α-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

PubMed

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-06-06

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex

PubMed Central

Sun, Wei; Yan, Qing; Vida, Thomas A.; Bean, Andrew J.

2003-01-01

Movement through the endocytic pathway occurs principally via a series of membrane fusion and fission reactions that allow sorting of molecules to be recycled from those to be degraded. Endosome fusion is dependent on SNARE proteins, although the nature of the proteins involved and their regulation has not been fully elucidated. We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes. A region of Hrs predicted to form a coiled coil required for binding the Q-SNARE, SNAP-25, mimicked the inhibition of endosome fusion produced by full-length Hrs, and was sufficient for endosome binding. SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain. Syntaxin 13 inhibited early endosomal fusion and botulinum toxin/E inhibition of early endosomal fusion was reversed by addition of SNAP-25(150–206), confirming a role for syntaxin 13, and establishing a role for SNAP-25 in endosomal fusion. Hrs inhibited formation of the syntaxin 13–SNAP-25–VAMP2 complex by displacing VAMP2 from the complex. These data suggest that SNAP-25 is a receptor for Hrs on early endosomal membranes and that the binding of Hrs to SNAP-25 on endosomal membranes inhibits formation of a SNARE complex required for homotypic endosome fusion. PMID:12847087

The Multifaceted Role of SNARE Proteins in Membrane Fusion

PubMed Central

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A.

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined. PMID:28163686

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

PubMed

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly.

PubMed

Rebane, Aleksander A; Wang, Bigeng; Ma, Lu; Qu, Hong; Coleman, Jeff; Krishnakumar, Shyam; Rothman, James E; Zhang, Yongli

2018-02-16

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 k B T, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

SNARE interactions in membrane trafficking: a perspective from mammalian central synapses.

PubMed

Kavalali, Ege T

2002-10-01

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are a large family of proteins that are present on all organelles involved in intracellular vesicle trafficking and secretion. The interaction of complementary SNAREs found on opposing membranes presents an attractive lock-and-key mechanism, which may underlie the specificity of vesicle trafficking. Moreover, formation of the tight complex between a vesicle membrane SNARE and corresponding target membrane SNAREs could drive membrane fusion. In synapses, this tight complex, also referred to as the synaptic core complex, is essential for neurotransmitter release. However, recent observations in knockout mice lacking major synaptic SNAREs challenge the prevailing notion on the executive role of these proteins in fusion and open up several questions about their exact role(s) in neurotransmitter release. Persistence of a form of regulated neurotransmitter release in these mutant mice also raises the possibility that other cognate or non-cognate SNAREs may partially compensate for the loss of a particular SNARE. Future analysis of SNARE function in central synapses will also have implications for the role of these molecules in other vesicle trafficking events such as endocytosis and vesicle replenishment. Such analysis can provide a molecular basis for synaptic processes including certain forms of short-term synaptic plasticity. Copyright 2002 Wiley Periodicals, Inc.

Single-molecule studies of the neuronal SNARE fusion machinery.

PubMed

Brunger, Axel T; Weninger, Keith; Bowen, Mark; Chu, Steven

2009-01-01

SNAREs are essential components of the machinery for Ca(2+)-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca(2+) sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis.

PubMed

Ma, Lu; Rebane, Aleksander A; Yang, Guangcan; Xi, Zhiqun; Kang, Yuhao; Gao, Ying; Zhang, Yongli

2015-12-23

Synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins couple their stage-wise folding/assembly to rapid exocytosis of neurotransmitters in a Munc18-1-dependent manner. The functions of the different assembly stages in exocytosis and the role of Munc18-1 in SNARE assembly are not well understood. Using optical tweezers, we observed four distinct stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains (or NTD, MD, CTD, and LD, respectively). We found that SNARE layer mutations differentially affect SNARE assembly. Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 initiates SNARE assembly and structures t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD) and stabilizes the half-zippered SNARE complex dependent upon the NRD. Our observations demonstrate distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1.

Inhibition of Calpains Protects Mn-Induced Neurotransmitter release disorders in Synaptosomes from Mice: Involvement of SNARE Complex and Synaptic Vesicle Fusion.

PubMed

Wang, Can; Xu, Bin; Ma, Zhuo; Liu, Chang; Deng, Yu; Liu, Wei; Xu, Zhao-Fa

2017-06-16

Overexposure to manganese (Mn) could disrupt neurotransmitter release via influencing the formation of SNARE complex, but the underlying mechanisms are still unclear. A previous study demonstrated that SNAP-25 is one of substrate of calpains. The current study investigated whether calpains were involved in Mn-induced disorder of SNARE complex. After mice were treated with Mn for 24 days, Mn deposition increased significantly in basal nuclei in Mn-treated and calpeptin pre-treated groups. Behaviorally, less time spent in the center of the area and decreased average velocity significantly in an open field test after 24 days of Mn exposure. With the increase in MnCl 2 dosage, intracellular Ca 2+ increased significantly, but pretreatment with calpeptin caused a dose-dependent decrease in calpains activity. There were fragments of N-terminal of SNAP-25 protein appearance in Mn-treated groups, but it is decreased with pretreatment of calpeptin. FM1-43-labeled synaptic vesicles also provided evidence that the treatment with Mn resulted in increasing first and then decreasing, which was consistent with Glu release and the 80 kDa protein levels of SNARE complexes. In summary, Mn induced the disorder of neurotransmitter release through influencing the formation of SNARE complex via cleaving SNAP-25 by overactivation of calpains in vivo.

Arabidopsis SNAREs SYP61 and SYP121 coordinate the trafficking of plasma membrane aquaporin PIP2;7 to modulate the cell membrane water permeability.

PubMed

Hachez, Charles; Laloux, Timothée; Reinhardt, Hagen; Cavez, Damien; Degand, Hervé; Grefen, Christopher; De Rycke, Riet; Inzé, Dirk; Blatt, Michael R; Russinova, Eugenia; Chaumont, François

2014-07-01

Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane. © 2014 American Society of Plant Biologists. All rights reserved.

Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.

PubMed

Zhang, Yongli

2017-07-01

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins. © 2017 The Protein Society.

Calcium-dependent regulation of SNARE-mediated membrane fusion by calmodulin.

PubMed

Di Giovanni, Jerome; Iborra, Cécile; Maulet, Yves; Lévêque, Christian; El Far, Oussama; Seagar, Michael

2010-07-30

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca(2+) sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca(2+)-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca(2+)/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K(D) = 500 nm) and syntaxin 1 (K(D) = 2 microm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca(2+) sensors act antagonistically in SNARE-mediated fusion.

CAPS and Munc13: CATCHRs that SNARE Vesicles.

PubMed

James, Declan J; Martin, Thomas F J

2013-12-04

CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS) and Munc13 (Mammalian Unc-13) proteins function to prime vesicles for Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes (MTCs) have been reported. MTCs coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.

Non-canonical role of the SNARE protein Ykt6 in autophagosome-lysosome fusion

PubMed Central

Takáts, Szabolcs; Glatz, Gábor; Szenci, Győző; Boda, Attila; Horváth, Gábor V.; Hegedűs, Krisztina; Kovács, Attila L.

2018-01-01

The autophagosomal SNARE Syntaxin17 (Syx17) forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS. Here we demonstrate that, unexpectedly, one more SNARE (Ykt6) is also required for autophagosome clearance in Drosophila. We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion. Indeed, Ykt6 localizes to lysosomes and autolysosomes, and forms a SNARE complex with Syx17 and Snap29. Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells. Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms. As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a Syx17-Snap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process. PMID:29694367

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis.

PubMed

Khodthong, Chuenchanok; Kabachinski, Greg; James, Declan J; Martin, Thomas F J

2011-08-03

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis. Copyright © 2011 Elsevier Inc. All rights reserved.

ATG14 controls SNARE-mediated autophagosome fusion with a lysosome.

PubMed

Liu, Rong; Zhi, Xiaoyong; Zhong, Qing

2015-01-01

Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for autophagosome fusion with a lysosome, but is dispensable for autophagosome biogenesis. These data support a key role of ATG14 in controlling autophagosome fusion with a lysosome.

The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

PubMed

Rizo, Josep; Südhof, Thomas C

2012-01-01

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein (GARP) Complex with the t-SNARE Syntaxin 6

PubMed Central

Abascal-Palacios, Guillermo; Schindler, Christina; Rojas, Adriana L; Bonifacino, Juan S.; Hierro, Aitor

2016-01-01

Summary The Golgi-Associated Retrograde Protein (GARP) is a tethering complex involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein we report the structural basis for the interaction of the human Ang2 subunit of GARP with Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a novel binding mode in which a di-tyrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction. The same binding determinants are found in other tethering proteins and syntaxins, suggesting a general interaction mechanism. PMID:23932592

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

PubMed Central

Boswell, Kristin L.; James, Declan J.; Esquibel, Joseph M.; Bruinsma, Stephen; Shirakawa, Ryutaro; Horiuchi, Hisanori

2012-01-01

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca2+, but Ca2+-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca2+ and restored Ca2+-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca2+-binding residues in C2A and C2B. Munc13-4 exhibited Ca2+-stimulated SNARE interactions dependent on C2A and Ca2+-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca2+-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca2+-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca2+ sensor at rate-limiting priming steps in granule exocytosis. PMID:22508512

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion.

PubMed

Boswell, Kristin L; James, Declan J; Esquibel, Joseph M; Bruinsma, Stephen; Shirakawa, Ryutaro; Horiuchi, Hisanori; Martin, Thomas F J

2012-04-16

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.

SNARE proteins synaptobrevin, SNAP-25 and syntaxin are involved in rapid and slow endocytosis at synapses

PubMed Central

Xu, Jianhua; Luo, Fujun; Zhang, Zhen; Xue, Lei; Wu, Xinsheng; Chiang, Hsueh-Cheng; Shin, Wonchul; Wu, Ling-Gang

2013-01-01

Rapid endocytosis, which takes only a few seconds, is widely observed in secretory cells. Although it is more efficient in recycling vesicles than slow clathrin-mediated endocytosis, its underlying mechanism, thought to be clathrin-independent, is largely unclear. Here we reported that cleavage of three SNARE proteins essential for exocytosis, including synaptobrevin, SNAP-25 and syntaxin, inhibited rapid endocytosis at the calyx of Held nerve terminal, suggesting the involvement of three SNARE proteins in rapid endocytosis. These SNARE proteins were also involved in slow endocytosis. In addition, SNAP-25 and syntaxin facilitated vesicle mobilization to the readily releasable pool, likely via their roles in endocytosis and/or exocytosis. We concluded that both rapid and slow endocytosis share the involvement of SNARE proteins. The dual role of three SNARE proteins in exo- and endocytosis suggests that SNARE proteins may be molecular substrates contributing to the exo-endocytosis coupling, which maintains exocytosis in secretory cells. PMID:23643538

Goose-neck snare-assisted transcatheter ASD closure: A safety procedure for large and complex ASDs.

PubMed

Butera, Gianfranco; Lovin, Nicusor; Basile, Domenica Paola; Carminati, Mario

2016-04-01

To report on a new technique that increases the safety of percutaneous atrial septal defect (ASD) closure using a goose-neck snare system. ASD transcatheter closure is a widespread procedure. However, in some cases, ASDs may be large and with soft rims. In these situation, a potential risk exists for device malposition or embolization. When transesophageal echocardiography (TEE) evaluation and balloon sizing showed large defects with floppy rims the chosen Amplatzer device was implanted in a standard way. In large defects with floppy rims, before release a 5-mm goose-neck snare with its 4 Fr catheter was placed across the delivery cable and fixed to catch the screwing mechanism of implanted Amplatzer device. The delivery cable was unscrewed and the device reached its final position without any tension. If the position was considered satisfactory the device was released from the goose-neck snare. Thirteen patients had a snare-assisted ASD transcatheter closure. Median device size was 24 mm (range 14-38 mm). Retrieval or repositioning of the device using the goose-neck snare was performed in four cases: in three patients, because of device malposition after delivery cable release and in one patient, because of unsuitability of closure of a second significant defect. Furthermore, in two subjects with multiple ASDs, a second fenestration looked quite significant with the device still attached to the delivery cable while it appeared smaller after release. Snare-assisted Amplatzer ASD device placement is a new method for ASD percutaneous closure and adds safety to the procedure. © 2016 Wiley Periodicals, Inc.

Energetics and Kinetics of trans-SNARE Zippering

NASA Astrophysics Data System (ADS)

Rebane, Aleksander A.; Shu, Tong; Krishnakumar, Shyam; Rothman, James E.; Zhang, Yongli

Synaptic exocytosis relies on assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins into a four-helix bundle to drive membrane fusion. Complementary SNAREs anchored to the synaptic vesicle (v-SNARE) and the plasma membrane (t-SNARE) associate from their N-termini, transiting a half-assembled intermediate (trans-SNARE), and ending at their C-termini with a rapid power stroke that leads to membrane fusion. Although cytosolic SNARE assembly has been characterized, it remains unknown how membranes modulate the energetics and kinetics of SNARE assembly. Here, we present optical tweezers measurements on folding of single membrane proteins in phospholipid bilayers. To our knowledge, this is the first such report. We measured the energetics, kinetics, and assembly intermediates of trans-SNAREs formed between a t-SNARE inserted into a bead-supported bilayer and a v-SNARE in a nanodisc. We found that the repulsive force of the apposed membranes increases the lifetime of the half-assembled intermediate. Our findings provide a single-molecule platform to study the regulation of trans-SNARE assembly by proteins that act on the half-assembled state, and thus reveal the mechanistic basis of the speed and high fidelity of synaptic transmission. This work was supported by US National Institutes of Health Grants F31 GM119312-01 (to A.A.R) and R01 GM093341 (to Y.Z.).

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking

PubMed Central

Choi, Bong-Kyu; Choi, Mal-Gi; Kim, Jae-Yeol; Yang, Yoosoo; Lai, Ying; Kweon, Dae-Hyuk; Lee, Nam Ki; Shin, Yeon-Kyun

2013-01-01

Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity. PMID:23431141

Entropic forces drive self-organization and membrane fusion by SNARE proteins

PubMed Central

Stratton, Benjamin S.; Warner, Jason M.; Rothman, James E.; O’Shaughnessy, Ben

2017-01-01

SNARE proteins are the core of the cell’s fusion machinery and mediate virtually all known intracellular membrane fusion reactions on which exocytosis and trafficking depend. Fusion is catalyzed when vesicle-associated v-SNAREs form trans-SNARE complexes (“SNAREpins”) with target membrane-associated t-SNAREs, a zippering-like process releasing ∼65 kT per SNAREpin. Fusion requires several SNAREpins, but how they cooperate is unknown and reports of the number required vary widely. To capture the collective behavior on the long timescales of fusion, we developed a highly coarse-grained model that retains key biophysical SNARE properties such as the zippering energy landscape and the surface charge distribution. In simulations the ∼65-kT zippering energy was almost entirely dissipated, with fully assembled SNARE motifs but uncomplexed linker domains. The SNAREpins self-organized into a circular cluster at the fusion site, driven by entropic forces that originate in steric–electrostatic interactions among SNAREpins and membranes. Cooperative entropic forces expanded the cluster and pulled the membranes together at the center point with high force. We find that there is no critical number of SNAREs required for fusion, but instead the fusion rate increases rapidly with the number of SNAREpins due to increasing entropic forces. We hypothesize that this principle finds physiological use to boost fusion rates to meet the demanding timescales of neurotransmission, exploiting the large number of v-SNAREs available in synaptic vesicles. Once in an unfettered cluster, we estimate ≥15 SNAREpins are required for fusion within the ∼1-ms timescale of neurotransmitter release. PMID:28490503

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

PubMed

James, Declan J; Khodthong, Chuenchanok; Kowalchyk, Judith A; Martin, Thomas F J

2008-07-28

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

PubMed Central

Kreutzberger, Alex J.B.; Kiessling, Volker; Tamm, Lukas K.

2015-01-01

Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays. PMID:26200867

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion.

PubMed

Yavuz, Halenur; Kattan, Iman; Hernandez, Javier Matias; Hofnagel, Oliver; Witkowska, Agata; Raunser, Stefan; Walla, Peter Jomo; Jahn, Reinhard

2018-04-17

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N-termini to their membrane-proximal C-termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates, where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

SNARE proteins underpin insulin-regulated GLUT4 traffic.

PubMed

Bryant, Nia J; Gould, Gwyn W

2011-06-01

Delivery of the glucose transporter type 4 (GLUT4) from an intracellular location to the cell surface in response to insulin represents a specialized form of membrane traffic, known to be impaired in the disease states of insulin resistance and type 2 diabetes. Like all membrane trafficking events, this translocation of GLUT4 requires members of the SNARE family of proteins. Here, we discuss two SNARE complexes that have been implicated in insulin-regulated GLUT4 traffic: one regulating the final delivery of GLUT4 to the cell surface in response to insulin and the other controlling GLUT4's intracellular trafficking. © 2011 John Wiley & Sons A/S.

The golgin protein Coy1 functions in intra-Golgi retrograde transport and interacts with the COG complex and Golgi SNAREs

PubMed Central

Anderson, Nadine S.; Mukherjee, Indrani; Bentivoglio, Christine M.; Barlowe, Charles

2017-01-01

Extended coiled-coil proteins of the golgin family play prominent roles in maintaining the structure and function of the Golgi complex. Here we further investigate the golgin protein Coy1 and document its function in retrograde transport between early Golgi compartments. Cells that lack Coy1 displayed a reduced half-life of the Och1 mannosyltransferase, an established cargo of intra-Golgi retrograde transport. Combining the coy1Δ mutation with deletions in other putative retrograde golgins (sgm1Δ and rud3Δ) caused strong glycosylation and growth defects and reduced membrane association of the conserved oligomeric Golgi (COG) complex. In contrast, overexpression of COY1 inhibited the growth of mutant strains deficient in fusion activity at the Golgi (sed5-1 and sly1-ts). To map Coy1 protein interactions, coimmunoprecipitation experiments revealed an association with the COG complex and with intra-Golgi SNARE proteins. These physical interactions are direct, as Coy1 was efficiently captured in vitro by Lobe A of the COG complex and the purified SNARE proteins Gos1, Sed5, and Sft1. Thus our genetic, in vivo, and biochemical data indicate a role for Coy1 in regulating COG complex-dependent fusion of retrograde-directed COPI vesicles. PMID:28794270

Green fluorescence protein-based content-mixing assay of SNARE-driven membrane fusion

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

Heo, Paul; Kong, Byoungjae; Jung, Young-Hun

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regardingmore » the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening. - Highlights: • SNARE proteins drive membrane fusion and open a pore for cargo release. • Biotinylated GFP and DyStrp was used as the reporter pair of fusion pore opening. • Procedure for efficient SNARE reconstitution and reporter encapsulation was established. • The FRET pair reported opening of a large fusion pore bigger than 5�

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

PubMed Central

Ko, Young-Joon; Lee, Miriam; Kang, KyeongJin; Song, Woo Keun; Jun, Youngsoo

2014-01-01

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms. PMID:24821814

A prospective comparison of cold snare polypectomy using traditional or dedicated cold snares for the resection of small sessile colorectal polyps

PubMed Central

Dwyer, Jeremy P.; Tan, Jonathan Y. C.; Urquhart, Paul; Secomb, Robyn; Bunn, Catherine; Reynolds, John; La Nauze, Richard; Kemp, William; Roberts, Stuart; Brown, Gregor

2017-01-01

Background and study aims  The evidence for efficacy and safety of cold snare polypectomy is limited. The aim of this study was to assess the completeness of resection and safety of cold snare polypectomy, using either traditional or dedicated cold snares. Patients and methods  This was a prospective, non-randomized study performed at a single tertiary hospital. Adult patients with at least one colorectal polyp (size ≤ 10 mm) removed by cold snare were included. In the first phase, all patients had polyps removed by traditional snare without diathermy. In the second phase, all patients had polyps removed by dedicated cold snare. Complete endoscopic resection was determined from histological examination of quadrantic polypectomy margin biopsies. Immediate or delayed bleeding within 2 weeks was recorded. Results  In total, 181 patients with 299 eligible polyps (n = 93 (173 polyps) traditional snare group, n = 88 (126 polyps) dedicated cold snare group) were included. Patient demographics and procedure indications were similar between groups. Mean polyp size was 6 mm in both groups ( P  = 0.25). Complete polyp resection was 165 /173 (95.4 %; 95 %CI 90.5 – 97.6 %) in the traditional snare group and 124/126 (98.4 %; 95 %CI 93.7 – 99.6 %) in the dedicated cold snare group ( P  = 0.16). Serrated polyps, compared with adenomatous polyps, had a higher rate of incomplete resection (7 % vs. 2 %, P  = 0.03). There was no statistically significant difference in the rate of immediate bleeding (3 % vs. 1 %, P  = 0.41) and there were no delayed hemorrhages or perforations. Conclusions  Cold snare polypectomy is effective and safe for the complete endoscopic resection of small (≤ 10 mm) colorectal polyps with either traditional or dedicated cold snares. PMID:29250580

Dilation of fusion pores by crowding of SNARE proteins

PubMed Central

Wu, Zhenyong; Bello, Oscar D; Thiyagarajan, Sathish; Auclair, Sarah Marie; Vennekate, Wensi; Krishnakumar, Shyam S; O'Shaughnessy, Ben; Karatekin, Erdem

2017-01-01

Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, 'flipped' t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion). DOI: http://dx.doi.org/10.7554/eLife.22964.001 PMID:28346138

The quantum physics of synaptic communication via the SNARE protein complex.

PubMed

Georgiev, Danko D; Glazebrook, James F

2018-07-01

Twenty five years ago, Sir John Carew Eccles together with Friedrich Beck proposed a quantum mechanical model of neurotransmitter release at synapses in the human cerebral cortex. The model endorsed causal influence of human consciousness upon the functioning of synapses in the brain through quantum tunneling of unidentified quasiparticles that trigger the exocytosis of synaptic vesicles, thereby initiating the transmission of information from the presynaptic towards the postsynaptic neuron. Here, we provide a molecular upgrade of the Beck and Eccles model by identifying the quantum quasiparticles as Davydov solitons that twist the protein α-helices and trigger exocytosis of synaptic vesicles through helical zipping of the SNARE protein complex. We also calculate the observable probabilities for exocytosis based on the mass of this quasiparticle, along with the characteristics of the potential energy barrier through which tunneling is necessary. We further review the current experimental evidence in support of this novel bio-molecular model as presented. Copyright © 2018 Elsevier Ltd. All rights reserved.

WNK4 inhibits plasma membrane targeting of NCC through regulation of syntaxin13 SNARE formation.

PubMed

Chung, Woo Young; Park, Hyun Woo; Han, Jung Woo; Lee, Min Goo; Kim, Joo Young

2013-12-01

WNK4, a serine/threonine kinase, plays a critical role in the expression of membrane proteins in the cell surface; however, the underlying mechanism of WNK4 is not clear. Here, we demonstrate that WNK4 inhibits the fusion of plasma membrane delivering vesicle with sorting/recycling endosome through disrupting SNARE formation of syntaxin13, an endosomal t-SNARE and VAMP2, the v-SNARE in plasma membrane delivering vesicle. Their interaction and co-localization were enhanced by hyperosmotic stimulation which is known for WNK4 activation. The kinase domain of WNK4 interacts with the transmembrane domain (TM) of syntaxin13 and this interaction was abolished when the TM was replaced with that of syntaxin16. Interestingly, cell fractionation using sucrose gradients revealed that WNK4 inhibited the formation of the syntaxin13/VAMP2 SNARE complex in the endosomal compartment, but not syntaxin16/VAMP2 or syntaxin13/VAMP7. Syntaxin13 was not phosphorylated by WNK4 and WNK4KI also showed the same binding strength and similar inhibitory regulation on SNARE formation of syntaxin13. Physiological relevance of this mechanism was proved with the expression of NCC (Na(+) C1(-) co-transporter) in the cell surface. The inhibiting activity of WNK4 on surface expression of NCC was abolished by syntaxin13 siRNA transfection. These results suggest that WNK4 attenuates PM targeting of NCC proteins through regulation of syntaxin13 SNARE complex formation with VAMP2 in recycling and sorting endosome. © 2013.

NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

PubMed

Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

2007-08-15

Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

Identification of SNAREs that mediate zymogen granule exocytosis

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

Pickett, James A.; Campos-Toimil, Manuel; Thomas, Paul

2007-08-03

A secretagogue-stimulated pancreatic acinar cell releases digestive enzymes from its apical pole. We attempted to identify the SNAREs involved in zymogen granule exocytosis. Antibodies against syntaxins 2 and 3, SNAP-23 and VAMP 8, and the corresponding recombinant SNAREs, inhibited amylase secretion from streptolysin O-permeabilised acini; other anti-SNARE antibodies and SNAREs had no effect. Botulinum neurotoxin C, which cleaved syntaxin 2 and (to a lesser extent) syntaxin 3, but not syntaxins 4, 7 or 8, also inhibited exocytosis. We propose that syntaxin 2, SNAP-23 and VAMP 8 mediate primary granule-plasma membrane fusion. Syntaxin 3 may be involved in secondary granule-granule fusion.

