berkule, Samantha B.; Dreyer, Benard P.; Fierman, Arthur H.; Huberman, Harris S.; Klass, Perri E.; Tomopoulos, Suzy; Yin, Hsiang Shonna; Morrow, Lesley M.; Mendelsohn, Alan L.

    2011-01-01

    Objective To determine whether maternal literacy level accounts for associations between educational level and the cognitive home environment in low-income families. Design Analysis of 369 mother-infant dyads participating in a long-term study related to early child development. Setting Urban public hospital. Participants Low-income mothers of 6-month-old infants. Main Exposure Maternal literacy level was assessed using the Woodcock-Johnson III/Bateria III Woodcock-Munoz Tests of Achievement, Letter-Word Identification Test. Maternal educational level was assessed by determining the last grade that had been completed by the mother. Main Outcome Measure The cognitive home environment (provision of learning materials, verbal responsivity, teaching, and shared reading) was assessed using StimQ, an office-based interview measure. Results In unadjusted analyses, a maternal literacy level of ninth grade or higher was associated with increases in scores for the overall StimQ and each of 4 subscales, whereas a maternal educational level of ninth grade or higher was associated with increases in scores for the overall StimQ and 3 of 4 subscales. In simultaneous multiple linear regression models including both literacy and educational levels, literacy continued to be associated with scores for the overall StimQ (adjusted mean difference, 3.7; 95% confidence interval, 1.7-5.7) and all subscales except teaching, whereas maternal educational level was no longer significantly associated with scores for the StimQ (1.8; 0.5-4.0) or any of its subscales. Conclusions Literacy level may be a more specific indicator of risk than educational level in low-income families. Studies of low-income families should include direct measures of literacy. Pediatricians should develop strategies to identify mothers with low literacy levels and promote parenting behaviors to foster cognitive development in these at-risk families. PMID:19736337

  11. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Location and size of symbol on label and labeling. 1302.04 Section 1302.04 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE LABELING AND PACKAGING REQUIREMENTS FOR CONTROLLED SUBSTANCES § 1302.04 Location and size of symbol on label...

  12. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 9 2011-04-01 2011-04-01 false Location and size of symbol on label and labeling. 1302.04 Section 1302.04 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE LABELING AND PACKAGING REQUIREMENTS FOR CONTROLLED SUBSTANCES § 1302.04 Location and size of symbol on label...

  13. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 9 2012-04-01 2012-04-01 false Location and size of symbol on label and labeling. 1302.04 Section 1302.04 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE LABELING AND PACKAGING REQUIREMENTS FOR CONTROLLED SUBSTANCES § 1302.04 Location and size of symbol on label...

  14. Impact of the graphic Canadian warning labels on adult smoking behaviour.

    PubMed

    Hammond, D; Fong, G T; McDonald, P W; Cameron, R; Brown, K S

    2003-12-01

    To assess the impact of graphic Canadian cigarette warning labels on current adult smokers. A random-digit-dial telephone survey was conducted with 616 adult smokers in south western Ontario, Canada in October/November 2001, with three month follow up. Smoking behaviour (quitting, quit attempts, and reduced smoking), intentions to quit, and salience of the warning labels. Virtually all smokers (91%) reported having read the warning labels and smokers demonstrated a thorough knowledge of their content. A strong positive relation was observed between a measure of cognitive processing-the extent to which smokers reported reading, thinking about, and discussing the new labels-and smokers' intentions to quit (odds ratio (OR) 1.11, 95% confidence interval (CI) 1.07 to 1.16; p < 0.001). Most important, cognitive processing predicted cessation behaviour at follow up. Smokers who had read, thought about, and discussed the new labels at baseline were more likely to have quit, made a quit attempt, or reduced their smoking three months later, after adjusting for intentions to quit and smoking status at baseline (OR 1.07, 95% CI 1.03 to 1.12; p < 0.001). Graphic cigarette warning labels serve as an effective population based smoking cessation intervention. The findings add to the growing literature on health warnings and provide strong support for the effectiveness of Canada's tobacco labelling policy.