Monorail snare technique for the retrieval of an adherent microcatheter from an onyx cast: technical case report.

PubMed

Kelly, Michael E; Turner, Raymond; Gonugunta, Vivek; Rasmussen, Peter A; Woo, Henry H; Fiorella, David

2008-07-01

Microcatheters retained after Onyx (eV3 Neurovascular, Inc., Irvine, CA) embolization represent a potential source of thromboembolic complications. Catheter retention depends on the degree of Onyx reflux and vessel tortuosity. To overcome this problem, we have adapted a previously described monorail snare technique for stretched coils to remove an adherent microcatheter from the occipital artery during Onyx embolization of a dural arteriovenous fistula. We used this technique successfully in a 62-year-old man with a posterior fossa dural arteriovenous fistula. An Echelon-10 microcatheter (eV3 Neurovascular, Inc.) system became adherent in the right occipital artery because of reflux and vessel tortuosity. Significant stretching of the microcatheter was observed during attempted removal. A 2-mm Amplatz Goose Neck microsnare (Microvena Corp., White Bear Lake, MN) was placed through a Rapid Transit microcatheter (Cordis Corp., Miami, FL). The hub of the indwelling Echelon microcatheter was cut off and the snare advanced over the outside of the microcatheter. The snare and Rapid Transit microcatheter were then advanced into the guiding catheter (6-French) as a unit over the indwelling Echelon microcatheter. Using the adherent Echelon as a "monorail" guide, the snare and Rapid Transit microcatheter were advanced distally into the occipital artery and the snare was retracted to engage the microcatheter. The microcatheters and snare were then easily removed because of the second vector of force placed by the snare system on the adherent microcatheter very close to the point of adherence. The monorail snare technique represents a simple and safe way to remove an adherent microcatheter from an Onyx cast during the embolization of dural arteriovenous fistulas. Prospective knowledge of this technique will facilitate more aggressive embolization without the reservation that a retained microcatheter could require surgical removal or anticoagulation.

Observing the conformation of individual SNARE proteins inside live cells

NASA Astrophysics Data System (ADS)

Weninger, Keith

2010-10-01

Protein conformational dynamics are directly linked to function in many instances. Within living cells, protein dynamics are rarely synchronized so observing ensemble-averaged behaviors can hide details of signaling pathways. Here we present an approach using single molecule fluorescence resonance energy transfer (FRET) to observe the conformation of individual SNARE proteins as they fold to enter the SNARE complex in living cells. Proteins were recombinantly expressed, labeled with small-molecule fluorescent dyes and microinjected for in vivo imaging and tracking using total internal reflection microscopy. Observing single molecules avoids the difficulties of averaging over unsynchronized ensembles. Our approach is easily generalized to a wide variety of proteins in many cellular signaling pathways.

Experimental estimation of snare detectability for robust threat monitoring.

PubMed

O'Kelly, Hannah J; Rowcliffe, J Marcus; Durant, Sarah; Milner-Gulland, E J

2018-02-01

Hunting with wire snares is rife within many tropical forest systems, and constitutes one of the severest threats to a wide range of vertebrate taxa. As for all threats, reliable monitoring of snaring levels is critical for assessing the relative effectiveness of management interventions. However, snares pose a particular challenge in terms of tracking spatial or temporal trends in their prevalence because they are extremely difficult to detect, and are typically spread across large, inaccessible areas. As with cryptic animal targets, any approach used to monitor snaring levels must address the issue of imperfect detection, but no standard method exists to do so. We carried out a field experiment in Keo Seima Wildlife Reserve in eastern Cambodia with the following objectives: (1) To estimate the detection probably of wire snares within a tropical forest context, and to investigate how detectability might be affected by habitat type, snare type, or observer. (2) To trial two sets of sampling protocols feasible to implement in a range of challenging field conditions. (3) To conduct a preliminary assessment of two potential analytical approaches to dealing with the resulting snare encounter data. We found that although different observers had no discernible effect on detection probability, detectability did vary between habitat type and snare type. We contend that simple repeated counts carried out at multiple sites and analyzed using binomial mixture models could represent a practical yet robust solution to the problem of monitoring snaring levels both inside and outside of protected areas. This experiment represents an important first step in developing improved methods of threat monitoring, and such methods are greatly needed in southeast Asia, as well as in as many other regions.

Frontotemporal dysregulation of the SNARE protein interactome is associated with faster cognitive decline in old age.

PubMed

Ramos-Miguel, Alfredo; Jones, Andrea A; Sawada, Ken; Barr, Alasdair M; Bayer, Thomas A; Falkai, Peter; Leurgans, Sue E; Schneider, Julie A; Bennett, David A; Honer, William G

2018-06-01

The molecular underpinnings associated with cognitive reserve remain poorly understood. Because animal models fail to fully recapitulate the complexity of human brain aging, postmortem studies from well-designed cohorts are crucial to unmask mechanisms conferring cognitive resistance against cumulative neuropathologies. We tested the hypothesis that functionality of the SNARE protein interactome might be an important resilience factor preserving cognitive abilities in old age. Cognition was assessed annually in participants from the Rush "Memory and Aging Project" (MAP), a community-dwelling cohort representative of the overall aging population. Associations between cognition and postmortem neurochemical data were evaluated in functional assays quantifying various species of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) machinery in samples from the inferior temporal (IT, n = 154) and middle-frontal (MF, n = 174) gyri. Using blue-native gel electrophoresis, we isolated and quantified several types of complexes containing the three SNARE proteins (syntaxin-1, SNAP25, VAMP), as well as the GABAergic/glutamatergic selectively expressed complexins-I/II (CPLX1/2), in brain tissue homogenates and reconstitution assays with recombinant proteins. Multivariate analyses revealed significant associations between IT and MF neurochemical data (SNARE proteins and/or complexes), and multiple age-related neuropathologies, as well as with multiple cognitive domains of MAP participants. Controlling for demographic variables, neuropathologic indices and total synapse density, we found that temporal 150-kDa SNARE species (representative of pan-synaptic functionality) and frontal CPLX1/CPLX2 ratio of 500-kDa heteromeric species (representative of inhibitory/excitatory input functionality) were, among all the immunocharacterized complexes, the strongest predictors of cognitive function nearest death. Interestingly, these two neurochemical

Architecture of the Synaptotagmin-SNARE Machinery for Neuronal Exocytosis

PubMed Central

Zhou, Qiangjun; Lai, Ying; Bacaj, Taulant; Zhao, Minglei; Lyubimov, Artem Y.; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Brewster, Aaron S.; Sauter, Nicholas K.; Cohen, Aina E.; Soltis, S. Michael; Alonso-Mori, Roberto; Chollet, Matthieu; Lemke, Henrik T.; Pfuetzner, Richard A.; Choi, Ucheor B.; Weis, William I.; Diao, Jiajie; Südhof, Thomas C.; Brunger, Axel T.

2015-01-01

Summary Synaptotagmin-1 and neuronal SNARE proteins play key roles in evoked synchronous neurotransmitter release. However, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many sidechains. The structures revealed several interfaces, including a large, specific, Ca2+-independent, and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms prior to Ca2+-triggering, and moves en bloc as Ca2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces. PMID:26280336

Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis

DOE PAGES

Zhou, Qiangjun; Lai, Ying; Bacaj, Taulant; ...

2015-08-17

Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. We report atomic-resolution crystal structures of Ca 2+- and Mg 2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca 2+-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca 2+-triggered neurotransmitter releasemore » in mouse hippocampal neuronal synapses and for Ca 2+-triggered vesicle fusion in a reconstituted system. Lastly, we propose that this interface forms before Ca 2+ triggering, moves en bloc as Ca 2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.« less

v-SNAREs control exocytosis of vesicles from priming to fusion.

PubMed

Borisovska, Maria; Zhao, Ying; Tsytsyura, Yaroslav; Glyvuk, Nataliya; Takamori, Shigeo; Matti, Ulf; Rettig, Jens; Südhof, Thomas; Bruns, Dieter

2005-06-15

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

Progress in understanding the neuronal SNARE function and its regulation.

PubMed

Yoon, T-Y; Shin, Y-K

2009-02-01

Vesicle budding and fusion underlies many essential biochemical deliveries in eukaryotic cells, and its core fusion machinery is thought to be built on one protein family named soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). Recent technical advances based on site-directed fluorescence labelling and nano-scale detection down to the single-molecule level rapidly unveiled the protein and the lipid intermediates along the fusion pathway as well as the molecular actions of fusion effectors. Here we summarize these new exciting findings in context with a new mechanistic model that reconciles two existing fusion models: the proteinaceous pore model and the hemifusion model. Further, we attempt to locate the points of action for the fusion effectors along the fusion pathway and to delineate the energetic interplay between the SNARE complexes and the fusion effectors.

Were snares and traps used in the Middle Stone Age and does it matter? A review and a case study from Sibudu, South Africa.

PubMed

Wadley, Lyn

2010-02-01

The concept of remote capture involved in the creation and use of snares and traps is one of several indicators that can be used for the recognition of enhanced working memory and complex cognition. It can be argued that this humble technology is a more reliable indicator of complex cognition than encounter hunting, for example with spears. It is difficult to recognize snares and traps archaeologically because they are generally made from materials that do not preserve well. To infer their presence in the past, it is therefore necessary to rely on circumstantial evidence such as mortality profiles, taxonomic diversity and high frequencies of creatures that are susceptible to capture in snares or traps. Clearly there are some problems with using snares to infer complex cognition because people do not necessarily choose meat-getting strategies with the lowest costs. Although snares make economic sense because they reduce search costs, their use by modern hunters is not associated with the type of status accorded to other means of hunting. Social demands, more than economic or environmental ones, may consequently have determined the amount of snaring and trapping that occurred in the past. Because of social attitudes, an absence of snaring need not mean that people were incapable of using this technique. At Sibudu, a South African Middle Stone Age site, snares or other non-selective capture techniques may have been used during the Howiesons Poort and perhaps also the Still Bay Industry. The circumstantial evidence consists of 1. high frequency representations of animals that prefer forested environments, including the tiny blue duiker (adult and juvenile) and the dangerous bushpig, 2. high frequencies of small mammals, 3. high taxonomic diversity and, 4. the presence of small carnivores. Importantly, the Howiesons Poort faunal assemblage is different from that in more recent Middle Stone Age occupations of the site.

Evolutionarily diverse SYP1 Qa-SNAREs jointly sustain pollen tube growth in Arabidopsis.

PubMed

Slane, Daniel; Reichardt, Ilka; El Kasmi, Farid; Bayer, Martin; Jürgens, Gerd

2017-11-01

Intracellular membrane fusion is effected by SNARE proteins that reside on adjacent membranes and form bridging trans-SNARE complexes. Qa-SNARE members of the Arabidopsis SYP1 family are involved in membrane fusion at the plasma membrane or during cell plate formation. Three SYP1 family members have been classified as pollen-specific as inferred from gene expression profiling studies, and two of them, SYP124 and SYP125, are confined to angiosperms. The SYP124 gene appears genetically unstable, whereas its sister gene SYP125 shows essentially no variation among Arabidopsis accessions. The third pollen-specific member SYP131 is sister to SYP132, which appears evolutionarily conserved in the plant lineage. Although evolutionarily diverse, the three SYP1 proteins are functionally overlapping in that only the triple mutant syp124 syp125 syp131 shows a specific and severe male gametophytic defect. While pollen development and germination appear normal, pollen tube growth is arrested during passage through the style. Our results suggest that angiosperm pollen tubes employ a combination of ancient and modern Qa-SNARE proteins to sustain their growth-promoting membrane dynamics during the reproductive process. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

PubMed Central

Wu, Zhenyong; Thiyagarajan, Sathish; O’Shaughnessy, Ben; Karatekin, Erdem

2017-01-01

Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically

COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal

PubMed Central

Xu, Peng; Hankins, Hannah M; MacDonald, Chris; Erlinger, Samuel J; Frazier, Meredith N; Diab, Nicholas S; Piper, Robert C; Jackson, Lauren P; MacGurn, Jason A

2017-01-01

The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI binds K63-linked polyubiquitin and this interaction is crucial for trafficking of a ubiquitinated yeast SNARE (Snc1). Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through the endocytic pathway to the Golgi for reuse in exocytosis. Removal of ubiquitin from Snc1, or deletion of a β'-COP subunit propeller domain that binds K63-linked polyubiquitin, disrupts Snc1 recycling causing aberrant accumulation in internal compartments. Moreover, replacement of the β'-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles in a non-degradative pathway. PMID:29058666

[Electrocautery snare efficacy in head and neck lesion treatment].

PubMed

Saito, Koichiro; Inagaki, Koji; Naganishi, Hideki; Takaoka, Takuji; Isogai, Yutaka; Ogawa, Kaoru

2009-11-01

The electrocautery snare has been widely used in gastroenterology and to remove bronchial and urinary bladder lesions, but rarely in head and lesion electrocautery. Since October 2006, we have used this instrument to treat 17 head and neck granuloma, papilloma, cyst, and cancer lesions under local or general anesthesia. Lesions were localized in the larynx, oropharynx, hypopharynx, or tracheostoma. The snare was used through a rhinolaryngovideoscope in most cases with a current of 15-30 watts produced by an electrosurgical generator. All procedures were easy, quick and successful, with minimal bleeding. No severe adverse effects were seen in any of our cases. The electrocautery snare was extremely useful in treating pedunculated lesions and in removing epiglottic cysts with a clear margin after excision of the mass without bleeding. Our results indicate the electrocautery snare to be useful and safe in treating selected head and neck lesion cases.

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

PubMed

Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

2016-01-13

Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6

NASA Astrophysics Data System (ADS)

Dai, Yawei; Seeger, Markus; Weng, Jingwei; Song, Song; Wang, Wenning; Tan, Yan-Wen

2016-08-01

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6

PubMed Central

Dai, Yawei; Seeger, Markus; Weng, Jingwei; Song, Song; Wang, Wenning; Tan, Yan-Wen

2016-01-01

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view. PMID:27493064

SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

PubMed Central

Lee, Miriam; Ko, Young-Joon; Moon, Yeojin; Han, Minsoo; Kim, Hyung-Wook; Lee, Sung Haeng; Kang, KyeongJin

2015-01-01

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion. PMID:26216899

Monorail snare technique for the recovery of stretched platinum coils: technical case report.

PubMed

Fiorella, David; Albuquerque, Felipe C; Deshmukh, Vivek R; McDougall, Cameron G

2005-07-01

Coil stretching represents a potentially hazardous technical complication not infrequently encountered during the embolization of cerebral aneurysms. Often, the stretched coil cannot be advanced into the aneurysm or withdrawn intact. The operator is then forced to attempt to retract the damaged coil, which may result in coil breakage, leaving behind a significant length of potentially thrombogenic stretched coil material within the parent vessel. To overcome this problem, we devised a technique to snare the distal, unstretched, intact portion of the platinum coil by use of the indwelling microcatheter and stretched portion of the coil as a monorail guide. We have used this technique successfully in four patients to snare coils stretched during cerebral aneurysm embolization. Three of these patients were undergoing Neuroform (Boston Scientific/Target, Fremont, CA) stent-supported coil embolization of unruptured aneurysms. In all cases, the snare was advanced easily to the targeted site for coil engagement by use of the microcatheter as a monorail guide. Once the intact distal segment of the coil was ensnared, coil removal was uneventful, with no disturbance of the remainder of the indwelling coil pack or Neuroform stent. A 2-mm Amplatz Goose Neck microsnare (Microvena Corp., White Bear Lake, MN) was placed through a Prowler-14 microcatheter (Cordis Corp., Miami, FL). The hub of the indwelling SL-10 microcatheter (Boston Scientific, Natick, MA) was then cut away with a scalpel, leaving the coil pusher wire intact, and removed. The open 2-mm snare was then advanced over the outside of the coil pusher wire and microcatheter. The snare and Prowler-14 microcatheter were then advanced into the guiding catheter (6- or 7-French) as a unit over the indwelling SL-10 microcatheter. By use of the SL-10 microcatheter and coil as a "monorail" guide, the snare was advanced over and beyond the microcatheter and the stretched portion of the coil until the snare was in position to

Single Molecule Measurements of Interaction Free Energies Between the Proteins Within Binary and Ternary SNARE Complexes

PubMed Central

Liu, W.; Montana, Vedrana; Parpura, Vladimir; Mohideen, U.

2010-01-01

We use an Atomic Force Microscope based single molecule measurements to evaluate the activation free energy in the interaction of SNARE proteins syntaxin 1A, SNAP25B and synaptobrevin 2 which regulate intracellular fusion of vesicles with target membranes. The dissociation rate of the binary syntaxin-synaptobrevin and the ternary syntaxin-SNAP25B-synaptobrevin complex was measured from the rupture force distribution as a function of the rate of applied force. The temperature dependence of the spontaneous dissociation rate was used to obtain the activation energy to the transition state of 19.8 ± 3.5 kcal/mol = 33 ± 6 kBT and 25.7 ± 3.0 kcal/mol = 43 ± 5 kBT for the binary and ternary complex, respectively. They are consistent with those measured previously for the ternary complex in lipid membranes and are of order expected for bilayer fusion and pore formation. The ΔG was 12.4–16.6 kcal/mol = 21–28 kBT and 13.8–18.0 kcal/mol = 23–30 kBT for the binary and ternary complex, respectively. The ternary complex was more stable by 1.4 kcal/mol = 2.3 kBT, consistent with the spontaneous dissociation rates. The higher adhesion energies and smaller molecular extensions measured with SNAP25B point to its possible unique and important physiological role in tethering/docking the vesicle in closer proximity to the plasma membrane and increasing the probability for fusion completion. PMID:20107522

MoVam7, a Conserved SNARE Involved in Vacuole Assembly, Is Required for Growth, Endocytosis, ROS Accumulation, and Pathogenesis of Magnaporthe oryzae

PubMed Central

Dou, Xianying; Wang, Qi; Qi, Zhongqiang; Song, Wenwen; Wang, Wei; Guo, Min; Zhang, Haifeng; Zhang, Zhengguang; Wang, Ping; Zheng, Xiaobo

2011-01-01

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in membrane fusion and vesicle transport of eukaryotic organisms including fungi. We previously identified MoSce22 as a homolog of Saccharomyces cerevisiae SNARE protein Sec22 to be involved in growth, stress resistance, and pathogenicity of Magnaporthe oryzae. Here, we provide evidences that MoVam7, an ortholog of S. cerevisiae SNARE protein Vam7, exerts conserved functions in vacuolar morphogenesis and functions in pathogenicity of M. oryzae. Staining with neutral red and FM4-64 revealed the presence of abnormal fragmented vacuoles and an absence of the Spitzenkörper body in the ΔMovam7 mutant. The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium. Additionally, treatments with cell wall perturbing agents indicated weakened cell walls and altered distributions of the cell wall component chitin. Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant. In summary, our studies indicate that MoVam7, like MoSec22, is a component of the SNARE complex whose functions in vacuole assembly also underlies the growth, conidiation, appressorium formation, and pathogenicity of M. oryzae. Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity. PMID:21283626

Use of a rectal snare to remove a hypopharyngeal haemangioma.

PubMed

Abo-Khatwa, M M; Abouel-Enin, S; Klimach, O; Osborne, J

2007-02-01

We describe in this case report a new technique for treatment of hypopharyngeal haemangioma, using the surgical diathermy snare. The snare was easily introduced through the direct laryngoscope, without any difficulties. The procedure was simple, rapid and involved minimal bleeding. We also discuss the histological types of haemangioma, clinical picture, radiological findings and other modalities of treatment.

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis*

PubMed Central

Ramakrishnan, Neeliyath A.; Drescher, Marian J.; Morley, Barbara J.; Kelley, Philip M.; Drescher, Dennis G.

2014-01-01

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (KD = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca2+. At 20 μm Ca2+, the dissociation rate was substantially lower, indicating increased binding (KD = ∼10−9) compared with 0 μm Ca2+ (KD = ∼10−8), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca2+, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion. PMID:24478316

The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

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

Zhou, Qiangjun; Zhou, Peng; Wang, Austin L.

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for themore » primed pre-fusion state. Ca 2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. In conclusion, the tripartite SNARE–complexin–synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.« less

The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

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

Zhou, Qiangjun; Zhou, Peng; Wang, Austin L.

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for themore » primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE–complexin–synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.« less

The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

DOE PAGES

Zhou, Qiangjun; Zhou, Peng; Wang, Austin L.; ...

2017-08-16

Synaptotagmin, complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for themore » primed pre-fusion state. Ca 2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. In conclusion, the tripartite SNARE–complexin–synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.« less

Tree-ring reconstruction of streamflow in the Snare River Basin, Northwest Territories, Canada

NASA Astrophysics Data System (ADS)

Martin, J. P.; Pisaric, M. F.

2017-12-01

Drought is a component of many ecosystems in North America causing environmental and socioeconomical impacts. In the ongoing context of climatic and environmental changes, drought-related issues are becoming problematic in northern Canada, which have not been associated with drought-like conditions in the past. Dryer than average conditions threatens the energy security of northern canadian communities, since this region relies on the production of hydroelectricity as an energy source. In the North Slave Region of Northwest Territory (NWT), water levels and streamflows were significantly lower in 2014/2015. The Government of the NWT had to spend nearly $50 million to purchase diesel fuel to generate enough electricity to supplement the reduced power generation of the Snare River hydroelectric system, hence the need to better understand the multi-decadal variability in streamflow. The aims of this presentation are i) to present jack pine and white spruce tree-ring chronologies of Southern NWT; ii) to reconstruct past streamflow of the Snare River Basin; iii) to evaluate the frequency and magnitude of extreme drought conditions, and iv) to identify which large-scale atmospheric or oceanic patterns are teleconnected to regional hydraulic conditions. Preliminary results show that the growth of jack pine and white spruce populations is better correlated with precipitation and temperature, respectively, than hydraulic conditions. Nonetheless, we present a robust streamflow reconstruction of the Snare River that is well correlated with the summer North Atlantic Oscillation (NAO) index, albeit the strength of the correlation is non-stationary. Spectral analysis corroborate the synchronicity between negative NAO conditions and drought conditions. From an operational standpoint, considering that the general occurrence of positive/negative NAO can be predicted, it the hope of the authors that these results can facilitate energetic planning in the Northwest Territories through

Wheat TaNPSN SNARE homologues are involved in vesicle-mediated resistance to stripe rust (Puccinia striiformis f. sp. tritici)

PubMed Central

Wang, Xiaodong; Wang, Xiaojie; Deng, Lin; Chang, Haitao; Dubcovsky, Jorge; Feng, Hao; Han, Qingmei; Huang, Lili; Kang, Zhensheng

2014-01-01

Subcellular localisation of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and their ability to form SNARE complexes are critical for determining the specificity of vesicle fusion. NPSN11, a Novel Plant SNARE (NPSN) gene, has been reported to be involved in the delivery of cell wall precursors to the newly formed cell plate during cytokinesis. However, functions of NPSN genes in plant–pathogen interactions are largely unknown. In this study, we cloned and characterized three NPSN genes (TaNPSN11, TaNPSN12, and TaNPSN13) and three plant defence-related SNARE homologues (TaSYP132, TaSNAP34, and TaMEMB12). TaSYP132 showed a highly specific interaction with TaNPSN11 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. We hypothesize that this interaction may indicate a partnership in vesicle trafficking. Expressions of the three TaNPSNs and TaSYP132 were differentially induced in wheat leaves when challenged by Puccinia striiformis f. sp. tritici (Pst). In virus-induced gene silencing (VIGS) assays, resistance of wheat (Triticum aestivum) cultivar Xingzi9104 to the Pst avirulent race CYR23 was reduced by knocking down TaNPSN11, TaNPSN13 and TaSYP132, but not TaNPSN12, implying diversified functions of these wheat SNARE homologues in prevention of Pst infection and hyphal elongation. Immuno-localization results showed that TaNPSN11 or its structural homologues were mainly distributed in vesicle structures near cell membrane toward Pst hypha. Taken together, our data suggests a role of TaNPSN11 in vesicle-mediated resistance to stripe rust. PMID:24963004

In Situ Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons.

PubMed

Almandoz-Gil, Leire; Persson, Emma; Lindström, Veronica; Ingelsson, Martin; Erlandsson, Anna; Bergström, Joakim

2018-01-01

The aggregation of alpha-synuclein (αSyn) is the pathological hallmark of Parkinson's disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of αSyn is to promote the soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was to visualize the co-localization between αSyn and the SNARE proteins (VAMP-2, SNAP-25, and syntaxin-1) for the first time using in situ proximity ligation assay (PLA). Cortical primary neurons were cultured from either non-transgenic or transgenic mice expressing human αSyn with the A30P mutation under the Thy-1 promoter. With an antibody recognizing both mouse and human αSyn, a PLA signal indicating close proximity between αSyn and the three SNARE proteins was observed both in the soma and throughout the processes. No differences in the extent of PLA signals were seen between non-transgenic and transgenic neurons. With an antibody specific against human αSyn, the PLA signal was mostly located to the soma and was only present in a few cells. Taken together, in situ PLA is a method that can be used to investigate the co-localization of αSyn and the SNARE proteins in primary neuronal cultures.

Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

PubMed Central

Kiessling, Volker; Liang, Binyong; Kreutzberger, Alex J. B.; Tamm, Lukas K.

2017-01-01

Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods. PMID:28360838

Overexpression of a SNARE protein AtBS14b alters BR response in Arabidopsis.

PubMed

Zhu, Zhong Xin; Ye, Hong Bo; Xuan, Yuan Hu; Yao, Da Nian

2014-12-01

N-ethyl-maleimide sensitive factor adaptor protein receptor (SNAREs) domain-containing proteins were known as key players in vesicle-associated membrane fusion. Genetic screening has revealed the function of SNAREs in different aspects of plant biology, but the role of many SNAREs are still unknown. In this study, we have characterized the role of Arabidopsis Qc-SNARE protein AtBS14b in brassinosteroids (BRs) signaling pathway. AtBS14b overexpression (AtBS14b ox) plants exhibited short hypocotyl and petioles lengths as well as insensitivity to exogenously supplied BR, while AtBS14b mutants did not show any visible BR-dependent morphological differences. BR biosynthesis enzyme BR6OX2 expression was slightly lower in AtBS14b ox than in wild type plants. Further BR-mediated repression of BR6OX2, CPD and DWF4 was inhibited in AtBS14b ox plants. AtBS14b-mCherry fusion protein localized in vesicular compartments surrounding plasma membrane in N. benthamiana leaves. In addition, isolation of AtBS14b-interacting BR signaling protein, which localized in plasma membrane, showed that AtBS14b directly interacted with membrane steroid binding protein 1 (MSBP1), but did not interact with BAK1 or BRI1. These data suggested that Qc-SNARE protein AtBS14b is the first SNARE protein identified that interacts with MSBP1, and the overexpression of AtBS14b modulates BR response in Arabidopsis.

Endoscopic mucosal resection for middle and large colorectal polyps with a double-loop snare.

PubMed

Yoshida, Naohisa; Saito, Yutaka; Hirose, Ryohei; Ogiso, Kiyoshi; Inada, Yutaka; Yagi, Nobuaki; Naito, Yuji; Otake, Yosuke; Nakajima, Takeshi; Matsuda, Takahisa; Yanagisawa, Akio; Itoh, Yoshito

2014-01-01

This study aimed to analyze the endoscopic mucosal resection (EMR) with a novel uniquely shaped, double-loop snare (Dualoop, Medico's Hirata Inc., Tokyo, Japan) for colorectal polyps. This was a clinical trial conducted in two referral centers, Kyoto Prefectural University of Medicine and National Cancer Center Hospital in Japan. First, the firmness of various snares including 'Dualoop' was experimentally analyzed with a pressure gauge. Five hundred and eighty nine consecutive polyps that underwent EMR with 'Dualoop' were compared to 339 polyps with the standard round snare. Lesion characteristics, en bloc resection, and complications were analyzed. 'Dualoop' had the most firmness among the various snares. The average tumor size was 9.3 mm (5-30), and en bloc resection was achieved in 95.4%. The rate of en bloc resection for middle polyps 15-19 mm in diameter was significantly higher with the 'Dualoop' than that with the round snare (97.9 vs. 80.0%, p < 0.05). The rate of en bloc resection was 64.7% for large polyps ≥20 mm in diameter using 'Dualoop'. Higher age, larger tumor size, and superficial polyps were associated with the failure of en bloc resection. EMR with 'Dualoop' was effective for resecting both middle and large polyps en-bloc. © 2014 S. Karger AG, Basel.

Snaring to control feral pigs sus scrofa in a remote Hawaiian rain forest

USGS Publications Warehouse

Anderson, Stephen J.; Stone, Charles P.

1993-01-01

Feral pig Sus scrofa control in Kipahulu Valley, a remote rain forest in Haleakala National Park, Maui, Hawaiian Islands, has been achieved with snares over a 45-month period. Initial pig densities in fenced management units of 6·2 km2 and 7·8 km2were estimated at 6 animals/km2 and 14·3 animals/km2 for the two units, based on population reconstruction from animals killed and aged. During the 45 months of the study, 1978 snares were set, and 1·6 million snare nights were logged. Snare density reached 96/km2 and 200/km2 for the two management units by the end of the study. A mean effort of 43 worker hours/pig was used to remove 53 pigs from the upper management unit, and a mean of 7 worker hours/pig to remove 175 animals from the more densely populated lower unit. Pig activity monitoring along transects provided a good measure of control effectiveness until densities of about 1 pig/km2 were achieved, after which transects became less useful than scouting for determining pig activity.

Exertional myopathy in a grizzly bear (Ursus arctos) captured by leghold snare.

PubMed

Cattet, Marc; Stenhouse, Gordon; Bollinger, Trent

2008-10-01

We diagnosed exertional myopathy (EM) in a grizzly bear (Ursus arctos) that died approximately 10 days after capture by leghold snare in west-central Alberta, Canada, in June 2003. The diagnosis was based on history, post-capture movement data, gross necropsy, histopathology, and serum enzyme levels. We were unable to determine whether EM was the primary cause of death because autolysis precluded accurate evaluation of all tissues. Nevertheless, comparison of serum aspartate aminotransferase and creatine kinase concentrations and survival between the affected bear and other grizzly bears captured by leghold snare in the same research project suggests EM also occurred in other bears, but that it is not generally a cause of mortality. We propose, however, occurrence of nonfatal EM in grizzly bears after capture by leghold snare has potential implications for use of this capture method, including negative effects on wildlife welfare and research data.

Preparation of the cortical reaction: maturation-dependent migration of SNARE proteins, clathrin, and complexin to the porcine oocyte's surface blocks membrane traffic until fertilization.

PubMed

Tsai, Pei-Shiue; van Haeften, Theo; Gadella, Bart M

2011-02-01

The cortical reaction is a calcium-dependent exocytotic process in which the content of secretory granules is released into the perivitellin space immediately after fertilization, which serves to prevent polyspermic fertilization. In this study, we investigated the involvement and the organization of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins in the docking and fusion of the cortical granule membrane with the oolemma in porcine oocytes. During meiotic maturation, secretory vesicles that were labeled with a granule-specific binding lectin, peanut agglutinin (PNA), migrated toward the oocyte's surface. This surface-orientated redistribution behavior was also observed for the oocyte-specific SNARE proteins SNAP23 and VAMP1 that colocalized with the PNA-labeled structures in the cortex area just under the oolemma and with the exclusive localization area of complexin (a trans-SNARE complex-stabilizing protein). The coming together of these proteins serves to prevent the spontaneous secretion of the docked cortical granules and to prepare the oocyte's surface for the cortical reaction, which should probably be immediately compensated for by a clathrin-mediated endocytosis. In vitro fertilization resulted in the secretion of the cortical granule content and the concomitant release of complexin and clathrin into the oocyte's cytosol, and this is considered to stimulate the observed endocytosis of SNARE-containing membrane vesicles.

Blockade of the SNARE Protein Syntaxin 1 Inhibits Glioblastoma Tumor Growth

PubMed Central

Ulloa, Fausto; Gonzàlez-Juncà, Alba; Meffre, Delphine; Barrecheguren, Pablo José; Martínez-Mármol, Ramón; Pazos, Irene; Olivé, Núria; Cotrufo, Tiziana; Seoane, Joan; Soriano, Eduardo

2015-01-01

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE proteins mediate membrane fusion events in cells and are essential for many cellular processes including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. Interestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM. PMID:25803850

Comparative analysis of plant genomes allows the definition of the "Phytolongins": a novel non-SNARE longin domain protein family

PubMed Central

2009-01-01

Background Subcellular trafficking is a hallmark of eukaryotic cells. Because of their pivotal role in the process, a great deal of attention has been paid to the SNARE proteins. Most R-SNAREs, or "longins", however, also possess a highly conserved, N-terminal fold. This "longin domain" is known to play multiple roles in regulating SNARE activity and targeting via interaction with other trafficking proteins. However, the diversity and complement of longins in eukaryotes is poorly understood. Results Our comparative genome survey identified a novel family of longin-related proteins, dubbed the "Phytolongins" because they are specific to land plants. Phytolongins share with longins the N-terminal longin domain and the C-terminal transmembrane domain; however, in the central region, the SNARE motif is replaced by a novel region. Phylogenetic analysis pinpoints the Phytolongins as a derivative of the plant specific VAMP72 longin sub-family and allows elucidation of Phytolongin evolution. Conclusion "Longins" have been defined as R-SNAREs composed of both a longin domain and a SNARE motif. However, expressed gene isoforms and splice variants of longins are examples of non-SNARE motif containing longins. The discovery of Phytolongins, a family of non-SNARE longin domain proteins, together with recent evidence on the conservation of the longin-like fold in proteins involved in both vesicle fusion (e.g. the Trs20 tether) and vesicle formation (e.g. σ and μ adaptin) highlight the importance of the longin-like domain in protein trafficking and suggest that it was one of the primordial building blocks of the eukaryotic membrane-trafficking machinery. PMID:19889231

Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation

PubMed Central

van den Bogaart, Geert; Thutupalli, Shashi; Risselada, Jelger H.; Meyenberg, Karsten; Holt, Matthew; Riedel, Dietmar; Diederichsen, Ulf; Herminghaus, Stephan; Grubmüller, Helmut; Jahn, Reinhard

2011-01-01

Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting the interaction of SNARE proteins between the synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is controversial, and the massive increase in membrane fusion efficiency of Ca2+-synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator: tethering the liposomes too far for SNARE nucleation in the absence of Ca2+, but brings the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively weak electrostatic interactions of synaptotagmin-1 with membranes substantially accelerate fusion. PMID:21642968

Günther Tulip inferior vena cava filter retrieval using a bidirectional loop-snare technique.

PubMed

Ross, Jordan; Allison, Stephen; Vaidya, Sandeep; Monroe, Eric

2016-01-01

Many advanced techniques have been reported in the literature for difficult Günther Tulip filter removal. This report describes a bidirectional loop-snare technique in the setting of a fibrin scar formation around the filter leg anchors. The bidirectional loop-snare technique allows for maximal axial tension and alignment for stripping fibrin scar from the filter legs, a commonly encountered complication of prolonged dwell times.

Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.

PubMed

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R; Chaumont, François

2012-08-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins

PubMed Central

Brunger, Axel T.; Cipriano, Daniel J.; Diao, Jiajie

2015-01-01

Abstract Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed. PMID:25788028

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

PubMed

Rogers, Jason V; Arlow, Tim; Inkellis, Elizabeth R; Koo, Timothy S; Rose, Mark D

2013-12-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

Reconstitution of SNARE proteins into solid-supported lipid bilayer stacks and X-ray structure analysis.

PubMed

Xu, Yihui; Kuhlmann, Jan; Brennich, Martha; Komorowski, Karlo; Jahn, Reinhard; Steinem, Claudia; Salditt, Tim

2018-02-01

SNAREs are known as an important family of proteins mediating vesicle fusion. For various biophysical studies, they have been reconstituted into supported single bilayers via proteoliposome adsorption and rupture. In this study we extended this method to the reconstitution of SNAREs into supported multilamellar lipid membranes, i.e. oriented multibilayer stacks, as an ideal model system for X-ray structure analysis (X-ray reflectivity and diffraction). The reconstitution was implemented through a pathway of proteomicelle, proteoliposome and multibilayer. To monitor the structural evolution in each step, we used small-angle X-ray scattering for the proteomicelles and proteoliposomes, followed by X-ray reflectivity and grazing-incidence small-angle scattering for the multibilayers. Results show that SNAREs can be successfully reconstituted into supported multibilayers, with high enough orientational alignment for the application of surface sensitive X-ray characterizations. Based on this protocol, we then investigated the effect of SNAREs on the structure and phase diagram of the lipid membranes. Beyond this application, this reconstitution protocol could also be useful for X-ray analysis of many further membrane proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparing scat detection dogs, cameras, and hair snares for surveying carnivores

USGS Publications Warehouse

Long, Robert A.; Donovan, T.M.; MacKay, Paula; Zielinski, William J.; Buzas, Jeffrey S.

2007-01-01

Carnivores typically require large areas of habitat, exist at low natural densities, and exhibit elusive behavior - characteristics that render them difficult to study. Noninvasive survey methods increasingly provide means to collect extensive data on carnivore occupancy, distribution, and abundance. During the summers of 2003-2004, we compared the abilities of scat detection dogs, remote cameras, and hair snares to detect black bears (Ursus americanus), fishers (Martes pennanti), and bobcats (Lynx rufus) at 168 sites throughout Vermont. All 3 methods detected black bears; neither fishers nor bobcats were detected by hair snares. Scat detection dogs yielded the highest raw detection rate and probability of detection (given presence) for each of the target species, as well as the greatest number of unique detections (i.e., occasions when only one method detected the target species). We estimated that the mean probability of detecting the target species during a single visit to a site with a detection dog was 0.87 for black bears, 0.84 for fishers, and 0.27 for bobcats. Although the cost of surveying with detection dogs was higher than that of remote cameras or hair snares, the efficiency of this method rendered it the most cost-effective survey method.

Mechanical Coupling via the Membrane Fusion SNARE Protein Syntaxin 1A: A Molecular Dynamics Study

PubMed Central

Knecht, Volker; Grubmüller, Helmut

2003-01-01

SNARE trans complexes between membranes likely promote membrane fusion. For the t-SNARE syntaxin 1A involved in synaptic transmission, the secondary structure and bending stiffness of the five-residue juxtamembrane linker is assumed to determine the required mechanical energy transfer from the cytosolic core complex to the membrane. These properties have here been studied by molecular dynamics and annealing simulations for the wild-type and a C-terminal-prolongated mutant within a neutral and an acidic bilayer, suggesting linker stiffnesses above 1.7 but below 50 × 10−3 kcal mol−1 deg−2. The transmembrane helix was found to be tilted by 15° and tightly anchored within the membrane with a stiffness of 4–5 kcal mol−1 Å−2. The linker turned out to be marginally helical and strongly influenced by its lipid environment. Charged lipids increased the helicity and H3 helix tilt stiffness. For the wild type, the linker was seen embedded deeply within the polar region of the bilayer, whereas the prolongation shifted the linker outward. This reduced its helicity and increased its average tilt, thereby presumably reducing fusion efficiency. Our results suggest that partially unstructured linkers provide considerable mechanical coupling; the energy transduced cooperatively by the linkers in a native fusion event is thus estimated to be 3–8 kcal/mol, implying a two-to-five orders of magnitude fusion rate increase. PMID:12609859

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating

PubMed Central

Rogers, Jason V.; Arlow, Tim; Inkellis, Elizabeth R.; Koo, Timothy S.; Rose, Mark D.

2013-01-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide–sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway. PMID:24152736

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121[W

PubMed Central

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S.; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R.; Chaumont, François

2012-01-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis. PMID:22942383

SNARE-mediated trafficking of {alpha}{sub 5}{beta}{sub 1} integrin is required for spreading in CHO cells

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

Skalski, Michael; Coppolino, Marc G.

2005-10-07

In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cellmore » spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of {alpha}{sub 5}{beta}{sub 1} integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.« less

Snare coupling of the pre-pectoral pacing lead delivery catheter to the femoral transseptal apparatus for endocardial cardiac resynchronization therapy : mid-term results.

PubMed

Patel, Mehul B; Worley, Seth J

2013-04-01

Limitations imposed by the coronary sinus venous anatomy triggered the transseptal approach for endocardial LV lead placement. The alignment of the interatrial septum (IAS) and its neighborhood anatomy does not favor transseptal puncture from the pre-pectoral area. Locating and advancing a pre-pectoral LV lead delivery catheter (PDC) through an opening created in the IAS via femoral transseptal puncture (FTP) is time consuming and technically difficult. We describe a method where the PDC is snare coupled to the femoral transseptal apparatus (FTA). When the FTA is advanced into the left atrium (LA) the coupled PDC follows. The catheter of a 25-mm loop snare kit is replaced with the PDC (SelectSite®). The snare loop is positioned in the right common iliac vein from the pre-pectoral access. The PDC is coupled to the FTA by advancing the transseptal apparatus through the open snare loop. After conventional FTP, the FTA is withdrawn back into the right atrium (RA) over an extra support wire positioned in the LA. The PDC with open snare loop is pulled over the FTA up to the RA. The PDC is advanced to close the snare loop on the extra support wire immediately distal to the tip of the dilator close to the puncture site. The PDC is deflected to align with the FTA. The snare coupled catheters are gently advanced across the IAS into the LA. The PDC is released from the FTA by advancing the snare and opening the loop; the snare is then removed from the PDC. The PDC is deflected and advanced into the left ventricle (LV). After positioning the 4.1 Fr lumen less LV lead, the PDC is sliced and removed. The PDC snare coupled to the FTA was advanced into the LA in all five patients, however, access was lost during catheter manipulation in the one right-sided case. Endocardial LV lead was successfully positioned in all five patients. Snare coupling the pre-pectoral SelectSite® catheter to the FTA is technically simple, reliable and a safe method for transseptal endocardial LV lead

Efficacy of lures and hair snares to detect lynx

Treesearch

Gregory W. McDaniel; Kevin S. McKelvey; John R. Squires; Leonard F. Ruggiero

2000-01-01

Resource managers lack an inexpensive and quantifiable method to detect lynx presence across large landscapes. We tested efficacy of a protocol based on hair snagging to detect presence of lynx (Lynx canadensis). We tested 2 key elements of the protocol: 1) a hair-snaring device and 2) commercial lures used to attract and elicit rubbing behavior in lynx. The...

Prospective randomized comparison of cold snare polypectomy and conventional polypectomy for small colorectal polyps.

PubMed

Ichise, Yasuyuki; Horiuchi, Akira; Nakayama, Yoshiko; Tanaka, Naoki

2011-01-01

The ideal method to remove small colorectal polyps is unknown. We compared removal by colon snare transection without electrocautery (cold snare polypectomy) with conventional electrocautery snare polypectomy (hot polypectomy) in terms of procedure duration, difficulty in retrieving polyps, bleeding, and post-polypectomy symptoms. Patients with colorectal polyps up to 8 mm in diameter were randomized to polypectomy by cold snare technique (cold group) or conventional polypectomy (conventional group). The principal outcome measures were abdominal symptoms within 2 weeks after polypectomy. Secondary outcome measures were the rates of retrieval of colorectal polyps and bleeding. Eighty patients were randomized: cold group, n = 40 (101 polyps) and conventional group, n = 40 (104 polyps). The patients' demographic characteristics and the number and size of polyps removed were similar between the two techniques. Procedure time was significantly shorter with cold polypectomy vs. conventional polypectomy (18 vs. 25 min, p < 0.0001). Complete polyp retrieval rates were identical [96% (97/101) vs. 96% (100/104)]. No bleeding requiring hemostasis occurred in either group. Abdominal symptoms shortly after polypectomy were more common with conventional polypectomy (i.e. 20%; 8/40) than with cold polypectomy (i.e. 2.5%; 1/40; p = 0.029). Cold polypectomy was superior to conventional polypectomy in terms of procedure time and post-polypectomy abdominal symptoms. The two methods were otherwise essentially identical in terms of bleeding risk and complete polyp retrieval. Cold polypectomy is therefore the preferred method for removal of small colorectal polyps. Copyright © 2011 S. Karger AG, Basel.

Endovascular technique using a snare and suture for retrieving a migrated peripherally inserted central catheter in the left pulmonary artery

PubMed Central

Teragawa, Hiroki; Sueda, Takashi; Fujii, Yuichi; Takemoto, Hiroaki; Toyota, Yasushi; Nomura, Shuichi; Nakagawa, Keigo

2013-01-01

We report a successful endovascular technique using a snare with a suture for retrieving a migrated broken peripherally inserted central catheter (PICC) in a chemotherapy patient. A 62-year-old male received monthly chemotherapy through a central venous port implanted into his right subclavian area. The patient completed chemotherapy without complications 1 mo ago; however, he experienced pain in the right subclavian area during his last chemotherapy session. Computed tomography on that day showed migration of a broken PICC in his left pulmonary artery, for which the patient was admitted to our hospital. We attempted to retrieve the ectopic PICC through the right jugular vein using a gooseneck snare, but were unsuccessful because the catheter was lodged in the pulmonary artery wall. Therefore, a second attempt was made through the right femoral vein using a snare with triple loops, but we could not grasp the migrated PICC. Finally, a string was tied to the top of the snare, allowing us to curve the snare toward the pulmonary artery by pulling the string. Finally, the catheter body was grasped and retrieved. The endovascular suture technique is occasionally extremely useful and should be considered by interventional cardiologists for retrieving migrated catheters. PMID:24109502

Acoustics of Idakkā: An Indian snare drum with definite pitch.

PubMed

Jose, Kevin; Chatterjee, Anindya; Gupta, Anurag

2018-05-01

The vibration of a homogeneous circular membrane backed by two taut strings is shown to yield several harmonic overtones for a wide range of physical and geometric parameters. Such a membrane is present at each end of the barrel of an idakkā, an Indian snare drum well known for its rich musicality. The audio recordings of the musical drum are analyzed and a case is made for the strong sense of pitch associated with the drum. A computationally inexpensive model of the string-membrane interaction is proposed assuming the strings to be without inertia. The interaction essentially entails wrapping/unwrapping of the string around a curve on the deforming membrane unlike the colliding strings in Western snare drums. The range of parameters for which harmonicity is achieved is examined and is found to be conforming with what is used in actual drum playing and construction.

Application of a snare technique in retrograde chronic total occlusion percutaneous coronary intervention – a step by step practical approach and an observational study

PubMed Central

Fang, Hsiu-Yu; Lee, Wei-Chieh; Fang, Chih-Yuan; Wu, Chiung-Jen

2016-01-01

Abstract Percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) has recently become popular among interventional cardiologists. CTO originating from the ostium has been one of the most difficult CTO lesions to treat with PCI for a number of reasons. Our aim was to illustrate a specific technique during retrograde CTO PCI referred to as the “snare technique.” We retrospectively examined the use of “snare technique” among 371 consecutive retrograde CTO PCIs performed at our institution between 2006 and 2015. “Snare technique” was used in 10 patients among the 371 retrograde CTO PCIs. The baseline clinical and angiographic characteristics of patients with or without “snare technique” were similar. The “snare technique” group had significantly fewer side branches at occlusion (30.0% vs 71.2%, P = 0.01) and a higher incidence of externalization (90% vs 25.5%, P < 0.001). The contrast volume was significantly lower in the “snare technique” group (285.0 ± 68.5 vs 379.2 ± 144.0, P = 0.04). The incidence of major complications, retrograde success, or final success did not differ between the groups. The “snare technique” is safe and feasible in retrograde CTO PCI, especially in cases of difficult coronary engagement in cases such as ostial occlusion, challenging coronary anatomy, or retrograde guidewire cannot get in antegrade guiding catheter. PMID:27741138

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

PubMed

Lai, Jeffrey K F; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Eskelinen, Eeva-Liisa; Faure, Mathias; Chan, Yoke Fun

2017-07-04

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N -ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.).

PubMed

Bao, Yong-Mei; Sun, Shu-Jing; Li, Meng; Li, Li; Cao, Wen-Lei; Luo, Jia; Tang, Hai-Juan; Huang, Ji; Wang, Zhou-Fei; Wang, Jian-Fei; Zhang, Hong-Sheng

2012-08-10

OsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which function as important components of the vesicle trafficking machinery in eukaryotic cells. In this paper, 12 Qc-SNARE genes were isolated from rice, and expression patterns of 9 genes were detected in various tissues and in seedlings challenged with oxidative stresses and inoculated with rice blast. The expression of OsSYP71 was clearly up-regulated under these stresses. Overexpression of OsSYP71 in rice showed more tolerance to oxidative stress and resistance to rice blast than wild-type plants. These results indicate that Qc-SNAREs play an important role in rice response to environmental stresses, and OsSYP71 is useful in engineering crop plants with enhanced tolerance to oxidative stress and resistance to rice blast. Copyright © 2012 Elsevier B.V. All rights reserved.