  15. 40 CFR 60.536 - Permanent label, temporary label, and owner's manual.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Performance for New Residential Wood Heaters § 60.536 Permanent label, temporary label, and owner's manual. (a... section. (2) Except for wood heaters subject to § 60.530 (e), (f), or (g), the permanent label shall... material expected to last the lifetime of the wood heater, (iv) Present required information in a manner so...

  16. 40 CFR 60.536 - Permanent label, temporary label, and owner's manual.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Performance for New Residential Wood Heaters § 60.536 Permanent label, temporary label, and owner's manual. (a... section. (2) Except for wood heaters subject to § 60.530 (e), (f), or (g), the permanent label shall... material expected to last the lifetime of the wood heater, (iv) Present required information in a manner so...

  17. 40 CFR 60.536 - Permanent label, temporary label, and owner's manual.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Performance for New Residential Wood Heaters § 60.536 Permanent label, temporary label, and owner's manual. (a... section. (2) Except for wood heaters subject to § 60.530 (e), (f), or (g), the permanent label shall... material expected to last the lifetime of the wood heater, (iv) Present required information in a manner so...

  18. 40 CFR 60.536 - Permanent label, temporary label, and owner's manual.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Performance for New Residential Wood Heaters § 60.536 Permanent label, temporary label, and owner's manual. (a... section. (2) Except for wood heaters subject to § 60.530 (e), (f), or (g), the permanent label shall... material expected to last the lifetime of the wood heater, (iv) Present required information in a manner so...

  19. 40 CFR 60.536 - Permanent label, temporary label, and owner's manual.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Performance for New Residential Wood Heaters § 60.536 Permanent label, temporary label, and owner's manual. (a... section. (2) Except for wood heaters subject to § 60.530 (e), (f), or (g), the permanent label shall... material expected to last the lifetime of the wood heater, (iv) Present required information in a manner so...

  20. MYB Labeling by Immunohistochemistry Is More Sensitive and Specific for Breast Adenoid Cystic Carcinoma than MYB Labeling by FISH.

    PubMed

    Poling, Justin S; Yonescu, Raluca; Subhawong, Andrea P; Sharma, Rajni; Argani, Pedram; Ning, Yi; Cimino-Mathews, Ashley

    2017-07-01

    Breast adenoid cystic carcinoma (ACC) is a primary breast carcinoma that, like salivary gland ACC, displays the t(6;9) translocation resulting in the MYB-NFIB gene fusion and immunopositivity for MYB by immunohistochemistry (IHC). However, it is not well established whether MYB immunoreactivity or rearrangement can be used to support a diagnosis of ACC in a malignant basaloid or benign cribriform breast lesion. Whole sections of primary breast ACC (n=11), collagenous spherulosis (CS; n=7), and microglandular adenosis (MGA; n=5) and tissue microarrays containing 16 basal-like, triple-negative breast carcinomas (TNBC) were labeled for MYB by IHC and underwent MYB fluorescence in situ hybridization using a break-apart probe. Strong, diffuse nuclear MYB labeling was seen in 100% ACC compared with no cases of basal-like TNBC, CS, or MGA (P=0.0001). Any degree of nuclear MYB labeling was seen in 100% ACC compared with 54% of all other cases (P=0.007), with any labeling seen in 71% CS, 63% basal-like TNBC, and 0% MGA. MYB rearrangement was detected in 89% (8/9) of evaluable ACC compared with 4% (1/26) of all other evaluable cases (P=0.0001), with a rearrangement detected in 1 (7%; n=1/15) evaluable basal-like TNBC. Strong, diffuse nuclear labeling for MYB is more sensitive than MYB fluorescence in situ hybridization for breast ACC and can be used to support a diagnosis of ACC in a cribriform or basaloid lesion in the breast. However, weak and focal labeling should be interpreted with caution as it can be seen in other benign cribriform and malignant basaloid lesions.