Two distinct secretion systems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzae

PubMed Central

Giraldo, Martha C.; Dagdas, Yasin F.; Gupta, Yogesh K.; Mentlak, Thomas A.; Yi, Mihwa; Martinez-Rocha, Ana Lilia; Saitoh, Hiromasa; Terauchi, Ryohei; Talbot, Nicholas J.; Valent, Barbara

2013-01-01

To cause plant diseases, pathogenic micro-organisms secrete effector proteins into host tissue to suppress immunity and support pathogen growth. Bacterial pathogens have evolved several distinct secretion systems to target effector proteins, but whether fungi, which cause the major diseases of most crop species, also require different secretory mechanisms is not known. Here we report that the rice blast fungus Magnaporthe oryzae possesses two distinct secretion systems to target effectors during plant infection. Cytoplasmic effectors, which are delivered into host cells, preferentially accumulate in the biotrophic interfacial complex, a novel plant membrane-rich structure associated with invasive hyphae. We show that the biotrophic interfacial complex is associated with a novel form of secretion involving exocyst components and the Sso1 t-SNARE. By contrast, effectors that are secreted from invasive hyphae into the extracellular compartment follow the conventional secretory pathway. We conclude that the blast fungus has evolved distinct secretion systems to facilitate tissue invasion. PMID:23774898

SNARE-mediated rapid lysosome fusion in membrane raft clustering and dysfunction of bovine coronary arterial endothelium

PubMed Central

Han, Wei-Qing; Xia, Min; Zhang, Chun; Zhang, Fan; Xu, Ming; Li, Ning-Jun

2011-01-01

The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91phox, and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function. PMID:21926345

CVAK104 is a Novel Regulator of Clathrin-mediated SNARE Sorting

PubMed Central

Borner, Georg H H; Rana, Amer A; Forster, Rebecca; Harbour, Michael; Smith, James C; Robinson, Margaret S

2007-01-01

Clathrin-coated vesicles (CCVs) mediate transport between the plasma membrane, endosomes and the trans Golgi network. Using comparative proteomics, we have identified coated-vesicle-associated kinase of 104 kDa (CVAK104) as a candidate accessory protein for CCV-mediated trafficking. Here, we demonstrate that the protein colocalizes with clathrin and adaptor protein-1 (AP-1), and that it is associated with a transferrin-positive endosomal compartment. Consistent with these observations, clathrin as well as the cargo adaptors AP-1 and epsinR can be coimmunoprecipitated with CVAK104. Small interfering RNA (siRNA) knockdown of CVAK104 in HeLa cells results in selective loss of the SNARE proteins syntaxin 8 and vti1b from CCVs. Morpholino-mediated knockdown of CVAK104 in Xenopus tropicalis causes severe developmental defects, including a bent body axis and ventral oedema. Thus, CVAK104 is an evolutionarily conserved protein involved in SNARE sorting that is essential for normal embryonic development. PMID:17587408

Usefulness of a multifunctional snare designed for colorectal hybrid endoscopic submucosal dissection (with video).

PubMed

Ohata, Ken; Muramoto, Takashi; Minato, Yohei; Chiba, Hideyuki; Sakai, Eiji; Matsuhashi, Nobuyuki

2018-02-01

Since colorectal endoscopic submucosal dissection (ESD) remains technically difficult, hybrid ESD was developed as an alternative therapeutic option to achieve en bloc resection of relatively large lesions. In this feasibility study, we evaluated the safety and efficacy of hybrid colorectal ESD using a newly developed multifunctional snare. From June to August 2016, we prospectively enrolled 10 consecutive patients with non-pedunculated intramucosal colorectal tumors 20 - 30 mm in diameter. All of the hybrid ESD steps were performed using the "SOUTEN" snare. The knob-shaped tip attached to the loop top helps to stabilize the needle-knife, making it less likely to slip during circumferential incision and enables partial submucosal dissection. All of the lesions were curatively resected by hybrid ESD, with a short mean procedure time (16.1 ± 4.8 minutes). The mean diameters of the resected specimens and tumors were 30.5 ± 4.9 and 26.0 ± 3.5 mm, respectively. No perforations occurred, while delayed bleeding occurred in 1 patient. In conclusion, hybrid ESD using a multifunctional snare enables easy, safe, and cost-effective resection of relatively large colorectal tumors to be achieved. UMIN000022545.

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane[OPEN

PubMed Central

Karnik, Rucha; Zhang, Ben; Waghmare, Sakharam; Aderhold, Christin; Grefen, Christopher; Blatt, Michael R.

2015-01-01

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners. PMID:25747882

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation

PubMed Central

Lai, Jeffrey K. F.; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Faure, Mathias

2017-01-01

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection. PMID:28677644

Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival

PubMed Central

Kaul, Sunil; Mittal, Sharad K.; Feigenbaum, Lionel; Kruhlak, Michael J.; Roche, Paul A.

2015-01-01

Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane fusion events required for intracellular membrane traffic. The ubiquitously expressed SNARE protein SNAP-23 regulates a wide variety of exocytosis events and is essential for mouse development. Germline deletion of SNAP-23 results in early embryonic lethality in mice, and for this reason we now describe mice and cell lines in which SNAP-23 can be conditionally-deleted using Cre-lox technology. Deletion of SNAP-23 in CD19-Cre expressing mice prevents B lymphocyte development and deletion of SNAP-23 using a variety of T lymphocyte-specific Cre mice prevents T lymphocyte development. Acute depletion of SNAP-23 in mouse fibroblasts leads to rapid apoptotic cell death. These data highlight the importance of SNAP-23 for cell survival and describe a mouse in which specific cell types can be eliminated by expression of tissue-specific Cre-recombinase. PMID:25706117

Calcium accelerates SNARE-mediated lipid mixing through modulating α-synuclein membrane interaction.

PubMed

Zhang, Zeting; Jiang, Xin; Xu, Danrui; Zheng, Wenwen; Liu, Maili; Li, Conggang

2018-04-04

α-Synuclein is involved in Parkinson's disease, and its interaction with cell membrane is vital to its pathological and physiological functions. We have shown that Ca 2+ can regulate α-synuclein membrane interaction, but the physiological role of Ca 2+ in modulating α-synuclein membrane interaction is still unexplored. Based on the previous findings that α-synuclein inhibits membrane fusion and its inhibitory effect is highly related to its membrane binding, here we employed solution state Nuclear Magnetic Resonance (NMR) spectroscopy and the ensemble fluorescence fusion assay to show that Ca 2+ can modulate the inhibitory effect of α-synuclein on SNARE-mediated membrane fusion through disrupting α-synuclein membrane interaction, resulting in acceleration of SNARE-mediated membrane fusion. These results suggest a modulatory effect of Ca 2+ on membrane mediated normal function of α-synuclein, which of importance for the study of the Parkinson's disease. Copyright © 2018 Elsevier B.V. All rights reserved.

EARP, a multisubunit tethering complex involved in endocytic recycling

PubMed Central

Schindler, Christina; Chen, Yu; Pu, Jing; Guo, Xiaoli; Bonifacino, Juan S.

2015-01-01

Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In particular, it is unknown if endocytic recycling requires the function of multisubunit tethering complexes, as is the case for other intracellular trafficking pathways. Herein we describe a tethering complex named Endosome-Associated Recycling Protein (EARP) that is structurally related to the previously described Golgi-Associated Retrograde Protein (GARP) complex. Both complexes share the Ang2, Vps52 and Vps53 subunits, but EARP comprises an uncharacterized protein, Syndetin, in place of the Vps54 subunit of GARP. This change determines differential localization of EARP to recycling endosomes and GARP to the Golgi complex. EARP interacts with the target-SNARE Syntaxin 6 and various cognate SNAREs. Depletion of Syndetin or Syntaxin 6 delays recycling of internalized transferrin to the cell surface. These findings implicate EARP in canonical membrane-fusion events in the process of endocytic recycling. PMID:25799061

The Arabidopsis R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane.

PubMed

Zhang, Ben; Karnik, Rucha; Wang, Yizhou; Wallmeroth, Niklas; Blatt, Michael R; Grefen, Christopher

2015-06-01

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K(+) channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K(+) channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K(+) channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a "hand-off" in channel control between the two SNARE proteins that is woven together with vesicle fusion. © 2015 American Society of Plant Biologists. All rights reserved.

Functional interaction of the SNARE protein NtSyp121 in Ca2+ channel gating, Ca2+ transients and ABA signalling of stomatal guard cells.

PubMed

Sokolovski, Sergei; Hills, Adrian; Gay, Robert A; Blatt, Michael R

2008-03-01

There is now growing evidence that membrane vesicle trafficking proteins, especially of the superfamily of SNAREs, are critical for cellular signalling in plants. Work from this laboratory first demonstrated that a soluble, inhibitory (dominant-negative) fragment of the SNARE NtSyp121 blocked K+ and Cl- channel responses to the stress-related hormone abscisic acid (ABA), but left open a question about functional impacts on signal intermediates, especially on Ca2+-mediated signalling events. Here, we report one mode of action for the SNARE mediated directly through alterations in Ca2+ channel gating and its consequent effects on cytosolic-free [Ca2+] ([Ca2+]i) elevation. We find that expressing the same inhibitory fragment of NtSyp121 blocks ABA-evoked stomatal closure, but only partially suppresses stomatal closure in the presence of the NO donor, SNAP, which promotes [Ca2+]i elevation independently of the plasma membrane Ca2+ channels. Consistent with these observations, Ca2+ channel gating at the plasma membrane is altered by the SNARE fragment in a manner effective in reducing the potential for triggering a rise in [Ca2+]i, and we show directly that its expression in vivo leads to a pronounced suppression of evoked [Ca2+]i transients. These observations offer primary evidence for the functional coupling of the SNARE with Ca2+ channels at the plant cell plasma membrane and, because [Ca2+]i plays a key role in the control of K+ and Cl- channel currents in guard cells, they underscore an important mechanism for SNARE integration with ion channel regulation during stomatal closure.

Molecular characterization and functional significance of the Vti family of SNARE proteins in tick salivary glands

PubMed Central

Villarreal, Ashley M.; Adamson, Steven W.; Browning, Rebecca E.; Khem Raj, B.C.; Sajid, Muhammad Sohail; Karim, Shahid

2013-01-01

Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and A. americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases. PMID:23499931

Histological Comparison of Cold versus Hot Snare Resections of the Colorectal Mucosa.

PubMed

Takayanagi, Daisuke; Nemoto, Daiki; Isohata, Noriyuki; Endo, Shungo; Aizawa, Masato; Utano, Kenichi; Kumamoto, Kensuke; Hojo, Hiroshi; Lefor, Alan Kawarai; Togashi, Kazutomo

2018-06-25

Delayed postpolypectomy bleeding occurs more frequently after hot resection than after cold resection. To elucidate the underlying mechanism, we performed a histological comparison of tissue after cold and hot snare resections. This is a prospective study, registered in the University Hospital Medical Information Network (UMIN000020104). This study was conducted at Aizu Medical Center, Fukushima Medical University, Japan. Fifteen patients scheduled to undergo resection of colorectal cancer were enrolled. On the day before surgery, 2 mucosal resections (hot and cold) of normal mucosa were performed on each patient using the same snare without saline injection. The difference was only the application of electrocautery or not. Resection sites were placed close to the cancer to be included in the surgical specimen. The primary outcome measure was the depth of destruction. Secondary outcome measures included the width of destruction, depth of the remaining submucosa, and number of vessels remaining at the resection sites. The number and diameter of vessels in undamaged submucosa were also evaluated. All cold resections were limited to the shallow submucosa, whereas 60% of hot resections advanced to the deep submucosa and 20% to the muscularis propria (p < 0.001). There was no significant difference in the width of destruction. The number of remaining large vessels after hot resections trended toward fewer (p = 0.15) with a decreased depth of remaining submucosa (p = 0.007). In the deep submucosa, the vessel diameter was larger (p < 0.001) and the number of large vessels was greater (p = 0.018). Histological assessment was not blinded to the 2 reviewers. Normal mucosa was used instead of adenomatous tissue. Hot resection caused damage to deeper layers involving more large vessels. This may explain the mechanism for the reduced incidence of hemorrhage after cold snare polypectomy. See Video Abstract at http://links.lww.com/DCR/A631.

Further refinements of the polyp snare for interuterine surgery--a new modality for treatment of myomas and polyps.

PubMed

McLucas, B

1995-01-01

Hysteroscopic treatment of 30 patients suffering from menorrhagia or post-partum complications was accomplished using an electrosurgical polyp snare. Using this method, 18 polyps and 12 myomas were successfully removed in less than twenty minutes without complications. Local anaesthesia was used in 12 patients. Three patients have presented with recurrence of menorrhagia, with a minimum of six months follow-up. Benefits of this technique compared to uterine resectoscopy include shorter operative time, decreased risk of fluid overload, and less risk of uterine perforation. The snare is difficult to use and a learning curve exists. Higher currents than that used for resection are required.

Rescue endoscopic bleeding control for nonvariceal upper gastrointestinal hemorrhage using clipping and detachable snaring.

PubMed

Lee, J H; Kim, B K; Seol, D C; Byun, S J; Park, K H; Sung, I K; Park, H S; Shim, C S

2013-06-01

Nonvariceal upper gastrointestinal (UGI) bleeding recurs after appropriate endoscopic therapy in 10 % - 15 % of cases. The mortality rate can be as high as 25 % when bleeding recurs, but there is no consensus about the best modality for endoscopic re-treatment. The aim of this study was to evaluate clipping and detachable snaring (CDS) for rescue endoscopic control of nonvariceal UGI hemorrhage. We report a case series of seven patients from a Korean tertiary center who underwent endoscopic hemostasis using the combined method of detachable snares with hemoclips. The success rate of endoscopic hemostasis with CDS was 86 %: six of the seven patients who had experienced primary endoscopic treatment failure or recurrent bleeding after endoscopic hemostasis were treated successfully. In conclusion, rescue endoscopic bleeding control by means of CDS is an option for controlling nonvariceal UGI bleeding when no other method of endoscopic treatment for recurrent bleeding and primary hemostatic failure is possible. © Georg Thieme Verlag KG Stuttgart · New York.

Myosin phosphatase and RhoA-activated kinase modulate neurotransmitter release by regulating SNAP-25 of SNARE complex

PubMed Central

Sipos, Adrienn; Darula, Zsuzsanna; Bécsi, Bálint; Nagy, Dénes; Iván, Judit; Erdődi, Ferenc

2017-01-01

Reversible phosphorylation of neuronal proteins plays an important role in the regulation of neurotransmitter release. Myosin phosphatase holoenzyme (MP) consists of a protein phosphatase-1 (PP1) catalytic subunit (PP1c) and a regulatory subunit, termed myosin phosphatase targeting subunit (MYPT1). The primary function of MP is to regulate the phosphorylation level of contractile proteins; however, recent studies have shown that MP is localized to neurons, and is also involved in the mediation of neuronal processes. Our goal was to investigate the effect of RhoA-activated kinase (ROK) and MP on the phosphorylation of one potential neuronal substrate, the synaptosomal-associated protein of 25 kDa (SNAP-25). SNAP-25 is a member of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, along with synaptobrevin and syntaxin, and the primary role of SNAP25 is to mediate vesicle fusion. We showed that MYPT1 interacts with SNAP-25, as revealed by immunoprecipitation and surface plasmon resonance based binding studies. Mass spectrometry analysis and in vitro phosphorylation/dephosphorylation assays demonstrated that ROK phosphorylates, while MP dephosphorylates, SNAP-25 at Thr138. Silencing MYPT1 in B50 neuroblastoma cells increased phosphorylation of SNAP-25 at Thr138. Inhibition of PP1 with tautomycetin increased, whereas inhibition of ROK by H1152, decreased the phosphorylation of SNAP-25 at Thr138 in B50 cells, in cortical synaptosomes, and in brain slices. In response to the transduction of the MP inhibitor, kinase-enhanced PP1 inhibitor (KEPI), into synaptosomes, an increase in phosphorylation of SNAP-25 and a decrease in the extent of neurotransmitter release were detected. The interaction between SNAP-25 and syntaxin increased with decreasing phosphorylation of SNAP-25 at Thr138, upon inhibition of ROK. Our data suggest that ROK/MP play a crucial role in vesicle trafficking, fusion, and neurotransmitter release by oppositely

Facilitating complex shape drawing in Williams syndrome and typical development.

PubMed

Hudson, Kerry D; Farran, Emily K

2013-07-01

Individuals with Williams syndrome (WS) produce drawings that are disorganised, likely due to an inability to replicate numerous spatial relations between parts. This study attempted to circumvent these drawing deficits in WS when copying complex combinations of one, two and three shapes. Drawing decisions were reduced by introducing a number of facilitators, for example, by using distinct colours and including facilitatory cues on the response sheet. Overall, facilitation improved drawing in the WS group to a comparable level of accuracy as typically developing participants (matched for non-verbal ability). Drawing accuracy was greatest in both groups when planning demands (e.g. starting location, line lengths and changes in direction) were reduced by use of coloured figures and providing easily distinguished and clearly grouped facilitatory cues to form each shape. This study provides the first encouraging evidence to suggest that drawing of complex shapes in WS can be facilitated; individuals with WS might be receptive to remediation programmes for drawing and handwriting. Copyright © 2013 Elsevier Ltd. All rights reserved.

The binding of Varp to VAMP7 traps VAMP7 in a closed, fusogenically inactive conformation

PubMed Central

Schäfer, Ingmar B.; Hesketh, Geoffrey G.; Bright, Nicholas A.; Gray, Sally R.; Pryor, Paul R.; Evans, Philip R; Luzio, J. Paul; Owen, David J.

2012-01-01

SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from Q-SNAREs embedded in one membrane and an R–SNARE embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE-binding regulator of SNARE complex formation. Varp co-localises with and binds to VAMP7, an R-SNARE involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7 SNARE motif is trapped between Varp and the VAMP7 longin domain and hence Varp kinetically inhibits VAMP7’s ability to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as the VAMP7 such as Rab32:GTP. PMID:23104059

Estimating black bear density using DNA data from hair snares

USGS Publications Warehouse

Gardner, B.; Royle, J. Andrew; Wegan, M.T.; Rainbolt, R.E.; Curtis, P.D.

2010-01-01

DNA-based mark-recapture has become a methodological cornerstone of research focused on bear species. The objective of such studies is often to estimate population size; however, doing so is frequently complicated by movement of individual bears. Movement affects the probability of detection and the assumption of closure of the population required in most models. To mitigate the bias caused by movement of individuals, population size and density estimates are often adjusted using ad hoc methods, including buffering the minimum polygon of the trapping array. We used a hierarchical, spatial capturerecapture model that contains explicit components for the spatial-point process that governs the distribution of individuals and their exposure to (via movement), and detection by, traps. We modeled detection probability as a function of each individual's distance to the trap and an indicator variable for previous capture to account for possible behavioral responses. We applied our model to a 2006 hair-snare study of a black bear (Ursus americanus) population in northern New York, USA. Based on the microsatellite marker analysis of collected hair samples, 47 individuals were identified. We estimated mean density at 0.20 bears/km2. A positive estimate of the indicator variable suggests that bears are attracted to baited sites; therefore, including a trap-dependence covariate is important when using bait to attract individuals. Bayesian analysis of the model was implemented in WinBUGS, and we provide the model specification. The model can be applied to any spatially organized trapping array (hair snares, camera traps, mist nests, etc.) to estimate density and can also account for heterogeneity and covariate information at the trap or individual level. ?? The Wildlife Society.

Knife-assisted snare resection: a novel technique for resection of scarred polyps in the colon.

PubMed

Chedgy, Fergus J Q; Bhattacharyya, Rupam; Kandiah, Kesavan; Longcroft-Wheaton, Gaius; Bhandari, Pradeep

2016-03-01

There have been significant advances in the management of complex colorectal polyps. Previous failed resection or polyp recurrence is associated with significant fibrosis, making endoscopic resection extremely challenging; the traditional approach to these lesions is surgery. The aim of this study was to evaluate the efficacy of a novel, knife-assisted snare resection (KAR) technique in the resection of scarred colonic polyps. This was a prospective cohort study of patients, in whom the KAR technique was used to resect scarred colonic polyps > 2  cm in size. Patients had previously undergone endoscopic mucosal resection (EMR) and developed recurrence, or EMR had been attempted but was aborted as a result of technical difficulty. A total of 42 patients underwent KAR of large (median 40  mm) scarred polyps. Surgery for benign disease was avoided in 38 of 41 patients (93 %). No life-threatening complications occurred. Recurrence was seen in six patients (16 %), five of whom underwent further endoscopic resection. The overall cure rate for KAR in complex scarred colonic polyps was 90 %. KAR of scarred colonic polyps by an expert endoscopist was an effective and safe technique with low recurrence rates. © Georg Thieme Verlag KG Stuttgart · New York.

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils

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

Carmo, Lívia A.S.; Dias, Felipe F.; Malta, Kássia K.

Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results: STX17more » was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. �

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

Yang, Yoosoo; Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791; Kim, Se-Hyun

Highlights: • Membrane fusion driven by SNARE complex is hindered by several polyphenols. • Distinctive inhibitory effect of each polyphenol on SNARE zippering in neuron was examined. • FRET between fluorescence protein-tagged SNAREs probed well SNARE zippering in PC12 cells. • Delphinidin and cyanidin inhibit N-terminal SNARE nucleation in Ca{sup 2+}-independent manner. • Myricetin inhibits Ca{sup 2+}-dependent transmembrane association of SNARE complex. - Abstract: Fusion of synaptic vesicles with the presynaptic plasma membrane in the neuron is mediated by soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) proteins. SNARE complex formation is a zippering-like process which initiates at the N-terminus andmore » proceeds to the C-terminal membrane-proximal region. Previously, we showed that this zippering-like process is regulated by several polyphenols, leading to the arrest of membrane fusion and the inhibition of neuroexocytosis. In vitro studies using purified SNARE proteins reconstituted in liposomes revealed that each polyphenol uniquely regulates SNARE zippering. However, the unique regulatory effect of each polyphenol in cells has not yet been examined. In the present study, we observed SNARE zippering in neuronal PC12 cells by measuring the fluorescence resonance energy transfer (FRET) changes of a cyan fluorescence protein (CFP) and a yellow fluorescence protein (YFP) fused to the N-termini or C-termini of SNARE proteins. We show that delphinidin and cyanidin inhibit the initial N-terminal nucleation of SNARE complex formation in a Ca{sup 2+}-independent manner, while myricetin inhibits Ca{sup 2+}-dependent transmembrane domain association of the SNARE complex in the cell. This result explains how polyphenols exhibit botulinum neurotoxin-like activity in vivo.« less

STS-119 EVA 3 GAT SSRMS LEE B Snare Lubrication OPS

NASA Image and Video Library

2009-03-23

S119-E-007105 (23 March 2009) --- Astronaut Richard Arnold, STS-119 mission specialist, participates in the mission's third scheduled session of extravehicular activity (EVA) as construction and maintenance continue on the International Space Station. During the six-hour, 27-minute spacewalk, Arnold and Joseph Acaba (out of frame), mission specialist, helped robotic arm operators relocate the Crew Equipment Translation Aid (CETA) cart from the Port 1 to Starboard 1 truss segment, installed a new coupler on the CETA cart, lubricated snares on the "B" end of the space station's robotic arm and performed a few "get ahead" tasks.

Complexation facilitated reduction of aromatic N-oxides by aqueous Fe(II)-tiron complex: reaction kinetics and mechanisms.

PubMed

Chen, Yiling; Zhang, Huichun

2013-10-01

Rapid reduction of carbadox (CDX), olaquindox and several other aromatic N-oxides were investigated in aqueous solution containing Fe(II) and tiron. Consistent with previous work, the 1:2 Fe(II)-tiron complex, FeL2(6-), is the dominant reactive species as its concentration linearly correlates with the observed rate constant kobs under various conditions. The N-oxides without any side chains were much less reactive, suggesting direct reduction of the N-oxides is slow. UV-vis spectra suggest FeL2(6-) likely forms 5- or 7-membered rings with CDX and olaquindox through the N and O atoms on the side chain. The formed inner-sphere complexes significantly facilitated electron transfer from FeL2(6-) to the N-oxides. Reduction products of the N-oxides were identified by HPLC/QToF-MS to be the deoxygenated analogs. QSAR analysis indicated neither the first electron transfer nor N-O bond cleavage is the rate-limiting step. Calculations of the atomic spin densities of the anionic N-oxides confirmed the extensive delocalization between the aromatic ring and the side chain, suggesting complex formation can significantly affect the reduction kinetics. Our results suggest the complexation facilitated N-oxide reduction by Fe(II)-tiron involves a free radical mechanism, and the subsequent deoxygenation might also benefit from the weak complexation of Fe(II) with the N-oxide O atom.

Rectal temperatures of immobilized, snare-trapped black bears in Great Dismal Swamp.

PubMed

Hellgren, E C; Vaughan, M R

1989-07-01

Rectal temperature was determined for 84 black bears (Ursus americanus) during 99 handlings in Great Dismal Swamp, Virginia and North Carolina (USA). All bears had been trapped with cable snares and immobilized with a 2:1 ketamine hydrochloridexylazine hydrochloride mixture. Temperatures were significantly greater in males and varied significantly by season. Immobilized bears began panting at rectal temperatures greater than 42.0 C. One death occurred at 43.0 C. We recommended cooling measures on black bears at rectal temperatures of greater than or equal to 40.0 C.

Assessment of Snared-Loop Technique When Standard Retrieval of Inferior Vena Cava Filters Fails

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

Doody, Orla, E-mail: orla_doody@hotmail.com; Noe, Geertje; Given, Mark F.

Purpose To identify the success and complications related to a variant technique used to retrieve inferior vena cava filters when simple snare approach has failed. Methods A retrospective review of all Cook Guenther Tulip filters and Cook Celect filters retrieved between July 2006 and February 2008 was performed. During this period, 130 filter retrievals were attempted. In 33 cases, the standard retrieval technique failed. Retrieval was subsequently attempted with our modified retrieval technique. Results The retrieval was successful in 23 cases (mean dwell time, 171.84 days; range, 5-505 days) and unsuccessful in 10 cases (mean dwell time, 162.2 days; range,more » 94-360 days). Our filter retrievability rates increased from 74.6% with the standard retrieval method to 92.3% when the snared-loop technique was used. Unsuccessful retrieval was due to significant endothelialization (n = 9) and caval penetration by the filter (n = 1). A single complication occurred in the group, in a patient developing pulmonary emboli after attempted retrieval. Conclusion The technique we describe increased the retrievability of the two filters studied. Hook endothelialization is the main factor resulting in failed retrieval and continues to be a limitation with these filters.« less

The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

PubMed

Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

2016-09-01

Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

Technical Note [cmUse of a Snare Wire to Perform Nephrostomy Access in the Presence of Obstructive Staghorn Calculi

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

Nosher, John L.; Siegel, Randall L.; Bodner, Leonard J.

1996-05-15

We describe a technique for gaining access to the central collecting system via a chosen calyx, utilizing an alternative entry point to that calyx. An Amplatz nitinol loop snare is then used to convert this access to a traditional approach.

Resection of giant right atrial lymphoma using vacuum-assisted cardiopulmonary bypass without snaring the inferior vena cava.

PubMed

Shin, Hankei; Mori, Mitsuharu; Matayoshi, Toru; Suzuki, Ryo; Yozu, Ryohei

2004-08-01

A 53-year-old man sustained hemodynamic collapse due to a huge right atrial tumor and was transferred to our hospital and underwent a life-saving emergency operation. The tumor arose from the inferolateral wall of the right atrium, occupying almost the whole right atrial cavity and obstructing not only the inflow of the right ventricle but also the orifice of the inferior vena cava. Venous cannulation via the right atrial wall and placing a snare around the inferior vena cava were impossible. With a cardiopulmonary bypass using vacuum-assisted venous drainage, the tumor was successfully resected and the tricuspid valve was replaced with a bioprosthetic valve without snaring the inferior vena cava. Postoperative histological examination demonstrated the tumor to be a large B-cell non-Hodgkin type malignant lymphoma. When the tumor is large and it is difficult to establish total cardiopulmonary bypass, the vacuum-assisted cardiopulmonary bypass is a useful option. This can achieve a bloodless operative field and is not blocked by the incoming air, due to the venous drainage being continually pressure-regulated.

Snare-assisted anterograde balloon mitral and aortic valvotomy using Inoue balloon catheter.

PubMed

Krishnan, Mangalath N; Syamkumar, M D; Sajeev, C G; Venugopal, K; Johnson, Francis; Vinaykumar, D; Velayudhan, C C; Jayakumar, T G

2007-01-02

We performed concurrent antegrade mitral and aortic valvotomy using Inoue dilatation catheter in 3 cases of combined rheumatic mitral and aortic stenosis. Following mitral valvotomy by standard procedure, aortic valve was crossed with the help of a floatation catheter. Stiff long length guide wire was fixed in descending aorta using a snare. Inoue catheter was threaded over the wire across the aortic valve and aortic valvotomy completed. Mitral valve area increased from mean 1 cm2 to 2 cm2; aortic gradient dropped from mean of 97 mm to 36 mm. Concurrent anterograde balloon mitral and aortic valvotomy may be effective and safe.

Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

PubMed Central

Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

2015-01-01

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes. PMID:25635869

Facilitating the Evaluation of Complexity in the Public Sector: Learning from the NHS in Scotland

ERIC Educational Resources Information Center

Connolly, John; Reid, Garth; Mooney, Allan

2015-01-01

It is necessary for public managers to be able to evaluate programmes in the context of complexity. This article offers key learning and reflections based on the experience of facilitating the evaluation of complexity with a range of public sector partners in Scotland. There have been several articles that consider evaluating complexity and…

Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila.

PubMed

Morelli, Elena; Ginefra, Pierpaolo; Mastrodonato, Valeria; Beznoussenko, Galina V; Rusten, Tor Erik; Bilder, David; Stenmark, Harald; Mironov, Alexandre A; Vaccari, Thomas

2014-01-01

How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29.

The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling.

PubMed

Harris, Kathryn P; Zhang, Yao V; Piccioli, Zachary D; Perrimon, Norbert; Littleton, J Troy

2016-05-25

Postsynaptic cells can induce synaptic plasticity through the release of activity-dependent retrograde signals. We previously described a Ca(2+)-dependent retrograde signaling pathway mediated by postsynaptic Synaptotagmin 4 (Syt4). To identify proteins involved in postsynaptic exocytosis, we conducted a screen for candidates that disrupted trafficking of a pHluorin-tagged Syt4 at Drosophila neuromuscular junctions (NMJs). Here we characterize one candidate, the postsynaptic t-SNARE Syntaxin 4 (Syx4). Analysis of Syx4 mutants reveals that Syx4 mediates retrograde signaling, modulating the membrane levels of Syt4 and the transsynaptic adhesion protein Neuroligin 1 (Nlg1). Syx4-dependent trafficking regulates synaptic development, including controlling synaptic bouton number and the ability to bud new varicosities in response to acute neuronal stimulation. Genetic interaction experiments demonstrate Syx4, Syt4, and Nlg1 regulate synaptic growth and plasticity through both shared and parallel signaling pathways. Our findings suggest a conserved postsynaptic SNARE machinery controls multiple aspects of retrograde signaling and cargo trafficking within the postsynaptic compartment.

A technique of snaring method for fitting a prosthetic valve into the annulus.

PubMed

Nagasaka, Shigeo; Kawata, Tetsuji; Matsuta, Masahiro; Taniguchi, Shigeki

2005-01-01

Tourniquetting technique to fit a prosthetic valve (PV) into the annulus in valve replacement surgery has been previously reported. We modified the previously reported method and designed a simpler tying technique. We performed 11 aortic (AVR: including four cases for calcified aortic stenosis (AS) with a small annulus and one cases for infective endocarditis with intramuscular abscess cavity), eight mitral valve replacements (MVR), and one tricuspid valve replacement (TVR: for corrected transposition of the great arteries). A PV was implanted using 2-0 polyester mattress sutures with a pledget. Each of the two tourniquets held a suture at the bottom of the annulus and at the opposite position to fit a PV. The sutures between each snare were tied down from the bottom to the top. In MVR, after seating of a PV with two tourniquets, we could make sure that no native tissue of any preserved mitral apparatus disturbed PV leaflet motion. In calcific AS, a PV had a good fitting into the annulus because of tourniquets applied to unseated part during tying sutures. In AVR for infective endocarditis, mattress sutures supported by a Teflon pledget were placed to close the abscess cavity. After snaring on one of these sutures, we tied down the sutures, ensuring that they did not cut through the friable tissues. In TVR, we found that native leaflets interfered with PV motion after seating down the prosthesis and those leaflets were resected before tying down the sutures. Postoperative transesophageal echocardiography showed no paravalvular leakage in any patients and excellent PV functions.

Coarse-Grain Simulations Reveal Movement of the Synaptobrevin C-Terminus in Response to Piconewton Forces

PubMed Central

Lindau, Manfred; Hall, Benjamin A.; Chetwynd, Alan; Beckstein, Oliver; Sansom, Mark S.P.

2012-01-01

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal transmembrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Application of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct application of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ∼70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex. PMID:23009845

Coarse-grain simulations reveal movement of the synaptobrevin C-terminus in response to piconewton forces.

PubMed

Lindau, Manfred; Hall, Benjamin A; Chetwynd, Alan; Beckstein, Oliver; Sansom, Mark S P

2012-09-05

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal transmembrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Application of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct application of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ∼70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

STS-119 Extravehicular Activity (EVA) 3 GAT SSRMS LEE B Snare Lubrication OPS

NASA Image and Video Library

2009-03-23

S119-E-007469 (23 March 2009) --- Astronaut Richard Arnold, STS-119 mission specialist, participates in the mission's third scheduled session of extravehicular activity (EVA) as construction and maintenance continue on the International Space Station. During the six-hour, 27-minute spacewalk, Arnold and Joseph Acaba (out of frame), mission specialist, helped robotic arm operators relocate the Crew Equipment Translation Aid (CETA) cart from the Port 1 to Starboard 1 truss segment, installed a new coupler on the CETA cart, lubricated snares on the "B" end of the space station's robotic arm and performed a few "get ahead" tasks.

STS-119 Extravehicular Activity (EVA) 3 GAT SSRMS LEE B Snare Lubrication OPS

NASA Image and Video Library

2009-03-23

S119-E-007398 (23 March 2009) --- Astronaut Richard Arnold, STS-119 mission specialist, participates in the mission's third scheduled session of extravehicular activity (EVA) as construction and maintenance continue on the International Space Station. During the six-hour, 27-minute spacewalk, Arnold and Joseph Acaba (out of frame), mission specialist, helped robotic arm operators relocate the Crew Equipment Translation Aid (CETA) cart from the Port 1 to Starboard 1 truss segment, installed a new coupler on the CETA cart, lubricated snares on the "B" end of the space station's robotic arm and performed a few "get ahead" tasks.

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

DOE PAGES

Colbert, Karen N.; Hattendorf, Douglas A.; Weiss, Thomas M.; ...

2013-07-15

In neurons, soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1–24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Inmore » addition, we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a–Syx1a complex.« less

The pollen-specific R-SNARE/longin PiVAMP726 mediates fusion of endo- and exocytic compartments in pollen tube tip growth

PubMed Central

Guo, Feng; McCubbin, Andrew G.

2012-01-01

The growing pollen tube apex is dedicated to balancing exo- and endocytic processes to form a rapidly extending tube. As perturbation of either tends to cause a morphological phenotype, this system provides tractable model for studying these processes. Vesicle-associated membrane protein 7s (VAMP7s) are members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that mediate cognate membrane fusion but their role in pollen tube growth has not been investigated. This manuscript identifies PiVAMP726 of Petunia inflata as a pollen-specific VAMP7 that localizes to the inverted cone of transport vesicles at the pollen tube tip. The endocytic marker FM4-64 was found to colocalize with yellow fluorescent protein (YFP)-PiVAMP726, which is consistent with PiVAMP726 containing an amino-acid motif implicated in endosomal localization, At high overexpression levels, YFP- PiVAMP726 inhibited growth and caused the formation of novel membrane compartments within the pollen tube tip. Functional dissection of PiVAMP726 implicated the N-terminal longin domain in negative regulation of the SNARE activity, but not localization of PiVAMP726. Expression of the constitutively active C-terminal SNARE domain alone, in pollen tubes, generated similar phenotypes to the full-length protein, but the truncated domain was more potent than the wild-type protein at both inhibiting growth and forming the novel membrane compartments. Both endo- and exocytic markers localized to these compartments in addition to YFP-PiVAMP726, leading to the speculation that PiVAMP726 might be involved in the recycling of endocytic vesicles in tip growth. PMID:22345643

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils.

PubMed

Carmo, Lívia A S; Dias, Felipe F; Malta, Kássia K; Amaral, Kátia B; Shamri, Revital; Weller, Peter F; Melo, Rossana C N

2015-10-01

SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. Copyright © 2015 Elsevier Inc. All rights reserved.

Magnetic navigation facilitates percutaneous coronary intervention for complex lesions.

PubMed

Sandhu, Gurpreet S; Sanon, Saurabh; Holmes, David R; Gulati, Rajiv; Brilakis, Emmanouil S; Lennon, Ryan J; Rihal, Charanjit S

2014-10-01

We sought to determine the utility of a magnetic navigation system (MNS) in treating a variety of coronary artery lesions including those that could not be revascularized with standard percutaneous coronary intervention (PCI). MNS may add value in the treatment of tortuous arteries and bifurcation lesions; however its widespread adoption has lagged because of cost and a lack of clear advantage over conventional PCI. We performed a retrospective analysis to determine whether MNS improved procedural success for highly complex lesions. One hundred and forty-eight patients underwent treatment with MNS at Mayo Clinic, Rochester. Clinical data, angiographic and procedural characteristics, lesion crossing success and outcomes were reviewed. Overall 133 patients underwent successful revascularization with 87% (143) of 164 lesions crossed using MNS alone. Another six lesions required a combination of MNS and conventional devices resulting in overall success of 91% (149/164). Eighteen complex lesions had previously failed PCI and 12 (67%) were successfully treated with MNS. Success after failed PCI was higher (88%) when a frequent user operated MNS, but occasional users also noted incremental success (30%). Twenty-five chronic total occlusions were included amongst these 164 lesions, with observed antegrade MNS lesion crossing rates of 78% for regular and 14% for occasional users. MNS is a useful adjunct to performance of PCI. This specialized technology has a clear learning curve and can facilitate treatment of highly complex lesions. © 2013 Wiley Periodicals, Inc.

Escaping the snare of chronological growth and launching a free curve alternative: general deviance as latent growth model.

PubMed

Wood, Phillip Karl; Jackson, Kristina M

2013-08-01

Researchers studying longitudinal relationships among multiple problem behaviors sometimes characterize autoregressive relationships across constructs as indicating "protective" or "launch" factors or as "developmental snares." These terms are used to indicate that initial or intermediary states of one problem behavior subsequently inhibit or promote some other problem behavior. Such models are contrasted with models of "general deviance" over time in which all problem behaviors are viewed as indicators of a common linear trajectory. When fit of the "general deviance" model is poor and fit of one or more autoregressive models is good, this is taken as support for the inhibitory or enhancing effect of one construct on another. In this paper, we argue that researchers consider competing models of growth before comparing deviance and time-bound models. Specifically, we propose use of the free curve slope intercept (FCSI) growth model (Meredith & Tisak, 1990) as a general model to typify change in a construct over time. The FCSI model includes, as nested special cases, several statistical models often used for prospective data, such as linear slope intercept models, repeated measures multivariate analysis of variance, various one-factor models, and hierarchical linear models. When considering models involving multiple constructs, we argue the construct of "general deviance" can be expressed as a single-trait multimethod model, permitting a characterization of the deviance construct over time without requiring restrictive assumptions about the form of growth over time. As an example, prospective assessments of problem behaviors from the Dunedin Multidisciplinary Health and Development Study (Silva & Stanton, 1996) are considered and contrasted with earlier analyses of Hussong, Curran, Moffitt, and Caspi (2008), which supported launch and snare hypotheses. For antisocial behavior, the FCSI model fit better than other models, including the linear chronometric growth curve

Cold-forceps avulsion with adjuvant snare-tip soft coagulation (CAST) is an effective and safe strategy for the management of non-lifting large laterally spreading colonic lesions.

PubMed

Tate, David J; Bahin, Farzan F; Desomer, Lobke; Sidhu, Mayenaaz; Gupta, Vikas; Bourke, Michael J

2018-01-01

 Non-lifting large laterally spreading colorectal lesions (LSLs) are challenging to resect endoscopically and often necessitate surgery. A safe, simple technique to treat non-lifting LSLs endoscopically with robust long-term outcomes has not been described.  In this single-center prospective observational study of consecutive patients referred for endoscopic mucosal resection (EMR) of LSLs ≥ 20 mm, LSLs not completely resectable by snare because of non-lifting underwent standardized completion of resection with cold-forceps avulsion and adjuvant snare-tip soft coagulation (CAST). Scheduled surveillance colonoscopies were performed at 4 - 6 months (SC1) and 18 months (SC2). Primary outcomes were endoscopic evidence of adenoma clearance and avoidance of surgery. The secondary outcome was safety.  From January 2012 to October 2016, 540 lifting LSLs (82.2 %) underwent complete snare excision at EMR. CAST was required for complete removal in 101 non-lifting LSLs (17.8 %): 63 naïve non-lifting lesions (NNLs; 62.7 %) and 38 previously attempted non-lifting lesions (PANLs; 37.3 %). PANLs were smaller ( P  < 0.001) and more likely to be non-granular ( P  = 0.001) than the lifting LSLs. NNLs were of similar size ( P  = 0.77) and morphology ( P  = 0.10) to the lifting LSLs. CAST was successful in all cases and adverse events were comparable to lifting LSLs resected by complete snare excision. Recurrence at SC1 was comparable for PANLs (15.2 %) and lifting LSLs (15.3 %; P  = 0.99), whereas NNLs recurred more frequently (27.5 %; P  = 0.049); however, surgery was no more common for either type of non-lifting LSL than for lifting LSLs.  CAST is a safe, effective, and surgery-sparing therapy for the majority of non-lifting LSLs. It is easy to use, inexpensive, and does not require additional equipment. © Georg Thieme Verlag KG Stuttgart · New York.

Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.

PubMed

DeRocco, Vanessa; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A; Weninger, Keith

2010-11-01

To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.

Four-color single molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

PubMed Central

DeRocco, Vanessa C.; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A.; Weninger, Keith

2010-01-01

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. PMID:21091445

Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses

PubMed Central

Sinha, Raunak; Ahmed, Saheeb; Jahn, Reinhard; Klingauf, Jurgen

2011-01-01

Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission. PMID:21844343

A SNARE-Like Superfamily Protein SbSLSP from the Halophyte Salicornia brachiata Confers Salt and Drought Tolerance by Maintaining Membrane Stability, K+/Na+ Ratio, and Antioxidant Machinery

PubMed Central

Singh, Dinkar; Yadav, Narendra Singh; Tiwari, Vivekanand; Agarwal, Pradeep K.; Jha, Bhavanath

2016-01-01

About 1000 salt-responsive ESTs were identified from an extreme halophyte Salicornia brachiata. Among these, a novel salt-inducible gene SbSLSP (Salicornia brachiata SNARE-like superfamily protein), showed up-regulation upon salinity and dehydration stress. The presence of cis-regulatory motifs related to abiotic stress in the putative promoter region supports our finding that SbSLSP gene is inducible by abiotic stress. The SbSLSP protein showed a high sequence identity to hypothetical/uncharacterized proteins from Beta vulgaris, Spinacia oleracea, Eucalyptus grandis, and Prunus persica and with SNARE-like superfamily proteins from Zostera marina and Arabidopsis thaliana. Bioinformatics analysis predicted a clathrin adaptor complex small-chain domain and N-myristoylation site in the SbSLSP protein. Subcellular localization studies indicated that the SbSLSP protein is mainly localized in the plasma membrane. Using transgenic tobacco lines, we establish that overexpression of SbSLSP resulted in elevated tolerance to salt and drought stress. The improved tolerance was confirmed by alterations in a range of physiological parameters, including high germination and survival rate, higher leaf chlorophyll contents, and reduced accumulation of Na+ ion and reactive oxygen species (ROS). Furthermore, overexpressing lines also showed lower water loss, higher cell membrane stability, and increased accumulation of proline and ROS-scavenging enzymes. Overexpression of SbSLSP also enhanced the transcript levels of ROS-scavenging and signaling enzyme genes. This study is the first investigation of the function of the SbSLSP gene as a novel determinant of salinity/drought tolerance. The results suggest that SbSLSP could be a potential candidate to increase salinity and drought tolerance in crop plants for sustainable agriculture in semi-arid saline soil. PMID:27313584

The FgVps39-FgVam7-FgSso1 Complex Mediates Vesicle Trafficking and Is Important for the Development and Virulence of Fusarium graminearum.

PubMed

Li, Bing; Liu, Luping; Li, Ying; Dong, Xin; Zhang, Haifeng; Chen, Huaigu; Zheng, Xiaobo; Zhang, Zhengguang

2017-05-01

Vesicle trafficking is an important event in eukaryotic organisms. Many proteins and lipids transported between different organelles or compartments are essential for survival. These processes are mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Rab-GTPases, and multisubunit tethering complexes such as class C core vacuole or endosome tethering and homotypic fusion or vacuole protein sorting (HOPS). Our previous study has demonstrated that FgVam7, which encodes a SNARE protein involving in vesicle trafficking, plays crucial roles in growth, asexual or sexual development, deoxynivalenol production, and pathogenicity in Fusarium graminearum. Here, the affinity purification approach was used to identify FgVam7-interacting proteins to explore its regulatory mechanisms during vesicle trafficking. The orthologs of yeast Vps39, a HOPS tethering complex subunit, and Sso1, a SNARE protein localized to the vacuole or endosome, were identified and selected for further characterization. In yeast two-hybrid and glutathione-S-transferase pull-down assays, FgVam7, FgVps39, and FgSso1 interacted with each other as a complex. The ∆Fgvps39 mutant generated by targeted deletion was significantly reduced in vegetative growth and asexual development. It failed to produce sexual spores and was defective in plant infection and deoxynivalenol production. Further cellular localization and cytological examinations suggested that FgVps39 is involved in vesicle trafficking from early or late endosomes to vacuoles in F. graminearum. Additionally, the ∆Fgvps39 mutant was defective in vacuole morphology and autophagy, and it was delayed in endocytosis. Our results demonstrate that FgVam7 interacts with FgVps39 and FgSso1 to form a unique complex, which is involved in vesicle trafficking and modulating the proper development of infection-related morphogenesis in F. graminearum.

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

PubMed

Yang, Yoosoo; Kong, Byoungjae; Jung, Younghoon; Park, Joon-Bum; Oh, Jung-Mi; Hwang, Jaesung; Cho, Jae Youl; Kweon, Dae-Hyuk

2018-01-01

Vesicle-associated V-soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans -SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.

PubMed

Chevalier, Adrien S; Chaumont, François

2015-05-01

Aquaporins are small channel proteins which facilitate the diffusion of water and small neutral molecules across biological membranes. Compared with animals, plant genomes encode numerous aquaporins, which display a large variety of subcellular localization patterns. More specifically, plant aquaporins of the plasma membrane intrinsic protein (PIP) subfamily were first described as plasma membrane (PM)-resident proteins, but recent research has demonstrated that the trafficking and subcellular localization of these proteins are complex and highly regulated. In the past few years, PIPs emerged as new model proteins to study subcellular sorting and membrane dynamics in plant cells. At least two distinct sorting motifs (one cytosolic, the other buried in the membrane) are required to direct PIPs to the PM. Hetero-oligomerization and interaction with SNAREs (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptors) also influence the subcellular trafficking of PIPs. In addition to these constitutive processes, both the progression of PIPs through the secretory pathway and their dynamics at the PM are responsive to changing environmental conditions. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

TFIIB-facilitated recruitment of preinitiation complexes by a TAF-independent mechanism.

PubMed

Hori, Roderick T; Xu, Shuping; Hu, Xianyuan; Pyo, Sung

2004-01-01

Gene activators contain activation domains that are thought to recruit limiting components of the transcription machinery to a core promoter. VP16, a viral gene activator, has served as a model for studying the mechanistic aspects of transcriptional activation from yeast to human. The VP16 activation domain can be divided into two modules--an N-terminal subdomain (VPN) and a C-terminal subdomain (VPC). This study demonstrates that VPC stimulates core promoters that are either independent or dependent on TAFs (TATA-box Binding Protein-Associated Factors). In contrast, VPN only activates the TAF-independent core promoter and this activity increases in a synergistic fashion when VPN is dimerized (VPN2). Compared to one copy of VPN (VPN1), VPN2 also displays a highly cooperative increase in binding hTFIIB. The increased TFIIB binding correlates with VPN2's increased ability to recruit a complex containing TFIID, TFIIA and TFIIB. However, VPN1 and VPN2 do not increase the assembly of a complex containing only TFIID and TFIIA. The VPN subdomain also facilitates assembly of a complex containing TBP:TFIIA:TFIIB, which lacks TAFs, and provides a mechanism that could function at TAF-independent promoters. Taken together, these results suggest the interaction between VPN and TFIIB potentially initiate a network of contacts allowing the activator to indirectly tether TFIID or TBP to DNA.

TFIIB-facilitated recruitment of preinitiation complexes by a TAF-independent mechanism

PubMed Central

Hori, Roderick T.; Xu, Shuping; Hu, Xianyuan; Pyo, Sung

2004-01-01

Gene activators contain activation domains that are thought to recruit limiting components of the transcription machinery to a core promoter. VP16, a viral gene activator, has served as a model for studying the mechanistic aspects of transcriptional activation from yeast to human. The VP16 activation domain can be divided into two modules—an N-terminal subdomain (VPN) and a C-terminal subdomain (VPC). This study demonstrates that VPC stimulates core promoters that are either independent or dependent on TAFs (TATA-box Binding Protein-Associated Factors). In contrast, VPN only activates the TAF-independent core promoter and this activity increases in a synergistic fashion when VPN is dimerized (VPN2). Compared to one copy of VPN (VPN1), VPN2 also displays a highly cooperative increase in binding hTFIIB. The increased TFIIB binding correlates with VPN2's increased ability to recruit a complex containing TFIID, TFIIA and TFIIB. However, VPN1 and VPN2 do not increase the assembly of a complex containing only TFIID and TFIIA. The VPN subdomain also facilitates assembly of a complex containing TBP:TFIIA:TFIIB, which lacks TAFs, and provides a mechanism that could function at TAF-independent promoters. Taken together, these results suggest the interaction between VPN and TFIIB potentially initiate a network of contacts allowing the activator to indirectly tether TFIID or TBP to DNA. PMID:15272087

Percutaneous closure of a giant left ventricular wall pseudoaneurysm: Anterograde approach with a double snare technique.