  1. Photoactivatable protein labeling by singlet oxygen mediated reactions.

    PubMed

    To, Tsz-Leung; Medzihradszky, Katalin F; Burlingame, Alma L; DeGrado, William F; Jo, Hyunil; Shu, Xiaokun

    2016-07-15

    Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. GPER-targeted, 99mTc-labeled, nonsteroidal ligands demonstrate selective tumor imaging and in vivo estrogen binding

    PubMed Central

    Nayak, Tapan K.; Ramesh, Chinnasamy; Hathaway, Helen J.; Norenberg, Jeffrey P.; Arterburn, Jeffrey B.; Prossnitz, Eric R.

    2014-01-01

    Our understanding of estrogen (E2) receptor biology has evolved in recent years with the discovery and characterization of a 7-transmembrane-spanning G protein-coupled estrogen receptor (GPER1/GPER/GPR30) and the development of GPER-selective functional chemical probes. GPER is highly expressed in certain breast, endometrial and ovarian cancers, establishing the importance of non-invasive methods to evaluate GPER expression in vivo. Herein, we developed 99mTc-labeled GPER ligands to demonstrate the in vivo status of GPER as an estrogen receptor and for GPER visualization in whole animals. A series of 99mTc(I)-labeled non-steroidal tetrahydro-3H-cyclopenta[c]quinolone derivatives was synthesized utilizing pyridin-2-yl hydrazine and picolylamine chelates. Radioligand receptor binding studies revealed binding affinities in the 10–30 nM range. Cell signaling assays previously demonstrated that derivatives retaining a ketone functionality displayed agonist properties whereas those lacking such a hydrogen bond acceptor were antagonists. In vivo biodistribution and imaging studies performed on mice bearing human endometrial and breast cancer cell xenografts yielded significant tumor uptake (0.4–1.1 %ID/g). Blocking studies revealed specific uptake in multiple organs (adrenals, uterus, mammary tissue) as well as tumor uptake with similar levels of competition by E2 and G-1, a GPER-selective agonist. In conclusion, we synthesized and evaluated a series of first generation 99mTc-labeled GPER-specific radioligands, demonstrating GPER as an estrogen-binding receptor for the first time in vivo using competitive binding principles, and establishing the utility of such ligands as tumor imaging agents. These results warrant further investigation into the role of GPER in estrogen-mediated carcinogenesis and as a target for diagnostic/therapeutic/ image-guided drug delivery. PMID:25030373

  3. A test of different menu labeling presentations.

    PubMed

    Liu, Peggy J; Roberto, Christina A; Liu, Linda J; Brownell, Kelly D

    2012-12-01

    Chain restaurants will soon need to disclose calorie information on menus, but research on the impact of calorie labels on food choices is mixed. This study tested whether calorie information presented in different formats influenced calories ordered and perceived restaurant healthfulness. Participants in an online survey were randomly assigned to a menu with either (1) no calorie labels (No Calories); (2) calorie labels (Calories); (3) calorie labels ordered from low to high calories (Rank-Ordered Calories); or (4) calorie labels ordered from low to high calories that also had red/green circles indicating higher and lower calorie choices (Colored Calories). Participants ordered items for dinner, estimated calories ordered, and rated restaurant healthfulness. Participants in the Rank-Ordered Calories condition and those in the Colored Calories condition ordered fewer calories than the No Calories group. There was no significant difference in calories ordered between the Calories and No Calories groups. Participants in each calorie label condition were significantly more accurate in estimating calories ordered compared to the No Calories group. Those in the Colored Calories group perceived the restaurant as healthier. The results suggest that presenting calorie information in the modified Rank-Ordered or Colored Calories formats may increase menu labeling effectiveness. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

    PubMed

    Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

    2006-01-01

    Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.