PubMed

Afonso Nogueira, Marta; Fiarresga, António; de Sousa, Lídia; Agapito, Ana; Galrinho, Ana; Cruz Ferreira, Rui

2016-01-01

Left ventricular pseudoaneurysm is a rare complication of acute myocardial infarction, cardiac surgery, trauma or infection. Since surgical repair is associated with high morbidity and mortality, percutaneous closure has been described as an alternative. In this regard, we present a case in which a symptomatic large left ventricular pseudoaneurysm was treated by percutaneous closure due to the patient's high surgical risk, using a double snare technique. Despite the technical difficulties, this procedure had a good final result followed by clinical success, confirming that this procedure is an effective alternative to surgery in high-risk patients. Copyright © 2016 Sociedade Portuguesa de Cardiologia. Publicado por Elsevier España, S.L.U. All rights reserved.

BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

PubMed Central

Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

2006-01-01

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

Septin dynamics are essential for exocytosis.

PubMed

Tokhtaeva, Elmira; Capri, Joe; Marcus, Elizabeth A; Whitelegge, Julian P; Khuzakhmetova, Venera; Bukharaeva, Ellya; Deiss-Yehiely, Nimrod; Dada, Laura A; Sachs, George; Fernandez-Salas, Ester; Vagin, Olga

2015-02-27

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Qa-SNAREs localized to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants

PubMed Central

Uemura, Tomohiro; Kim, Hyeran; Saito, Chieko; Ebine, Kazuo; Ueda, Takashi; Schulze-Lefert, Paul; Nakano, Akihiko

2012-01-01

In all eukaryotic cells, a membrane-trafficking system connects the post-Golgi organelles, such as the trans-Golgi network (TGN), endosomes, vacuoles, and the plasma membrane. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. Unlike its roles in animal and yeast cells, the TGN has also been reported to function like early endosomal compartments in plant cells. However, the physiological roles of the TGN functions in plants are not understood. Here, we report a study of the SYP4 group (SYP41, SYP42, and SYP43), which represents the plant orthologs of the Tlg2/syntaxin16 Qa-SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) that localizes on the TGN in yeast and animal cells. The SYP4 group regulates the secretory and vacuolar transport pathways in the post-Golgi network and maintains the morphology of the Golgi apparatus and TGN. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance responses to a fungal pathogen. We also reveal a plant cell-specific higher-order role of the SYP4 group in the protection of chloroplasts from salicylic acid-dependent biotic stress. PMID:22307646

Wide-field piecemeal cold snare polypectomy of large sessile serrated polyps without a submucosal injection is safe.

PubMed

Tate, David J; Awadie, Halim; Bahin, Farzan F; Desomer, Lobke; Lee, Ralph; Heitman, Steven J; Goodrick, Kathleen; Bourke, Michael J

2018-03-01

BACKGROUND AND STUDY AIMS : Large series suggest endoscopic mucosal resection is safe and effective for the removal of large (≥ 10 mm) sessile serrated polyps (SSPs), but it exposes the patient to the risks of electrocautery, including delayed bleeding. We examined the feasibility and safety of piecemeal cold snare polypectomy (pCSP) for the resection of large SSPs.  Sequential large SSPs (10 - 35 mm) without endoscopic evidence of dysplasia referred over 12 months to a tertiary endoscopy center were considered for pCSP. A thin-wire snare was used in all cases. Submucosal injection was not performed. High definition imaging of the defect margin was used to ensure the absence of residual serrated tissue. Adverse events were assessed at 2 weeks and surveillance was planned for between 6 and 12 months.  41 SSPs were completely removed by pCSP in 34 patients. The median SSP size was 15 mm (interquartile range [IQR] 14.5 - 20 mm; range 10 - 35 mm). The median procedure duration was 4.5 minutes (IQR 1.4 - 6.3 minutes). There was no evidence of perforation or significant intraprocedural bleeding. At 2-week follow-up, there were no significant adverse events, including delayed bleeding and post polypectomy syndrome. First follow-up has been undertaken for 15 /41 lesions at a median of 6 months with no evidence of recurrence.  There is potential for pCSP to become the standard of care for non-dysplastic large SSPs. This could reduce the burden of removing SSPs on patients and healthcare systems, particularly by avoidance of delayed bleeding. © Georg Thieme Verlag KG Stuttgart · New York.

Selected SNARE proteins are essential for the polarized membrane insertion of igf-1 receptor and the regulation of initial axonal outgrowth in neurons.

PubMed

Grassi, Diego; Plonka, Florentyna Bustos; Oksdath, Mariana; Guil, Alvaro Nieto; Sosa, Lucas J; Quiroga, Santiago

2015-01-01

The establishment of polarity necessitates initial axonal outgrowth and, therefore, the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles (PPVs) primarily at the neuronal growth cone. Little is known about the SNAREs family proteins involved in the regulation of PPV fusion with the neuronal plasmalemma at early stages of differentiation. We show here that five SNARE proteins (VAMP2, VAMP4, VAMP7, Syntaxin6 and SNAP23) were expressed by hippocampal pyramidal neurons before polarization. Expression silencing of three of these proteins (VAMP4, Syntaxin6 and SNAP23) repressed axonal outgrowth and the establishment of neuronal polarity, by inhibiting IGF-1 receptor exocytotic polarized insertion, necessary for neuronal polarization. In addition, stimulation with IGF-1 triggered the association of VAMP4, Syntaxin6 and SNAP23 to vesicular structures carrying the IGF-1 receptor and overexpression of a negative dominant form of Syntaxin6 significantly inhibited exocytosis of IGF-1 receptor containing vesicles at the neuronal growth cone. Taken together, our results indicated that VAMP4, Syntaxin6 and SNAP23 functions are essential for regulation of PPV exocytosis and the polarized insertion of IGF-1 receptor and, therefore, required for initial axonal elongation and the establishment of neuronal polarity.

LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

PubMed Central

Bennett, Terry L.; Kraft, Shannon M.; Reaves, Barbara J.; Mima, Joji; O’Brien, Kevin M.; Starai, Vincent J.

2013-01-01

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis. PMID:23437241

Implementation of a web-based national child health-care programme in a local context: A complex facilitator role.

PubMed

Tell, Johanna; Olander, Ewy; Anderberg, Peter; Berglund, Johan Sanmartin

2018-02-01

The aim of this study was to investigate child health-care coordinators' experiences of being a facilitator for the implementation of a new national child health-care programme in the form of a web-based national guide. The study was based on eight remote, online focus groups, using Skype for Business. A qualitative content analysis was performed. The analysis generated three categories: adapt to a local context, transition challenges and led by strong incentives. There were eight subcategories. In the latent analysis, the theme 'Being a facilitator: a complex role' was formed to express the child health-care coordinators' experiences. Facilitating a national guideline or decision support in a local context is a complex task that requires an advocating and mediating role. For successful implementation, guidelines and decision support, such as a web-based guide and the new child health-care programme, must match professional consensus and needs and be seen as relevant by all. Participation in the development and a strong bottom-up approach was important, making the web-based guide and the programme relevant to whom it is intended to serve, and for successful implementation. The study contributes valuable knowledge when planning to implement a national web-based decision support and policy programme in a local health-care context.

Blocking dephosphorylation at Serine 120 residue in t-SNARE SNAP-23 leads to massive inhibition in exocytosis from mast cells.

PubMed

Naskar, Pieu; Naqvi, Nilofer; Puri, Niti

2018-03-01

Mast cells (MCs) respond to allergen challenge by release of pre-stored inflammatory mediators from their secretory granules, on cross-linking of Fc(epsilon) receptor I (Fc(epsilon)RI) receptors. The target-SNARE (t-SNARE) SNAP-23 has been shown to play an important role in MC exocytosis and undergoes transient phosphorylation at Serine 95 (S95) and Serine 120 (S120), concomitant with mediator release. During current study we explored the importance of transient nature of phosphorylation at S120 in MC exocytosis. A phosphomimetic SNAP-23-S120D mutant of rodent SNAP-23 was cloned into EGFP vector and its effect on the exocytosis and the mechanisms involved was studied in RBL-2H3 MC line. Secretion reporter assay with SNAP-23-S120D transfected MCs revealed a very significant inhibition of exocytosis, and reduced ruffling in response to Fc(epsilon)RI cross-linking. Further, the effect of this mutation on localization of SNAP-23 in MCs was studied. Immunofluorescence microscopy studies and membrane-cytosol fractionation of green fluorescent protein-tagged SNAP- 23-S120D (GFP-SNAP-23-S120D) transfected MCs showed that a large proportion of GFP-SNAP-23-S120D was residing in cytosol unlike wild-type SNAP-23, in resting and activated MCs and even the membrane associated portion was on internal lysosomal membranes than plasma membrane. These studies imply that dephosphorylation of S120 is important for SNAP-23 membrane association dynamics and subsequently MC degranulation.

Endoglucanase Peripheral Loops Facilitate Complexation of Glucan Chains on Cellulose via Adaptive Coupling to the Emergent Substrate Structures

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

Lin, Yuchun; Beckham, Gregg T.; Himmel, Michael E.

We examine how the catalytic domain of a glycoside hydrolase family 7 endoglucanase catalytic domain (Cel7B CD) facilitates complexation of cellulose chains from a crystal surface. With direct relevance to the science of biofuel production, this problem also represents a model system of biopolymer processing by proteins in Nature. Interactions of Cel7B CD with a cellulose microfibril along different paths of complexation are characterized by mapping the atomistic fluctuations recorded in free-energy simulations onto the parameters of a coarse-grain model. The resulting patterns of protein-biopolymer couplings also uncover the sequence signatures of the enzyme in peeling off glucan chains frommore » the microfibril substrate. We show that the semiopen active site of Cel7B CD exhibits similar barriers and free energies of complexation over two distinct routes; namely, scooping of a chain into the active-site cleft and threading from the chain end into the channel. On the other hand, the complexation energetics strongly depends on the surface packing of the targeted chain and the resulting interaction sites with the enzyme. A revealed principle is that Cel7B CD facilitates cellulose deconstruction via adaptive coupling to the emergent substrate. The flexible, peripheral segments of the protein outside of the active-site cleft are able to accommodate the varying features of cellulose along the simulated paths of complexation. The general strategy of linking physics-based molecular interactions to protein sequence could also be helpful in elucidating how other protein machines process biopolymers.« less

Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1.

PubMed

Morgera, Francesca; Sallah, Margaret R; Dubuke, Michelle L; Gandhi, Pallavi; Brewer, Daniel N; Carr, Chavela M; Munson, Mary

2012-01-01

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons

PubMed Central

Soo Hoo, Linda; Banna, Chris D.; Radeke, Carolyn M.; Sharma, Nikunj; Albertolle, Mary E.; Low, Seng Hui; Weimbs, Thomas; Vandenberg, Carol A.

2016-01-01

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons. PMID:27662481

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

PubMed

Soo Hoo, Linda; Banna, Chris D; Radeke, Carolyn M; Sharma, Nikunj; Albertolle, Mary E; Low, Seng Hui; Weimbs, Thomas; Vandenberg, Carol A

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons.

Cynefin as Reference Framework to Facilitate Insight and Decision-Making in Complex Contexts of Biomedical Research.

PubMed

Kempermann, Gerd

2017-01-01

The Cynefin scheme is a concept of knowledge management, originally devised to support decision making in management, but more generally applicable to situations, in which complexity challenges the quality of insight, prediction, and decision. Despite the fact that life itself, and especially the brain and its diseases, are complex to the extent that complexity could be considered their cardinal feature, complex problems in biomedicine are often treated as if they were actually not more than the complicated sum of solvable sub-problems. Because of the emergent properties of complex contexts this is not correct. With a set of clear criteria Cynefin helps to set apart complex problems from "simple/obvious," "complicated," "chaotic," and "disordered" contexts in order to avoid misinterpreting the relevant causality structures. The distinction comes with the insight, which specific kind of knowledge is possible in each of these categories and what are the consequences for resulting decisions and actions. From student's theses over the publication and grant writing process to research politics, misinterpretation of complexity can have problematic or even dangerous consequences, especially in clinical contexts. Conceptualization of problems within a straightforward reference language like Cynefin improves clarity and stringency within projects and facilitates communication and decision-making about them.

The ERdj5-Sel1L complex facilitates cholera toxin retrotranslocation

PubMed Central

Williams, Jeffrey M.; Inoue, Takamasa; Banks, Lindsey; Tsai, Billy

2013-01-01

Cholera toxin (CT) traffics from the host cell surface to the endoplasmic reticulum (ER), where the toxin's catalytic CTA1 subunit retrotranslocates to the cytosol to induce toxicity. In the ER, CT is captured by the E3 ubiquitin ligase Hrd1 via an undefined mechanism to prepare for retrotranslocation. Using loss-of-function and gain-of-function approaches, we demonstrate that the ER-resident factor ERdj5 promotes CTA1 retrotranslocation, in part, via its J domain. This Hsp70 cochaperone regulates binding between CTA and the ER Hsp70 BiP, a chaperone previously implicated in toxin retrotranslocation. Importantly, ERdj5 interacts with the Hrd1 adaptor Sel1L directly through Sel1L's N-terminal lumenal domain, thereby linking ERdj5 to the Hrd1 complex. Sel1L itself also binds CTA and facilitates toxin retrotranslocation. By contrast, EDEM1 and OS-9, two established Sel1L binding partners, do not play significant roles in CTA1 retrotranslocation. Our results thus identify two ER factors that promote ER-to-cytosol transport of CTA1. They also indicate that ERdj5, by binding to Sel1L, triggers BiP–toxin interaction proximal to the Hrd1 complex. We postulate this scenario enables the Hrd1-associated retrotranslocation machinery to capture the toxin efficiently once the toxin is released from BiP. PMID:23363602

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

PubMed Central

Fong, Yu Hang; Wong, Ho Chun; Yuen, Man Hon; Lau, Pak Ho; Chen, Yu Wai; Wong, Kam-Bo

2013-01-01

Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease. PMID:24115911

Sustainable development goals for global health: facilitating good governance in a complex environment.

PubMed

Haffeld, Just

2013-11-01

Increasing complexity is following in the wake of rampant globalization. Thus, the discussion about Sustainable Development Goals (SDGs) requires new thinking that departs from a critique of current policy tools in exploration of a complexity-friendly approach. This article argues that potential SDGs should: treat stakeholders, like states, business and civil society actors, as agents on different aggregate levels of networks; incorporate good governance processes that facilitate early involvement of relevant resources, as well as equitable participation, consultative processes, and regular policy and programme implementation reviews; anchor adoption and enforcement of such rules to democratic processes in accountable organizations; and include comprehensive systems evaluations, including procedural indicators. A global framework convention for health could be a suitable instrument for handling some of the challenges related to the governance of a complex environment. It could structure and legitimize government involvement, engage stakeholders, arrange deliberation and decision-making processes with due participation and regular policy review, and define minimum standards for health services. A monitoring scheme could ensure that agents in networks comply according to whole-systems targets, locally defined outcome indicators, and process indicators, thus resolving the paradox of government control vs. local policy space. A convention could thus exploit the energy created in the encounter between civil society, international organizations and national authorities. Copyright © 2013 Reproductive Health Matters. Published by Elsevier Ltd. All rights reserved.

SNARE-dependent upregulation of KCC2 activity following metabotropic zinc receptor (mZnR/GPR39) activation in rat cortical neurons in vitro

PubMed Central

Saadi, Robert A.; He, Kai; Hartnett, Karen A.; Kandler, Karl; Hershfinkel, Michal; Aizenman, Elias

2012-01-01

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABAA receptor channels. In a recent study, we showed that Zn2+ regulates GABAA reversal potentials in the hippocampus by enhancing the activity of KCC2 via an increase in its surface expression. Zn2+ initiates this process by activating the Gq-coupled metabotropic Zn2+ receptor mZnR/GPR39. Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture and then tested the hypothesis that the increase in KCC2 activity is mediated through a SNARE-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca2+ indicator and exposing cells to Zn2+, which triggered consistent Ca2+ responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist MCPG. Importantly, Zn2+ treatment under these conditions did not increase the intracellular concentrations of Zn2+ itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH4+ influx via the co-transporter using an intracellular pH sensitive fluorescent indicator. We observed that Zn2+ pretreatment induced a Ca2+-dependent increase in KCC2 activity. The effects of Zn2+ on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39−/− mice, suggesting that Zn2+ acts via mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and SNAP-25. Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn2+-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons

ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes.

PubMed

Diao, Jiajie; Liu, Rong; Rong, Yueguang; Zhao, Minglei; Zhang, Jing; Lai, Ying; Zhou, Qiangjun; Wilz, Livia M; Li, Jianxu; Vivona, Sandro; Pfuetzner, Richard A; Brunger, Axel T; Zhong, Qing

2015-04-23

Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.

Matrix metalloproteinase-1 facilitates MSC migration via cleavage of IGF-2/IGFBP2 complex.

PubMed

Guan, Shou P; Lam, Alan T L; Newman, Jennifer P; Chua, Kevin L M; Kok, Catherine Y L; Chong, Siao T; Chua, Melvin L K; Lam, Paula Y P

2018-01-01

The specific mechanism underlying the tumor tropism of human mesenchymal stem cells (MSCs) for cancer is not well defined. We previously showed that the migration potential of MSCs correlated with the expression and protease activity of matrix metalloproteinase (MMP)-1. Furthermore, highly tumor-tropic MSCs expressed higher levels of MMP-1 and insulin-like growth factor (IGF)-2 than poorly migrating MSCs. In this study, we examined the functional roles of IGF-2 and MMP-1 in mediating the tumor tropism of MSCs. Exogenous addition of either recombinant IGF-2 or MMP-1 could stimulate MSC migration. The correlation between IGF-2, MMP-1 expression, and MSC migration suggests that MMP-1 may play a role in regulating MSC migration via the IGF-2 signaling cascade. High concentrations of IGF binding proteins (IGFBPs) can inhibit IGF-stimulated functions by blocking its binding to its receptors and proteolysis of IGFBP is an important mechanism for the regulation of IGF signaling. We thus hypothesized that MMP-1 acts as an IGFBP2 proteinase, resulting in the cleavage of IGF-2/IGFBP2 complex and extracellular release of free IGF-2. Indeed, our results showed that conditioned media from highly migrating MSCs, which expressed high levels of MMP-1, cleaved the IGF-2/IGFBP2 complex. Taken together, these results showed that the MMP-1 secreted by highly tumor-tropic MSCs cleaved IGF-2/IGFBP2 complex. Free IGF-2 released from the complex may facilitate MSC migration toward tumor.

[A case of polypoid bronchial neurofibroma originating from right B2b successfully treated by bronchoscopic snaring forceps and Nd-YAG laser therapy].

PubMed

Takiguchi, Y; Uchiyama, T; Sato, K; Tatsumi, K; Kimura, H; Nagao, K; Fujisawa, T; Ohwada, H; Hiroshima, K; Kuriyama, T

1993-12-01

A 34-year-old man with persistent cough was admitted to our hospital. Bronchoscopic examination revealed a polypoid tumor with smooth surface which almost completely obstructed the right main bronchus. The tumor was removed by transbronchial snaring forceps and histologically confirmed as neurofibroma. Residual tumor was excised by biopsy forceps and further endoscopic Nd-YAG laser vaporization was performed. This is the first case in our country in which bronchoscopic treatment was performed for bronchial neurofibroma. Bronchoscopic removal might be the preferred treatment in the present case, although long-term follow-up is also required.

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

PubMed

Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

2016-01-15

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/DNA complex following virus entry.

PubMed

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L; Sapp, Martin

2012-09-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.

Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

PubMed Central

Bienkowska-Haba, Malgorzata; Williams, Carlyn; Kim, Seong Man; Garcea, Robert L.

2012-01-01

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles. PMID:22761365

Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

PubMed

Jouhet, Juliette; Gray, John C

2009-10-01

Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

PubMed Central

Nikolaus, Joerg; Karatekin, Erdem

2016-01-01

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. PMID:27585113

Oligomerisation of Synaptobrevin-2 Studied by Native Mass Spectrometry and Chemical Cross-Linking

NASA Astrophysics Data System (ADS)

Wittig, Sabine; Haupt, Caroline; Hoffmann, Waldemar; Kostmann, Susann; Pagel, Kevin; Schmidt, Carla

2018-06-01

Synaptobrevin-2 is a key player in signal transmission in neurons. It forms, together with SNAP25 and Syntaxin-1A, the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and mediates exocytosis of synaptic vesicles with the pre-synaptic membrane. While Synaptobrevin-2 is part of a four-helix bundle in this SNARE complex, it is natively unstructured in the absence of lipids or other SNARE proteins. Partially folded segments, presumably SNARE complex formation intermediates, as well as formation of Synaptobrevin-2 dimers and oligomers, were identified in previous studies. Here, we employ three Synaptobrevin-2 variants—the full-length protein Syb(1-116), the soluble, cytosolic variant Syb(1-96) as well as a shorter version Syb(49-96) containing structured segments but omitting a trigger site for SNARE complex formation—to study oligomerisation in the absence of interaction partners or when incorporated into the lipid bilayer of liposomes. Combining native mass spectrometry with chemical cross-linking, we find that the truncated versions show increased oligomerisation. Our findings from both techniques agree well and confirm the presence of oligomers in solution while membrane-bound Synaptobrevin-2 is mostly monomeric. Using ion mobility mass spectrometry, we could further show that lower charge states of Syb(49-96) oligomers, which most likely represent solution structures, follow an isotropic growth curve suggesting that they are intrinsically disordered. From a technical point of view, we show that the combination of native ion mobility mass spectrometry with chemical cross-linking is well-suited for the analysis of protein homo-oligomers. [Figure not available: see fulltext.

Creating Knock-outs of Conserved Oligomeric Golgi complex subunits using CRISPR-mediated gene editing paired with a selection strategy based on glycosylation defects associated with impaired COG complex function

PubMed Central

Blackburn, Jessica Bailey; Lupashin, Vladimir V.

2017-01-01

Summary The Conserved Oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex’s role in glycosylation homeostasis to obtain COG complex subunit knock-outs (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis. PMID:27632008

How the strengths of Lisp-family languages facilitate building complex and flexible bioinformatics applications.

PubMed

Khomtchouk, Bohdan B; Weitz, Edmund; Karp, Peter D; Wahlestedt, Claes

2018-05-01

We present a rationale for expanding the presence of the Lisp family of programming languages in bioinformatics and computational biology research. Put simply, Lisp-family languages enable programmers to more quickly write programs that run faster than in other languages. Languages such as Common Lisp, Scheme and Clojure facilitate the creation of powerful and flexible software that is required for complex and rapidly evolving domains like biology. We will point out several important key features that distinguish languages of the Lisp family from other programming languages, and we will explain how these features can aid researchers in becoming more productive and creating better code. We will also show how these features make these languages ideal tools for artificial intelligence and machine learning applications. We will specifically stress the advantages of domain-specific languages (DSLs): languages that are specialized to a particular area, and thus not only facilitate easier research problem formulation, but also aid in the establishment of standards and best programming practices as applied to the specific research field at hand. DSLs are particularly easy to build in Common Lisp, the most comprehensive Lisp dialect, which is commonly referred to as the 'programmable programming language'. We are convinced that Lisp grants programmers unprecedented power to build increasingly sophisticated artificial intelligence systems that may ultimately transform machine learning and artificial intelligence research in bioinformatics and computational biology.

How the strengths of Lisp-family languages facilitate building complex and flexible bioinformatics applications

PubMed Central

Khomtchouk, Bohdan B; Weitz, Edmund; Karp, Peter D; Wahlestedt, Claes

2018-01-01

Abstract We present a rationale for expanding the presence of the Lisp family of programming languages in bioinformatics and computational biology research. Put simply, Lisp-family languages enable programmers to more quickly write programs that run faster than in other languages. Languages such as Common Lisp, Scheme and Clojure facilitate the creation of powerful and flexible software that is required for complex and rapidly evolving domains like biology. We will point out several important key features that distinguish languages of the Lisp family from other programming languages, and we will explain how these features can aid researchers in becoming more productive and creating better code. We will also show how these features make these languages ideal tools for artificial intelligence and machine learning applications. We will specifically stress the advantages of domain-specific languages (DSLs): languages that are specialized to a particular area, and thus not only facilitate easier research problem formulation, but also aid in the establishment of standards and best programming practices as applied to the specific research field at hand. DSLs are particularly easy to build in Common Lisp, the most comprehensive Lisp dialect, which is commonly referred to as the ‘programmable programming language’. We are convinced that Lisp grants programmers unprecedented power to build increasingly sophisticated artificial intelligence systems that may ultimately transform machine learning and artificial intelligence research in bioinformatics and computational biology. PMID:28040748

Loss of Drosophila Vps16A enhances autophagosome formation through reduced Tor activity.

PubMed

Takáts, Szabolcs; Varga, Ágnes; Pircs, Karolina; Juhász, Gábor

2015-01-01

The HOPS tethering complex facilitates autophagosome-lysosome fusion by binding to Syx17 (Syntaxin 17), the autophagosomal SNARE. Here we show that loss of the core HOPS complex subunit Vps16A enhances autophagosome formation and slows down Drosophila development. Mechanistically, Tor kinase is less active in Vps16A mutants likely due to impaired endocytic and biosynthetic transport to the lysosome, a site of its activation. Tor reactivation by overexpression of Rheb suppresses autophagosome formation and restores growth and developmental timing in these animals. Thus, Vps16A reduces autophagosome numbers both by indirectly restricting their formation rate and by directly promoting their clearance. In contrast, the loss of Syx17 blocks autophagic flux without affecting the induction step in Drosophila.

Escaping the snare of chronological growth and launching a free curve alternative: General deviance as latent growth model

PubMed Central

WOOD, PHILLIP KARL; JACKSON, KRISTINA M.

2014-01-01

Researchers studying longitudinal relationships among multiple problem behaviors sometimes characterize autoregressive relationships across constructs as indicating “protective” or “launch” factors or as “developmental snares.” These terms are used to indicate that initial or intermediary states of one problem behavior subsequently inhibit or promote some other problem behavior. Such models are contrasted with models of “general deviance” over time in which all problem behaviors are viewed as indicators of a common linear trajectory. When fit of the “general deviance” model is poor and fit of one or more autoregressive models is good, this is taken as support for the inhibitory or enhancing effect of one construct on another. In this paper, we argue that researchers consider competing models of growth before comparing deviance and time-bound models. Specifically, we propose use of the free curve slope intercept (FCSI) growth model (Meredith & Tisak, 1990) as a general model to typify change in a construct over time. The FCSI model includes, as nested special cases, several statistical models often used for prospective data, such as linear slope intercept models, repeated measures multivariate analysis of variance, various one-factor models, and hierarchical linear models. When considering models involving multiple constructs, we argue the construct of “general deviance” can be expressed as a single-trait multimethod model, permitting a characterization of the deviance construct over time without requiring restrictive assumptions about the form of growth over time. As an example, prospective assessments of problem behaviors from the Dunedin Multidisciplinary Health and Development Study (Silva & Stanton, 1996) are considered and contrasted with earlier analyses of Hussong, Curran, Moffitt, and Caspi (2008), which supported launch and snare hypotheses. For antisocial behavior, the FCSI model fit better than other models, including the linear

Technological complexity and the global dispersal of modern humans.

PubMed

Hoffecker, John F; Hoffecker, Ian T

2017-11-01

Anatomically modern humans (Homo sapiens) dispersed out of Africa roughly 120,000 years ago and again after 75,000 years ago. The early dispersal was geographically restricted to the Arabian Peninsula, Levant, and possibly parts of southern Asia. The later dispersal was ultimately global in scope, including areas not previously occupied by Homo. One explanation for the contrast between the two out-of-Africa dispersals is that the modern humans who expanded into Eurasia 120,000 years ago lacked the functionally and structurally complex technology of recent hunter-gatherers. This technology, which includes, for example, mechanical projectiles, snares and traps, and sewn clothing, provides not only expanded dietary breadth and increased rates of foraging efficiency and success in places where plant and animal productivity is low, but protection from cold weather in places where winter temperatures are low. The absence of complex technology before 75,000 years ago also may explain why modern humans in the Levant did not develop sedentary settlements and agriculture 120,000 years ago (i.e., during the Last Interglacial). © 2017 Wiley Periodicals, Inc.

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

PubMed Central

Jankowsky, E; Schwenzer, B

1996-01-01

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

Evaluation of nutria (Myocastor coypus) detection methods in Maryland, USA

USGS Publications Warehouse

Pepper, Margaret A.; Herrmann, Valentine; Hines, James; Nichols, James D.; Kendrot, Stephen R

2017-01-01

Nutria (Myocaster coypus), invasive, semi-aquatic rodents native to South America, were introduced into Maryland near Blackwater National Wildlife Refuge (BNWR) in 1943. Irruptive population growth, expansion, and destructive feeding habits resulted in the destruction of thousands of acres of emergent marshes at and surrounding BNWR. In 2002, a partnership of federal, state and private entities initiated an eradication campaign to protect remaining wetlands from further damage and facilitate the restoration of coastal wetlands throughout the Chesapeake Bay region. Program staff removed nearly 14,000 nutria from five infested watersheds in a systematic trapping and hunting program between 2002 and 2014. As part of ongoing surveillance activities, the Chesapeake Bay Nutria Eradication Project uses a variety of tools to detect and remove nutria. Project staff developed a floating raft, or monitoring platform, to determine site occupancy. These platforms are placed along waterways and checked periodically for evidence of nutria visitation. We evaluated the effectiveness of monitoring platforms and three associated detection methods: hair snares, presence of scat, and trail cameras. Our objectives were to (1) determine if platform placement on land or water influenced nutria visitation rates, (2) determine if the presence of hair snares influenced visitation rates, and (3) determine method-specific detection probabilities. Our analyses indicated that platforms placed on land were 1.5–3.0 times more likely to be visited than those placed in water and that platforms without snares were an estimated 1.7–3.7 times more likely to be visited than those with snares. Although the presence of snares appears to have discouraged visitation, seasonal variation may confound interpretation of these results. Scat was the least effective method of determining nutria visitation, while hair snares were as effective as cameras. Estimated detection probabilities provided by occupancy

Combined use of clips and nylon snare ("tulip-bundle") as a rescue endoscopic bleeding control in a mallory-weiss syndrome.

PubMed

Ivekovic, Hrvoje; Radulovic, Bojana; Jankovic, Suzana; Markos, Pave; Rustemovic, Nadan

2014-01-01

Mallory-Weiss syndrome (MWS) accounts for 6-14% of all cases of upper gastrointestinal bleeding. Prognosis of patients with MWS is generally good, with a benign course and rare recurrence of bleeding. However, no strict recommendations exist in regard to the mode of action after a failure of primary endoscopic hemostasis. We report a case of an 83-year-old male with MWS and rebleeding after the initial endoscopic treatment with epinephrine and clips. The final endoscopic control of bleeding was achieved by a combined application of clips and a nylon snare in a "tulip-bundle" fashion. The patient had an uneventful postprocedural clinical course and was discharged from the hospital five days later. To the best of our knowledge, this is the first case report showing the "tulip-bundle" technique as a rescue endoscopic bleeding control in the esophagus.

Developmentally regulated switch in alternatively spliced SNAP-25 isoforms alters facilitation of synaptic transmission.

PubMed

Bark, Christina; Bellinger, Frederick P; Kaushal, Ashutosh; Mathews, James R; Partridge, L Donald; Wilson, Michael C

2004-10-06

Although the basic molecular components that promote regulated neurotransmitter release are well established, the contribution of these proteins as regulators of the plasticity of neurotransmission and refinement of synaptic connectivity during development is elaborated less fully. For example, during the period of synaptic growth and maturation in brain, the expression of synaptosomal protein 25 kDa (SNAP-25), a neuronal t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) essential for action potential-dependent neuroexocytosis, is altered through alternative splicing of pre-mRNA transcripts. We addressed the role of the two splice-variant isoforms of SNAP-25 with a targeted mouse mutation that impairs the shift from SNAP-25a to SNAP-25b. Most of these mutant mice die between 3 and 5 weeks of age, which coincides with the time when SNAP-25b expression normally reaches mature levels in brain and synapse formation is essentially completed. The altered expression of these SNAP-25 isoforms influences short-term synaptic function by affecting facilitation but not the initial probability of release. This suggests that mechanisms controlling alternative splicing between SNAP-25 isoforms contribute to a molecular switch important for survival that helps to guide the transition from immature to mature synaptic connections, as well as synapse regrowth and remodeling after neural injury.

Facilitating surgeon understanding of complex anatomy using a three-dimensional printed model.

PubMed

Cromeens, Barrett P; Ray, William C; Hoehne, Brad; Abayneh, Fikir; Adler, Brent; Besner, Gail E

2017-08-01

3-dimensional prints (3DP) anecdotally facilitate surgeon understanding of anatomy and decision-making. However, the actual benefit to surgeons or patients has not been quantified. This study investigates how surgeon understanding of complex anatomy is altered by a 3DP compared to computed tomography (CT) scan or CT + digital reconstruction (CT + DR). Key anatomic features were segmented from a CT-abdomen/pelvis of pygopagus twins to build a DR and printed in color on a 3D printer. Pediatric surgery trainees and attendings (n = 21) were tested regarding anatomy identification and their understanding of point-to-point distances, scale, and shape. There was no difference between media regarding point-to-point distances. The 3DP led to an increased number of correct answers for questions of scale and shape compared to CT (P < 0.05). CT + DR performance was intermediate but not statistically different from 3DP or CT. Identification of anatomy was inconsistent between media; however, answers were significantly closer to correct when using the 3DP. Participants completed the test faster with the 3DP (6.6 ± 0.5 min) (P < 0.05) than with CT (18.9 ± 2.5 min) or CT + 3DR (14.9 ± 1.5 min). Although point-to-point measurements were not different, 3DP increased the understanding of shape, scale, and anatomy. It enabled understanding significantly faster than other media. In difficult surgical cases with complex anatomy and a need for efficient multidisciplinary coordination, 3D printed models should be considered for surgical planning. Copyright © 2017 Elsevier Inc. All rights reserved.

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

PubMed Central

VanRheenen, Susan M.; Cao, Xiaochun; Sapperstein, Stephanie K.; Chiang, Elbert C.; Lupashin, Vladimir V.; Barlowe, Charles; Waters, M. Gerard

1999-01-01

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well. PMID:10562277

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

PubMed

Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

2017-09-29

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Using social media to facilitate knowledge transfer in complex engineering environments: a primer for educators

NASA Astrophysics Data System (ADS)

Murphy, Glen; Salomone, Sonia

2013-03-01

While highly cohesive groups are potentially advantageous they are also often correlated with the emergence of knowledge and information silos based around those same functional or occupational clusters. Consequently, an essential challenge for engineering organisations wishing to overcome informational silos is to implement mechanisms that facilitate, encourage and sustain interactions between otherwise disconnected groups. This paper acts as a primer for those seeking to gain an understanding of the design, functionality and utility of a suite of software tools generically termed social media technologies in the context of optimising the management of tacit engineering knowledge. Underpinned by knowledge management theory and using detailed case examples, this paper explores how social media technologies achieve such goals, allowing for the transfer of knowledge by tapping into the tacit and explicit knowledge of disparate groups in complex engineering environments.

Gradual Hunterian ligation for infected prosthetic bypass.

PubMed

Egun, A; Slade, D; McCollum, C N

2000-04-01

To review gradual snare occlusion for the management of complex or recurrent graft infection. Medical records of patients treated with gradual snare occlusion following graft infection were reviewed for indication for operation, type of bypass and graft material used. In addition, infecting organism, grade of infection (Szilágyi) and outcome were recorded. Four femoropopliteal, two extra-anatomic (axillofemoral) and aortobifemoral bypasses were included in this study. All had chronic infection (Szilágyi grade III) with onset of 4 to 24 months and two of which were recurrent. The causative organisms were coagulase-negative staphylococci, Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus in three patients, with no organism isolated in the remaining cases. There was no loss of limb following gradual snare occlusion but there was only one death due to aortic stump rupture 2 weeks later. Gradual snare occlusion is an alternative for the management of chronic or recurrent graft infection. Copyright 1999 Harcourt Publishers Ltd.

Argumentation: A Methodology to Facilitate Critical Thinking.

PubMed

Makhene, Agnes

2017-06-20

Caring is a difficult nursing activity that involves a complex nature of a human being in need of complex decision-making and problem solving through the critical thinking process. It is mandatory that critical thinking is facilitated in general and in nursing education particularly in order to render care in diverse multicultural patient care settings. This paper aims to describe how argumentation can be used to facilitate critical thinking in learners. A qualitative, exploratory and descriptive design that is contextual was used. Purposive sampling method was used to draw a sample and Miles and Huberman methodology of qualitative analysis was used to analyse data. Lincoln and Guba's strategies were employed to ensure trustworthiness, while Dhai and McQuoid-Mason's principles of ethical consideration were used. Following data analysis the findings were integrated within literature which culminated into the formulation of guidelines that can be followed when using argumentation as a methodology to facilitate critical thinking.

UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans

PubMed Central

Park, Seungmee; Bin, Na-Ryum; Wong, Raymond; Sitarska, Ewa; Sugita, Kyoko; Ma, Ke; Algouneh, Arash; Turlova, Ekaterina; Wang, Siyan; Siriya, Pranay; Kalia, Lorraine; Feng, Zhong-Ping; Monnier, Philippe P.; Zhen, Mei; Gao, Shangbang

2017-01-01

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1. SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

PubMed Central

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-01

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes

NASA Astrophysics Data System (ADS)

Brouwer, Ineke; Giniatullina, Asiya; Laurens, Niels; van Weering, Jan R. T.; Bald, Dirk; Wuite, Gijs J. L.; Groffen, Alexander J.

2015-09-01

Ca2+-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca2+-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca2+ trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca2+-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca2+ and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca2+-secretion coupling.

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

PubMed

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

PubMed

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Functional diversification of Arabidopsis SEC1-related SM proteins in cytokinetic and secretory membrane fusion.

PubMed

Karnahl, Matthias; Park, Misoon; Krause, Cornelia; Hiller, Ulrike; Mayer, Ulrike; Stierhof, York-Dieter; Jürgens, Gerd

2018-06-12

Sec1/Munc18 (SM) proteins contribute to membrane fusion by interacting with Qa-SNAREs or nascent trans -SNARE complexes. Gymnosperms and the basal angiosperm Amborella have only a single SEC1 gene related to the KEULE gene in Arabidopsis However, the genomes of most angiosperms including Arabidopsis encode three SEC1-related SM proteins of which only KEULE has been functionally characterized as interacting with the cytokinesis-specific Qa-SNARE KNOLLE during cell-plate formation. Here we analyze the closest paralog of KEULE named SEC1B. In contrast to the cytokinesis defects of keule mutants, sec1b mutants are homozygous viable. However, the keule sec1b double mutant was nearly gametophytically lethal, displaying collapsed pollen grains, which suggests substantial overlap between SEC1B and KEULE functions in secretion-dependent growth. SEC1B had a strong preference for interaction with the evolutionarily ancient Qa-SNARE SYP132 involved in secretion and cytokinesis, whereas KEULE interacted with both KNOLLE and SYP132. This differential interaction with Qa-SNAREs is likely conferred by domains 1 and 2a of the two SM proteins. Comparative analysis of all four possible combinations of the relevant SEC1 Qa-SNARE double mutants revealed that in cytokinesis, the interaction of SEC1B with KNOLLE plays no role, whereas the interaction of KEULE with KNOLLE is prevalent and functionally as important as the interactions of both SEC1B and KEU with SYP132 together. Our results suggest that functional diversification of the two SEC1-related SM proteins during angiosperm evolution resulted in enhanced interaction of SEC1B with Qa-SNARE SYP132, and thus a predominant role of SEC1B in secretion.

Membrane fusion and exocytosis.

PubMed

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Work-Facilitating Information Visualization Techniques for Complex Wastewater Systems

NASA Astrophysics Data System (ADS)

Ebert, Achim; Einsfeld, Katja

The design and the operation of urban drainage systems and wastewater treatment plants (WWTP) have become increasingly complex. This complexity is due to increased requirements concerning process technology, technical, environmental, economical, and occupational safety aspects. The plant operator has access not only to some timeworn filers and measured parameters but also to numerous on-line and off-line parameters that characterize the current state of the plant in detail. Moreover, expert databases and specific support pages of plant manufactures are accessible through the World Wide Web. Thus, the operator is overwhelmed with predominantly unstructured data.

Writing To Facilitate Learning in Microbiology.

ERIC Educational Resources Information Center

Fisher, Linda E.

This paper describes a microbiology course that utilizes writing to facilitate learning of complex concepts, for communicating experimental results, and as a diagnostic tool for the instructor in monitoring the students' understanding of material on an on-going basis. In-class writing assignments that summarize subject units are accompanied by a…

Diversity in structure and function of tethering complexes: evidence for different mechanisms in vesicular transport regulation.

PubMed

Kümmel, D; Heinemann, U

2008-04-01

The term 'tethering factor' has been coined for a heterogeneous group of proteins that all are required for protein trafficking prior to vesicle docking and SNARE-mediated membrane fusion. Two groups of tethering factors can be distinguished, long coiled-coil proteins and multi-subunit complexes. To date, eight such protein complexes have been identified in yeast, and they are required for different trafficking steps. Homologous complexes are found in all eukaryotic organisms, but conservation seems to be less strict than for other components of the trafficking machinery. In fact, for most proposed multi-subunit tethers their ability to actually bridge two membranes remains to be shown. Here we discuss recent progress in the structural and functional characterization of tethering complexes and present the emerging view that the different complexes are quite diverse in their structure and the molecular mechanisms underlying their function. TRAPP and the exocyst are the structurally best characterized tethering complexes. Their comparison fails to reveal any similarity on a struc nottural level. Furthermore, the interactions with regulatory Rab GTPases vary, with TRAPP acting as a nucleotide exchange factor and the exocyst being an effector. Considering these differences among the tethering complexes as well as between their yeast and mammalian orthologs which is apparent from recent studies, we suggest that tethering complexes do not mediate a strictly conserved process in vesicular transport but are diverse regulators acting after vesicle budding and prior to membrane fusion.

Barriers to and facilitators of implementing complex workplace dietary interventions: process evaluation results of a cluster controlled trial.

PubMed

Fitzgerald, Sarah; Geaney, Fiona; Kelly, Clare; McHugh, Sheena; Perry, Ivan J

2016-04-21

Ambiguity exists regarding the effectiveness of workplace dietary interventions. Rigorous process evaluation is vital to understand this uncertainty. This study was conducted as part of the Food Choice at Work trial which assessed the comparative effectiveness of a workplace environmental dietary modification intervention and an educational intervention both alone and in combination versus a control workplace. Effectiveness was assessed in terms of employees' dietary intakes, nutrition knowledge and health status in four large manufacturing workplaces. The study aimed to examine barriers to and facilitators of implementing complex workplace interventions, from the perspectives of key workplace stakeholders and researchers involved in implementation. A detailed process evaluation monitored and evaluated intervention implementation. Interviews were conducted at baseline (27 interviews) and at 7-9 month follow-up (27 interviews) with a purposive sample of workplace stakeholders (managers and participating employees). Topic guides explored factors which facilitated or impeded implementation. Researchers involved in recruitment and data collection participated in focus groups at baseline and at 7-9 month follow-up to explore their perceptions of intervention implementation. Data were imported into NVivo software and analysed using a thematic framework approach. Four major themes emerged; perceived benefits of participation, negotiation and flexibility of the implementation team, viability and intensity of interventions and workplace structures and cultures. The latter three themes either positively or negatively affected implementation, depending on context. The implementation team included managers involved in coordinating and delivering the interventions and the researchers who collected data and delivered intervention elements. Stakeholders' perceptions of the benefits of participating, which facilitated implementation, included managers' desire to improve company

Altered mechanisms underlying the abnormal glutamate release in amyotrophic lateral sclerosis at a pre-symptomatic stage of the disease.

PubMed

Bonifacino, Tiziana; Musazzi, Laura; Milanese, Marco; Seguini, Mara; Marte, Antonella; Gallia, Elena; Cattaneo, Luca; Onofri, Franco; Popoli, Maurizio; Bonanno, Giambattista

2016-11-01

Abnormal Glu release occurs in the spinal cord of SOD1(G93A) mice, a transgenic animal model for human ALS. Here we studied the mechanisms underlying Glu release in spinal cord nerve terminals of SOD1(G93A) mice at a pre-symptomatic disease stage (30days) and found that the basal release of Glu was more elevated in SOD1(G93A) with respect to SOD1 mice, and that the surplus of release relies on synaptic vesicle exocytosis. Exposure to high KCl or ionomycin provoked Ca(2+)-dependent Glu release that was likewise augmented in SOD1(G93A) mice. Equally, the Ca(2+)-independent hypertonic sucrose-induced Glu release was abnormally elevated in SOD1(G93A) mice. Also in this case, the surplus of Glu release was exocytotic in nature. We could determine elevated cytosolic Ca(2+) levels, increased phosphorylation of Synapsin-I, which was causally related to the abnormal Glu release measured in spinal cord synaptosomes of pre-symptomatic SOD1(G93A) mice, and increased phosphorylation of glycogen synthase kinase-3 at the inhibitory sites, an event that favours SNARE protein assembly. Western blot experiments revealed an increased number of SNARE protein complexes at the nerve terminal membrane, with no changes of the three SNARE proteins and increased expression of synaptotagmin-1 and β-Actin, but not of an array of other release-related presynaptic proteins. These results indicate that the abnormal exocytotic Glu release in spinal cord of pre-symptomatic SOD1(G93A) mice is mainly based on the increased size of the readily releasable pool of vesicles and release facilitation, supported by plastic changes of specific presynaptic mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Elongation factor Ts directly facilitates the formation and disassembly of the Escherichia coli elongation factor Tu·GTP·aminoacyl-tRNA ternary complex.

PubMed

Burnett, Benjamin J; Altman, Roger B; Ferrao, Ryan; Alejo, Jose L; Kaur, Navdep; Kanji, Joshua; Blanchard, Scott C

2013-05-10

Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. EF-Tu·GTP·aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). EF-Ts directly facilitates the formation and disassociation of ternary complex. This system demonstrates a novel function of EF-Ts. Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis.

Prenatal complex rhythmic music sound stimulation facilitates postnatal spatial learning but transiently impairs memory in the domestic chick.

PubMed

Kauser, H; Roy, S; Pal, A; Sreenivas, V; Mathur, R; Wadhwa, S; Jain, S

2011-01-01

Early experience has a profound influence on brain development, and the modulation of prenatal perceptual learning by external environmental stimuli has been shown in birds, rodents and mammals. In the present study, the effect of prenatal complex rhythmic music sound stimulation on postnatal spatial learning, memory and isolation stress was observed. Auditory stimulation with either music or species-specific sounds or no stimulation (control) was provided to separate sets of fertilized eggs from day 10 of incubation. Following hatching, the chicks at age 24, 72 and 120 h were tested on a T-maze for spatial learning and the memory of the learnt task was assessed 24 h after training. In the posthatch chicks at all ages, the plasma corticosterone levels were estimated following 10 min of isolation. The chicks of all ages in the three groups took less (p < 0.001) time to navigate the maze over the three trials thereby showing an improvement with training. In both sound-stimulated groups, the total time taken to reach the target decreased significantly (p < 0.01) in comparison to the unstimulated control group, indicating the facilitation of spatial learning. However, this decline was more at 24 h than at later posthatch ages. When tested for memory after 24 h of training, only the music-stimulated chicks at posthatch age 24 h took a significantly longer (p < 0.001) time to traverse the maze, suggesting a temporary impairment in their retention of the learnt task. In both sound-stimulated groups at 24 h, the plasma corticosterone levels were significantly decreased (p < 0.001) and increased thereafter at 72 h (p < 0.001) and 120 h which may contribute to the differential response in spatial learning. Thus, prenatal auditory stimulation with either species-specific or complex rhythmic music sounds facilitates spatial learning, though the music stimulation transiently impairs postnatal memory. 2011 S. Karger AG, Basel.

Conformational fluctuation of Synaptotagmin-1 observed with single molecule fluorescence resonance energy transfer (smFRET)

NASA Astrophysics Data System (ADS)

Choi, Ucheor; Weninger, Keith

2008-10-01

Calcium dependent neurotransmitter release at the synapses involves a synaptic vesicle protein synaptotagmin-1, a calcium sensor, to regulate exocytosis. It has been known that Synaptotagmin-1 interacts with assembled SNARE complexes, but it is unclear how their molecular mechanisms are coupled. X-ray studies in the absence of calcium revealed a closed conformation of synaptotagmin-1 and with calcium bound to the C2 domains of synaptotagmin-3 found extensive interactions holding the domains open. Suggesting the two conformations can be the key to the two functions of synaptotagmin in regulating neurotransmission. Here we use single molecule fluorescence resonance energy transfer (smFRET) to study synaptotagmin interactions with SNARE complexes and the spontaneous conformational changes of synaptotagmin-1 when calcium is induced.

The Race that Precedes Coactivation: Development of Multisensory Facilitation in Children

ERIC Educational Resources Information Center

Barutchu, Ayla; Crewther, David P.; Crewther, Sheila G.

2009-01-01

Rationale: The facilitating effect of multisensory integration on motor responses in adults is much larger than predicted by race-models and is in accordance with the idea of coactivation. However, the development of multisensory facilitation of endogenously driven motor processes and its relationship to the development of complex cognitive skills…

UreE-UreG Complex Facilitates Nickel Transfer and Preactivates GTPase of UreG in Helicobacter pylori*♦

PubMed Central

Yang, Xinming; Li, Hongyan; Lai, Tsz-Pui; Sun, Hongzhe

2015-01-01

The pathogenicity of Helicobacter pylori relies heavily on urease, which converts urea to ammonia to neutralize the stomach acid. Incorporation of Ni2+ into the active site of urease requires a battery of chaperones. Both metallochaperones UreE and UreG play important roles in the urease activation. In this study, we demonstrate that, in the presence of GTP and Mg2+, UreG binds Ni2+ with an affinity (Kd) of ∼0.36 μm. The GTPase activity of Ni2+-UreG is stimulated by both K+ (or NH4+) and HCO3− to a biologically relevant level, suggesting that K+/NH4+ and HCO3− might serve as GTPase elements of UreG. We show that complexation of UreE and UreG results in two protein complexes, i.e. 2E-2G and 2E-G, with the former being formed only in the presence of both GTP and Mg2+. Mutagenesis studies reveal that Arg-101 on UreE and Cys-66 on UreG are critical for stabilization of 2E-2G complex. Combined biophysical and bioassay studies show that the formation of 2E-2G complex not only facilitates nickel transfer from UreE to UreG, but also enhances the binding of GTP. This suggests that UreE might also serve as a structural scaffold for recruitment of GTP to UreG. Importantly, we demonstrate for the first time that UreE serves as a bridge to grasp Ni2+ from HypA, subsequently donating it to UreG. The study expands our horizons on the molecular details of nickel translocation among metallochaperones UreE, UreG, and HypA, which further extends our knowledge on the urease maturation process. PMID:25752610

Composite cell sheet for periodontal regeneration: crosstalk between different types of MSCs in cell sheet facilitates complex periodontal-like tissue regeneration.