  5. Polyelectrolyte coating of ferumoxytol nanoparticles for labeling of dendritic cells

    NASA Astrophysics Data System (ADS)

    Celikkin, Nehar; Jakubcová, Lucie; Zenke, Martin; Hoss, Mareike; Wong, John Erik; Hieronymus, Thomas

    2015-04-01

    Engineered magnetic nanoparticles (MNPs) are emerging to be used as cell tracers, drug delivery vehicles, and contrast agents for magnetic resonance imaging (MRI) for enhanced theragnostic applications in biomedicine. In vitro labeling of target cell populations with MNPs and their implantation into animal models and patients shows promising outcomes in monitoring successful cell engraftment, differentiation and migration by using MRI. Dendritic cells (DCs) are professional antigen-presenting cells that initiate adaptive immune responses. Thus, DCs have been the focus of cellular immunotherapy and are increasingly applied in clinical trials. Here, we addressed the coating of different polyelectrolytes (PE) around ferumoxytol particles using the layer-by-layer technique. The impact of PE-coated ferumoxytol particles for labeling of DCs and Flt3+ DC progenitors was then investigated. The results from our studies revealed that PE-coated ferumoxytol particles can be readily employed for labeling of DC and DC progenitors and thus are potentially suitable as contrast agents for MRI tracking.

  6. A transversal approach for patch-based label fusion via matrix completion

    PubMed Central

    Sanroma, Gerard; Wu, Guorong; Gao, Yaozong; Thung, Kim-Han; Guo, Yanrong; Shen, Dinggang

    2015-01-01

    Recently, multi-atlas patch-based label fusion has received an increasing interest in the medical image segmentation field. After warping the anatomical labels from the atlas images to the target image by registration, label fusion is the key step to determine the latent label for each target image point. Two popular types of patch-based label fusion approaches are (1) reconstruction-based approaches that compute the target labels as a weighted average of atlas labels, where the weights are derived by reconstructing the target image patch using the atlas image patches; and (2) classification-based approaches that determine the target label as a mapping of the target image patch, where the mapping function is often learned using the atlas image patches and their corresponding labels. Both approaches have their advantages and limitations. In this paper, we propose a novel patch-based label fusion method to combine the above two types of approaches via matrix completion (and hence, we call it transversal). As we will show, our method overcomes the individual limitations of both reconstruction-based and classification-based approaches. Since the labeling confidences may vary across the target image points, we further propose a sequential labeling framework that first labels the highly confident points and then gradually labels more challenging points in an iterative manner, guided by the label information determined in the previous iterations. We demonstrate the performance of our novel label fusion method in segmenting the hippocampus in the ADNI dataset, subcortical and limbic structures in the LONI dataset, and mid-brain structures in the SATA dataset. We achieve more accurate segmentation results than both reconstruction-based and classification-based approaches. Our label fusion method is also ranked 1st in the online SATA Multi-Atlas Segmentation Challenge. PMID:26160394

  7. Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.

    PubMed

    Hacker, Stephan M; Welter, Moritz; Marx, Andreas

    2015-03-09

    This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD. Copyright © 2015 John Wiley & Sons, Inc.

  8. Replacing maladaptive speech with verbal labeling responses: an analysis of generalized responding.

    PubMed Central

    Foxx, R M; Faw, G D; McMorrow, M J; Kyle, M S; Bittle, R G

    1988-01-01

    We taught three mentally handicapped students to answer questions with verbal labels and evaluated the generalized effects of this training on their maladaptive speech (e.g., echolalia) and correct responding to untrained questions. The students received cues-pause-point training on an initial question set followed by generalization assessments on a different set in another setting. Probes were conducted on novel questions in three other settings to determine the strength and spread of the generalization effect. A multiple baseline across subjects design revealed that maladaptive speech was replaced with correct labels (answers) to questions in the training and all generalization settings. These results replicate and extend previous research that suggested that cues-pause-point procedures may be useful in replacing maladaptive speech patterns by teaching students to use their verbal labeling repertoires. PMID:3225258

  9. Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid 1

    PubMed Central

    Baldi, Bruce G.; Maher, Barbara R.; Slovin, Janet Pernise; Cohen, Jerry D.

    1991-01-01

    We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [15N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[15N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[15N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants. PMID:16668112

  10. 78 FR 24211 - Draft Guidance for Industry on Safety Considerations for Container Labels and Carton Labeling...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-24

    ... container labels and carton labeling design, is the second in a series of three planned guidance documents...] Draft Guidance for Industry on Safety Considerations for Container Labels and Carton Labeling Design To... entitled ``Safety Considerations for Container Labels and Carton Labeling Design to Minimize Medication...

  11. Evaluating the Impact of Menu Labeling on Food Choices and Intake

    PubMed Central

    Larsen, Peter D.; Agnew, Henry; Baik, Jenny; Brownell, Kelly D.

    2010-01-01

    Objectives. We assessed the impact of restaurant menu calorie labels on food choices and intake. Methods. Participants in a study dinner (n = 303) were randomly assigned to either (1) a menu without calorie labels (no calorie labels), (2) a menu with calorie labels (calorie labels), or (3) a menu with calorie labels and a label stating the recommended daily caloric intake for an average adult (calorie labels plus information). Food choices and intake during and after the study dinner were measured. Results. Participants in both calorie label conditions ordered fewer calories than those in the no calorie labels condition. When calorie label conditions were combined, that group consumed 14% fewer calories than the no calorie labels group. Individuals in the calorie labels condition consumed more calories after the study dinner than those in both other conditions. When calories consumed during and after the study dinner were combined, participants in the calorie labels plus information group consumed an average of 250 fewer calories than those in the other groups. Conclusions. Calorie labels on restaurant menus impacted food choices and intake; adding a recommended daily caloric requirement label increased this effect, suggesting menu label legislation should require such a label. Future research should evaluate menu labeling's impact on children's food choices and consumption. PMID:20019307

  12. Effect of whole yeast cell product supplementation (CitriStim®) on immune responses and cecal microflora species in pullet and layer chickens during an experimental coccidial challenge.

    PubMed

    Markazi, Ashley D; Perez, Victor; Sifri, Mamduh; Shanmugasundaram, Revathi; Selvaraj, Ramesh K

    2017-07-01

    Three separate experiments were conducted to study the effects of whole yeast cell product supplementation in pullets and layer hens. Body weight gain, fecal and intestinal coccidial oocyst counts, cecal microflora species, cytokine mRNA amounts, and CD4+ and CD8+ T-cell populations in the cecal tonsils were analyzed following an experimental coccidial infection. In Experiment I, day-old Leghorn layer chicks were fed 3 experimental diets with 0, 0.1, or 0.2% whole yeast cell product (CitriStim®, ADM, Decatur, IL). At 21 d of age, birds were challenged with 1 × 105 live coccidial oocysts. Supplementation with whole yeast cell product decreased the fecal coccidial oocyst count at 7 (P = 0.05) and 8 (P < 0.01) d post-challenge. In Experiment II, 27-week old Leghorn layer hens were fed 3 experimental diets with 0, 0.05 or 0.1% whole yeast cell product and challenged with 1 × 105 live coccidial oocysts on d 25 of whole yeast cell product feeding. Supplementation with whole yeast cell product decreased the coccidial oocyst count in the intestinal content (P < 0.01) at 5, 13, and 38 d post-coccidial challenge. Supplementation with whole yeast cell product increased relative proportion of Lactobacillus (P < 0.01) in the cecal tonsils 13 d post-coccidial challenge. Supplementation with whole yeast cell product decreased CD8+ T cell percentages (P < 0.05) in the cecal tonsils at 5 d post-coccidial challenge. In Experiment III, 32-week-old Leghorn layer hens were fed 3 experimental diets with 0, 0.1, or 0.2% whole yeast cell product and challenged with 1 × 105 live coccidial oocysts on d 66 of whole yeast cell product feeding. At 5 d post-coccidial challenge, whole yeast cell product supplementation down-regulated (P = 0.01) IL-10 mRNA amount. It could be concluded that supplementing whole yeast cell product can help minimize coccidial infection in both growing pullets and layer chickens. © 2017 Poultry Science Association Inc.

  13. First performance evaluation of software for automatic segmentation, labeling and reformation of anatomical aligned axial images of the thoracolumbar spine at CT.

    PubMed

    Scholtz, Jan-Erik; Wichmann, Julian L; Kaup, Moritz; Fischer, Sebastian; Kerl, J Matthias; Lehnert, Thomas; Vogl, Thomas J; Bauer, Ralf W

    2015-03-01

    To evaluate software for automatic segmentation, labeling and reformation of anatomical aligned axial images of the thoracolumbar spine on CT in terms of accuracy, potential for time savings and workflow improvement. 77 patients (28 women, 49 men, mean age 65.3±14.4 years) with known or suspected spinal disorders (degenerative spine disease n=32; disc herniation n=36; traumatic vertebral fractures n=9) underwent 64-slice MDCT with thin-slab reconstruction. Time for automatic labeling of the thoracolumbar spine and reconstruction of double-angulated axial images of the pathological vertebrae was compared with manually performed reconstruction of anatomical aligned axial images. Reformatted images of both reconstruction methods were assessed by two observers regarding accuracy of symmetric depiction of anatomical structures. In 33 cases double-angulated axial images were created in 1 vertebra, in 28 cases in 2 vertebrae and in 16 cases in 3 vertebrae. Correct automatic labeling was achieved in 72 of 77 patients (93.5%). Errors could be manually corrected in 4 cases. Automatic labeling required 1min in average. In cases where anatomical aligned axial images of 1 vertebra were created, reconstructions made by hand were significantly faster (p<0.05). Automatic reconstruction was time-saving in cases of 2 and more vertebrae (p<0.05). Both reconstruction methods revealed good image quality with excellent inter-observer agreement. The evaluated software for automatic labeling and anatomically aligned, double-angulated axial image reconstruction of the thoracolumbar spine on CT is time-saving when reconstructions of 2 and more vertebrae are performed. Checking results of automatic labeling is necessary to prevent errors in labeling. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. Second Language Use Facilitates Implicit Emotion Regulation via Content Labeling

    PubMed Central

    Morawetz, Carmen; Oganian, Yulia; Schlickeiser, Ulrike; Jacobs, Arthur M.; Heekeren, Hauke R.

    2017-01-01

    Previous studies reported that negative stimuli induced less affect in bilinguals when stimuli were presented in bilinguals’ second, weaker language (L2) than when they were presented in their native language (L1). This effect of L2 use was attributed to increased emotional distance as well as to increased levels of cognitive control during L2 use. Here we investigated how explicit (cognitive reappraisal, i.e., reinterpreting the meaning of the emotional stimulus to alter its emotional impact) and implicit (content labeling, i.e., categorizing the content of the image; and emotion labeling, i.e., naming the emotion induced by the emotional stimulus) emotion regulation strategies are altered in an L2 (English) context in German native speakers with medium to high proficiency in their L2. While previous studies used linguistic stimuli, such as words, to induce affect, here we used images to test whether reduced affect could also be observed for non-linguistic stimuli when presented in an L2 context. We hypothesized that the previously implicated increase in emotional distance and cognitive control in an L2 would result in an L2 advantage in emotion regulation (i.e., leading to less negative emotions compared to an L1 context), by strengthening the effect of linguistic re-evaluation on the evoked emotions. Using a classic emotion regulation paradigm, we examined changes in subjective emotional state ratings during reappraisal, emotion labeling and content labeling in a L1 and L2 context. We found that the strength of evoked affective responses did not depend on the language context in which an image was presented. Crucially, content labeling in L2 was more effective than in L1, whereas emotion labeling did not differ between languages. Overall, evoked responses were regulated most effectively through explicit emotion regulation (reappraisal) in L1 and L2 context. These results demonstrate an L2 advantage effect for emotion regulation through content labeling and

  15. Allelic divergence and cultivar-specific SSR alleles revealed by capillary electrophoresis using fluorescence-labeled SSR markers in sugarcane

    USDA-ARS?s Scientific Manuscript database

    Though sugarcane cultivars (Saccharum spp. hybrids) are complex aneu-polyploid hybrids, genetic evaluation and tracking of clone- or cultivar-specific alleles become possible due to capillary electrophoregrams (CE) using fluorescence-labeled SSR primer pairs. Twenty-four sugarcane cultivars, 12 each...

  16. Off-Label Drug Use

    MedlinePlus

    ... their drugs for off-label uses. Off-label marketing is very different from off-label use. Why ... at a higher risk for medication errors, side effects, and unwanted drug reactions. It’s important that the ...

  17. Chemo-enzymatic synthesis of isotopically labeled nicotinamide riboside.

    PubMed

    Tran, Ai; Yokose, Ryota; Cen, Yana

    2018-05-15

    As a cofactor for numerous reactions, NAD+ is found widely dispersed across many maps of cellular metabolism. This core redox role alone makes the biosynthesis of NAD+ of great interest. Recent studies have revealed new biological roles for NAD+ as a substrate for diverse enzymes that regulate a broad spectrum of key cellular tasks. These NAD+-consuming enzymes further highlight the importance of understanding NAD+ biosynthetic pathways. In this study, we developed a chemo-enzymatic synthesis of isotopically labeled NAD+ precursor, nicotinamide riboside (NR). The synthesis of NR isotopomers allowed us to unambiguously determine that NR is efficiently converted to NAD+ in the cellular environment independent of degradation to nicotinamide, and it is incorporated into NAD+ in its intact form. The versatile synthetic method along with the isotopically labeled NRs will provide powerful tools to further decipher the important yet complicated NAD+ metabolism.

  18. Pesticide Label Review Training

    EPA Pesticide Factsheets

    This training will help ensure that reviewers evaluate labels according to four core principles. It also will help pesticide registrants developing labels understand what EPA expects of pesticide labels, and what the Agency generally finds acceptable.

  19. A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870.

    PubMed

    Edgar, Alexander J; Trost, Matthias; Watts, Colin; Zaru, Rossana

    2014-02-01

    Protein kinase inhibitors frequently have interesting effects that cannot be fully ascribed to the intended target kinase(s) but identifying additional targets that might explain the effects is not straightforward. By comparing two different inhibitors of the Rsk (p90 ribosomal S6 kinase) kinases, we found that the increasingly used compound BI-D1870 had biological effects in murine DCs (dendritic cells) that could not be solely ascribed to Rsk or other documented targets. We assessed the ability of BI-D1870 and a second Rsk inhibitor, BIX 02565 to protect enzyme active sites from reaction with biotinylated nucleotide acyl phosphates. Using SILAC (stable isotope labelling by amino acids in cell culture)-labelled DC lysates as a source of enzyme targets, we identify several kinases that interact with BI-D1870 but not with BIX 02565. We confirmed that these kinases, including Slk, Lok and Mst1, are inhibited by BI-D1870 but to a much lesser extent by BIX 02565 and that phosphorylation of some of their substrates is blocked by BI-D1870 in living cells. Our results suggest that the BI-D1870 inhibitor should be used with caution. The SILAC-based methodology we used should be useful for further comparative unbiased profiling of the target spectrum of kinase inhibitors with interesting biological effects under conditions that closely mimic those found in cells. © 2014 The author(s).

  20. Topics in Library Technology: Labeling Techniques *

    PubMed Central

    Truelson, Stanley D.

    1966-01-01

    Labels which do not fit on the spines of books should be placed on the upper rather than lower left corner of the front cover, because the upper corner becomes visible first when a volume is tilted from the shelf. None of the past methods of marking call numbers on the spines or covers of books—direct hand lettering by pen, brush, or stylus; affixing cold release characters; embossing by hot type; or gluing labels which are handlettered, typed, or printed—nor even present automatic data processing systems have offered all the advantages of the relatively new Se-Lin labeling system: legibility, reasonable speed of application, automatic protective covering, permanent bonding, and no need for a skilled letterer. Labels seem unaesthetic to some librarians, but their advantages outweigh this consideration. When only one or a few copies of the same call number are required, Se-Lin is the best system now available for libraries marking over 1,000 books a year. PMID:5901359