PubMed

Zhang, Hao; Liu, Shiyu; Zhu, Bin; Xu, Qiu; Ding, Yin; Jin, Yan

2016-11-14

Tissue-engineering strategies based on mesenchymal stem cells (MSCs) and cell sheets have been widely used for periodontal tissue regeneration. However, given the complexity in periodontal structure, the regeneration methods using a single species of MSC could not fulfill the requirement for periodontal regeneration. We researched the interaction between the periodontal ligament stem cells (PDLSCs) and jaw bone marrow-derived mesenchymal stem cells (JBMMSCs), and constructed a composite cell sheet comprising both of the above MSCs to regenerate complex periodontium-like structures in nude mice. Our results show that by co-culturing PDLSCs and JBMMSCs, the expressions of bone and extracellular matrix (ECM)-related genes and proteins were significantly improved in both MSCs. Further investigations showed that, compared to the cell sheet using PDLSCs or JBMMSCs, the composite stem cell sheet (CSCS), which comprises these two MSCs, expressed higher levels of bone- and ECM-related genes and proteins, and generated a composite structure more similar to the native periodontal tissue physiologically in vivo. In conclusion, our results demonstrate that the crosstalk between PDLSCs and JBMMSCs in cell sheets facilitate regeneration of complex periodontium-like structures, providing a promising new strategy for physiological and functional regeneration of periodontal tissue.

Does Adaptive Scaffolding Facilitate Students' Ability to Regulate their Learning with Hypermedia?

ERIC Educational Resources Information Center

Azevedo, Roger; Cromley, Jennifer G.; Seibert, Diane

2004-01-01

Is adaptive scaffolding effective in facilitating students' ability to regulate their learning of complex science topics with hypermedia? We examined the role of different scaffolding instructional interventions in facilitating students' shift to more sophisticated mental models as indicated by both performance and process data. Undergraduate…

Early Adolescent Friendship Selection Based on Externalizing Behavior: the Moderating Role of Pubertal Development. The SNARE Study.

PubMed

Franken, Aart; Prinstein, Mitchell J; Dijkstra, Jan Kornelis; Steglich, Christian E G; Harakeh, Zeena; Vollebergh, Wilma A M

2016-11-01

This study examined friendship (de-)selection processes in early adolescence. Pubertal development was examined as a potential moderator. It was expected that pubertal development would be associated with an increased tendency for adolescents to select their friends based on their similarities in externalizing behavior engagement (i.e., delinquency, alcohol use, and tobacco use). Data were used from the first three waves of the SNARE (Social Network Analysis of Risk behavior in Early adolescence) study (N = 1144; 50 % boys; M age  = 12.7; SD = 0.47), including students who entered the first year of secondary school. The hypothesis was tested using Stochastic Actor-Based Modeling in SIENA. While taking the network structure into account, and controlling for peer influence effects, the results supported this hypothesis. Early adolescents with higher pubertal development were as likely as their peers to select friends based on similarity in externalizing behavior and especially likely to remain friends with peers who had a similar level of externalizing behavior, and thus break friendship ties with dissimilar friends in this respect. As early adolescents are actively engaged in reorganizing their social context, adolescents with a higher pubertal development are especially likely to lose friendships with peers who do not engage in externalizing behavior, thus losing an important source of adaptive social control (i.e., friends who do not engage in externalizing behavior).

DigiMemo: Facilitating the Note Taking Process

ERIC Educational Resources Information Center

Kurt, Serhat

2009-01-01

Everyone takes notes daily for various reasons. Note taking is very popular in school settings and generally recognized as an effective learning strategy. Further, note taking is a complex process because it requires understanding, selection of information and writing. Some new technological tools may facilitate the note taking process. Among such…

Components of the SNARE-containing regulon are co-regulated in root cells undergoing defense

PubMed Central

Klink, Vincent P.; Sharma, Keshav; Pant, Shankar R.; McNeece, Brant; Niraula, Prakash; Lawrence, Gary W.

2017-01-01

ABSTRACT The term regulon has been coined in the genetic model plant Arabidopsis thaliana, denoting a structural and physiological defense apparatus defined genetically through the identification of the penetration (pen) mutants. The regulon is composed partially by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) syntaxin PEN1. PEN1 has homology to a Saccharomyces cerevisae gene that regulates a Secretion (Sec) protein, Suppressor of Sec 1 (Sso1p). The regulon is also composed of the β-glucosidase (PEN2) and an ATP binding cassette (ABC) transporter (PEN3). While important in inhibiting pathogen infection, limited observations have been made regarding the transcriptional regulation of regulon genes until now. Experiments made using the model agricultural Glycine max (soybean) have identified co-regulated gene expression of regulon components. The results explain the observation of hundreds of genes expressed specifically in the root cells undergoing the natural process of defense. Data regarding additional G. max genes functioning within the context of the regulon are presented here, including Sec 14, Sec 4 and Sec 23. Other examined G. max homologs of membrane fusion genes include an endosomal bromo domain-containing protein1 (Bro1), syntaxin6 (SYP6), SYP131, SYP71, SYP8, Bet1, coatomer epsilon (ε-COP), a coatomer zeta (ζ-COP) paralog and an ER to Golgi component (ERGIC) protein. Furthermore, the effectiveness of biochemical pathways that would function within the context of the regulon ave been examined, including xyloglucan xylosyltransferase (XXT), reticuline oxidase (RO) and galactinol synthase (GS). The experiments have unveiled the importance of the regulon during defense in the root and show how the deposition of callose relates to the process. PMID:28010187

Extended cold snare polypectomy for small colorectal polyps increases the R0 resection rate.

PubMed

Abe, Yasuhiro; Nabeta, Haruaki; Koyanagi, Ryota; Nakamichi, Taro; Hirashima, Hayato; Lefor, Alan Kawarai; Shinozaki, Satoshi

2018-02-01

 Despite widespread use of cold snare polypectomy (CSP), the R0 resection rate is not well documented. We perform extended CSP, resecting polyps with a > 1 mm circumferential margin. The aim of this study is to compare the R0 resection rate of extended CSP with conventional CSP and to assess safety. From April 2014 to September 2016, 712 non-pedunculated colorectal polyps, < 10 mm in size, resected using CSP from 316 patients were retrospectively analyzed.  We divided lesions into conventional CSP (n = 263) and extended CSP groups (n = 449). The baseline characteristics of these two groups were not significantly different in univariate or multivariate analyses. Sessile polyps comprised 94 % (668/712), and the remaining were flat-elevated polyps. Mean size of polyps (±standard deviation) was 4.2 ± 1.5 mm. The most frequent pathology was low grade adenoma (97 %, 689/712). The R0 resection rate was significantly higher in the extended CSP group (439/449 [98 %]) than in the conventional CSP group (222/263 [84 %], P  < 0.001). There was no delayed bleeding or perforation in either group (conventional CSP group, 0/263, 95 % confidence interval: 0.0 - 1.4 % and extended CSP group, 0/449, 95 % confidence interval: 0.0 - 0.8 %).  Extended CSP results in a higher R0 resection rate compared with conventional CSP. Extended CSP did not result in a higher rate of delayed bleeding or perforation. Extended CSP is a safe and promising procedure for endoscopic resection of non-pedunculated colorectal polyps < 10 mm in size.

Mutual Alignment Comparison Facilitates Abstraction and Transfer of a Complex Scientific Concept

ERIC Educational Resources Information Center

Orton, Judy M.; Anggoro, Florencia K.; Jee, Benjamin D.

2012-01-01

Learning about a scientific concept often occurs in the context of unfamiliar examples. Mutual alignment analogy--a type of analogical comparison in which the analogues are only partially understood--has been shown to facilitate learning from unfamiliar examples . In the present study, we examined the role of mutual alignment analogy in the…

Facilitating Facilitators: Enhancing PBL through a Structured Facilitator Development Program

ERIC Educational Resources Information Center

Salinitri, Francine D.; Wilhelm, Sheila M.; Crabtree, Brian L.

2015-01-01

With increasing adoption of the problem-based learning (PBL) model, creative approaches to enhancing facilitator training and optimizing resources to maintain effective learning in small groups is essential. We describe a theoretical framework for the development of a PBL facilitator training program that uses the constructivist approach as the…

PEG-lipid-PLGA hybrid nanoparticles loaded with berberine-phospholipid complex to facilitate the oral delivery efficiency.

PubMed

Yu, Fei; Ao, Mingtao; Zheng, Xiao; Li, Nini; Xia, Junjie; Li, Yang; Li, Donghui; Hou, Zhenqing; Qi, Zhongquan; Chen, Xiao Dong

2017-11-01

The natural product berberine (BBR), present in various plants, arouses great interests because of its numerous pharmacological effects. However, the further development and application of BBR had been hampered by its poor oral bioavailability. In this work, we report on polymer-lipid hybrid nanoparticles (PEG-lipid-PLGA NPs) loaded with BBR phospholipid complex using a solvent evaporation method for enhancing the oral BBR efficiency. The advantage of this new drug delivery system is that the BBR-soybean phosphatidylcholine complex (BBR-SPC) could be used to enhance the liposolubility of BBR and improve the affinity with the biodegradable polymer to increase the drug-loading capacity and controlled/sustained release. The entrapment efficiency of the PEG-lipid-PLGA NPs/BBR-SPC was observed to approach approximately 89% which is more than 2.4 times compared with that of the PEG-lipid-PLGA NPs/BBR. To the best of our knowledge, this is the first report on using polymer material for effective encapsulation of BBR to improve its oral bioavailability. The prepared BBR delivery systems demonstrated a uniform spherical shape, a well-dispersed core-shell structure and a small particle size (149.6 ± 5.1 nm). The crystallographic and thermal analysis has indicated that the BBR dispersed in the PEG-lipid-PLGA NPs matrix is in an amorphous form. More importantly, the enhancement in the oral relative bioavailability of the PEG-lipid-PLGA NPs/BBR-SPC was ∼343% compared with that of BBR. These positive results demonstrated that PEG-lipid-PLGA NPs/BBR-SPC may have the potential for facilitating the oral drug delivery of BBR.

An exploration of teaching presence in online interprofessional education facilitation.

PubMed

Evans, Sherryn Maree; Ward, Catherine; Reeves, Scott

2017-07-01

Although the prevalence of online asynchronous interprofessional education (IPE) has increased in the last decade, little is known about the processes of facilitation in this environment. The teaching presence element of the Community of Inquiry Framework offers an approach to analyze the contributions of online facilitators, however, to date it has only been used on a limited basis in health professions education literature. Using an exploratory case study design, we explored the types of contributions made by IPE facilitators to asynchronous interprofessional team discussions by applying the notion of teaching presence. Using a purposeful sampling approach, we analyzed 14 facilitators' contributions to asynchronous team discussion boards in an online IPE course. We analyzed data using directed content analysis based on the key indicators of teaching presence. The online IPE facilitators undertook the three critical pedagogical functions identified in teaching presence: facilitating discourse, direct instruction, and instructional design and organization. While our data fitted well with a number of key activities embedded in these three functions, further modification of the teaching presence concept was needed to describe our facilitators' teaching presence. This study provides an initial insight into the key elements of online asynchronous IPE facilitation. Further research is required to continue to illuminate the complexity of online asynchronous IPE facilitation.

Endoscopic treatment by snare electrocoagulation prior to Nd:YAG laser photocoagulation in 85 voluminous colorectal villous adenomas.

PubMed

Aubert, A; Meduri, B; Fritsch, J; Aime, F; Baglin, A; Barbagelata, M

1991-05-01

The association of endoscopic resection with Nd:YAG laser photocoagulation was used to treat benign colorectal villous adenomas. Eight-five patients were included: 49 with surgical contraindications, 35 for whom surgical resection appeared to be too hazardous, and 1 who refused surgery. Forty-five tumors had an axial extension between 1 and 3 cm, and 40 tumors had an axial extension of at least 4 cm. Diathermic snare resection was performed to remove large tumoral fragments prior to laser photocoagulation of the residual flat lesions. Treatments were repeated every 15 days until total tumor destruction was achieved. A carcinoma was detected in biopsy specimens obtained during endoscopic treatment of five patients. Two patients were lost to follow-up. Treatment results could be analyzed in 78 patients. Successful treatment was achieved in 67 patients. Tumor destruction was complete in 77 percent of patients who had lesions of at least 4 cm diameter and in 93 percent of patients with smaller lesions. The axial extension of the tumor was the main factor affecting the results of treatment. No major complications occurred. During the average 103-week follow-up period, 21 percent of the patients with total tumor destruction had a recurrence. The risk of recurrence was correlated with the number of initial treatment sessions and previous surgery treatment. It would appear that the treatment with endoscopic resection prior to Nd:YAG laser photocoagulation is a safe and effective method in the destruction of colorectal villous adenomas.

Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells

PubMed Central

Hagiwara-Chatani, Natsumi; Shirai, Kota; Kido, Takumi; Horigome, Tomoatsu; Yasue, Akihiro; Adachi, Naoki; Hirai, Yohei

2017-01-01

Embryonic stem (ES) and induced pluripotent stem (iPS) cells are attractive tools for regenerative medicine therapies. However, aberrant cell populations that display flattened morphology and lose ground-state pluripotency often appear spontaneously, unless glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase (MEK1/2) are inactivated. Here, we show that membrane translocation of the t-SNARE protein syntaxin-4 possibly is involved in this phenomenon. We found that mouse ES cells cultured without GSK3β/MEK1/2 inhibitors (2i) spontaneously extrude syntaxin-4 at the cell surface and that artificial expression of cell surface syntaxin-4 induces appreciable morphological changes and mesodermal differentiation through dephosphorylation of Akt. Transcriptome analyses revealed several candidate elements responsible for this, specifically, an E-to P-cadherin switch and a marked downregulation of Zscan4 proteins, which are DNA-binding proteins essential for ES cell pluripotency. Embryonic carcinoma cell lines F9 and P19CL6, which maintain undifferentiated states independently of Zscan4 proteins, exhibited similar cellular behaviors upon stimulation with cell surface syntaxin-4. The functional ablation of E-cadherin and overexpression of P-cadherin reproduced syntaxin-4-induced cell morphology, demonstrating that the E- to P-cadherin switch executes morphological signals from cell surface syntaxin-4. Thus, spontaneous membrane translocation of syntaxin-4 emerged as a critical element for maintenance of the stem-cell niche. PMID:28057922

Therapeutic use of botulinum toxin in migraine: mechanisms of action

PubMed Central

Ramachandran, Roshni; Yaksh, Tony L

2014-01-01

Migraine pain represents sensations arising from the activation of trigeminal afferents, which innervate the meningeal vasculature and project to the trigeminal nucleus caudalis (TNC). Pain secondary to meningeal input is referred to extracranial regions innervated by somatic afferents that project to homologous regions in the TNC. Such viscerosomatic convergence accounts for referral of migraine pain arising from meningeal afferents to particular extracranial dermatomes. Botulinum toxins (BoNTs) delivered into extracranial dermatomes are effective in and approved for treating chronic migraine pain. Aside from their well-described effect upon motor endplates, BoNTs are also taken up in local afferent nerve terminals where they cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, and prevent local terminal release. However, a local extracranial effect of BoNT cannot account for allthe effects of BoNT upon migraine. We now know that peripherally delivered BoNTs are taken up in sensory afferents and transported to cleave SNARE proteins in the ganglion and TNC, prevent evoked afferent release and downstream activation. Such effects upon somatic input (as from the face) likewise would not alone account for block of input from converging meningeal afferents. This current work suggests that BoNTs may undergo transcytosis to cleave SNAREs in second-order neurons or in adjacent afferent terminals. Finally, while SNAREs mediate exocytotic release, they are also involved in transport of channels and receptors involved in facilitated pain states. The role of such post-synaptic effects of BoNT action in migraine remains to be determined. PMID:24819339

Working alone or in the presence of others: exploring social facilitation in baggage X-ray security screening tasks.

PubMed

Yu, Rui-feng; Wu, Xin

2015-01-01

This study investigated whether the mere presence of a human audience would evoke a social facilitation effect in baggage X-ray security screening tasks. A 2 (target presence: present vs. absent) ×  2 (task complexity: simple vs. complex) ×  2 (social presence: alone vs. human audience) within-subject experiment simulating a real baggage screening task was conducted. This experiment included 20 male participants. The participants' search performance in this task was recorded. The results showed that the presence of a human audience speeded up responses in simple tasks and slowed down responses in complex tasks. However, the social facilitation effect produced by the presence of a human audience had no effect on response accuracy. These findings suggested that the complexity of screening tasks should be considered when designing work organisation modes for security screening tasks. Practitioner summary: This study investigated whether the presence of a human audience could evoke a social facilitation effect in baggage X-ray security screening tasks. An experimental simulation was conducted. The results showed that the presence of a human audience facilitated the search performance of simple tasks and inhibited the performance of complex tasks.

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity*

PubMed Central

van der Kant, Rik; Jonker, Caspar T. H.; Wijdeven, Ruud H.; Bakker, Jeroen; Janssen, Lennert; Klumperman, Judith; Neefjes, Jacques

2015-01-01

Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail in yeast where their sequential membrane targeting and assembly is well understood. Mammalian CORVET and HOPS subunits significantly differ from their yeast homologues, and novel proteins with high homology to CORVET/HOPS subunits have evolved. However, an analysis of the molecular interactions between these subunits in mammals is lacking. Here, we provide a detailed analysis of interactions within the mammalian CORVET and HOPS as well as an additional endosomal-targeting complex (VIPAS39-VPS33B) that does not exist in yeast. We show that core interactions within CORVET and HOPS are largely conserved but that the membrane-targeting module in HOPS has significantly changed to accommodate binding to mammalian-specific RAB7 interacting lysosomal protein (RILP). Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-associated mutations in VPS33B selectively disrupt recruitment to late endosomes by RILP or binding to its partner VIPAS39. Within the shared core of CORVET/HOPS, we find that VPS11 acts as a molecular switch that binds either CORVET-specific TGFBRAP1 or HOPS-specific VPS39/RILP thereby allowing selective targeting of these tethering complexes to early or late endosomes to time fusion events in the endo/lysosomal pathway. PMID:26463206

Teaching Note--Constructing Critical Conversations: A Model for Facilitating Classroom Dialogue for Critical Learning

ERIC Educational Resources Information Center

Kang, Hye-Kyung; O'Neill, Peggy

2018-01-01

Discussions of power and privilege, oppression, and structural inequities in classrooms can produce complex understanding and critical analysis when facilitated effectively. In this article we present the critical conversations model for facilitating conversations that open up space for discussing such issues and encourage the development of…

Facilitated recycling protects human RNA polymerase III from repression by Maf1 in vitro.

PubMed

Cabart, Pavel; Lee, JaeHoon; Willis, Ian M

2008-12-26

Yeast cells synthesize approximately 3-6 million molecules of tRNA every cell cycle at a rate of approximately 2-4 transcripts/gene/s. This high rate of transcription is achieved through many rounds of reinitiation by RNA polymerase (pol) III on stable DNA-bound complexes of the initiation factor TFIIIB. Studies in yeast have shown that the rate of reinitiation is increased by facilitated recycling, a process that involves the repeated reloading of the polymerase on the same transcription unit. However, when nutrients become limiting or stress conditions are encountered, RNA pol III transcription is rapidly repressed through the action of the conserved Maf1 protein. Here we examine the relationship between Maf1-mediated repression and facilitated recycling in a human RNA pol III in vitro system. Using an immobilized template transcription assay, we demonstrate that facilitated recycling is conserved from yeast to humans. We assessed the ability of recombinant human Maf1 to inhibit different steps in transcription before and after preinitiation complex assembly. We show that recombinant Maf1 can inhibit the recruitment of TFIIIB and RNA pol III to immobilized templates. However, RNA pol III bound to preinitiation complexes or in elongation complexes is protected from repression by Maf1 and can undergo several rounds of initiation. This indicates that recombinant Maf1 is unable to inhibit facilitated recycling. The data suggest that additional biochemical steps may be necessary for rapid Maf1-dependent repression of RNA pol III transcription.

Increased lipolysis and altered lipid homeostasis protect y-synuclein null mutant mice from diet-induced obesity

USDA-ARS?s Scientific Manuscript database

Synucleins are a family of homologous proteins principally known for their involvement in neurodegeneration. In neurons a-synuclein promotes assembly of SNARE complexes required for fusion of synaptic vesicles with the plasma membrane during neurotransmitter release. Y-synuclein is highly expressed ...

Cex1p facilitates Rna1p-mediated dissociation of the Los1p-tRNA-Gsp1p-GTP export complex.

PubMed

McGuire, Andrew T; Mangroo, Dev

2012-02-01

Nuclear tRNA export plays an essential role in key cellular processes such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage and development. Like other nuclear export processes, assembly of the nuclear tRNA export complex in the nucleus is dependent on Ran-GTP/Gsp1p-GTP, and dissociation of the export receptor-tRNA-Ran-GTP/Gsp1p-GTP complex in the cytoplasm requires RanBP1/Yrb1p and RanGAP/Rna1p to activate the GTPase activity of Ran-GTP/Gsp1p-GTP. The Saccharomyces cerevisiae Cex1p and Human Scyl1 have also been proposed to participate in unloading of the tRNA export receptors at the cytoplasmic face of the nuclear pore complex (NPC). Here, we provide evidence suggesting that Cex1p is required for activation of the GTPase activity of Gsp1p and dissociation of the receptor-tRNA-Gsp1p export complex in S. cerevisiae. The data suggest that Cex1p recruits Rna1p from the cytoplasm to the NPC and facilitates Rna1p activation of the GTPase activity of Gsp1p by enabling Rna1p to gain access to Gsp1p-GTP bound to the export receptor tRNA complex. It is possible that this tRNA unloading mechanism is conserved in evolutionarily diverse organisms and that other Gsp1p-GTP-dependent export processes use a pathway-specific component to recruit Rna1p to the NPC. © 2011 John Wiley & Sons A/S.

Membrane microdomains and the cytoskeleton constrain AtHIR1 dynamics and facilitate the formation of an AtHIR1-associated immune complex.

PubMed

Lv, Xueqin; Jing, Yanping; Xiao, Jianwei; Zhang, Yongdeng; Zhu, Yingfang; Julian, Russell; Lin, Jinxing

2017-04-01

Arabidopsis hypersensitive-induced reaction (AtHIR) proteins function in plant innate immunity. However, the underlying mechanisms by which AtHIRs participate in plant immunity remain elusive. Here, using VA-TIRFM and FLIM-FRET, we revealed that AtHIR1 is present in membrane microdomains and co-localizes with the membrane microdomain marker REM1.3. Single-particle tracking analysis revealed that membrane microdomains and the cytoskeleton, especially microtubules, restrict the lateral mobility of AtHIR1 at the plasma membrane and facilitate its oligomerization. Furthermore, protein proximity index measurements, fluorescence cross-correlation spectroscopy, and biochemical experiments demonstrated that the formation of the AtHIR1 complex upon pathogen perception requires intact microdomains and cytoskeleton. Taken together, these findings suggest that microdomains and the cytoskeleton constrain AtHIR1 dynamics, promote AtHIR1 oligomerization, and increase the efficiency of the interactions of AtHIR1 with components of the AtHIR1 complex in response to pathogens, thus providing valuable insight into the mechanisms of defense-related responses in plants. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner.

PubMed

Diao, Aipo; Frost, Laura; Morohashi, Yuichi; Lowe, Martin

2008-03-14

During membrane traffic, transport carriers are first tethered to the target membrane prior to undergoing fusion. Mechanisms exist to connect tethering with fusion, but in most cases, the details remain poorly understood. GM130 is a member of the golgin family of coiled-coil proteins tat is involved in membrane tethering at the endoplasmic reticulum (ER) to Golgi intermediate compartment and cis-Golgi. Here, we demonstrate that GM130 interacts with syntaxin 5, a t-SNARE also localized to the early secretory pathway. Binding to syntaxin 5 is specific, direct, and mediated by the membrane-proximal region of GM130. Interestingly, interaction with syntaxin 5 is inhibited by the binding of the vesicle docking protein p115 to a distal binding site in GM130. The interaction between GM130 and the small GTPase Rab1 is also inhibited by p115 binding. Our findings suggest a mechanism for coupling membrane tethering and fusion at the ER to Golgi intermediate compartment and cis-Golgi, with GM130 playing a central role in linking these processes. Consistent with this hypothesis, we find that depletion of GM130 by RNA interference slows the rate of ER to Golgi trafficking in vivo. The interactions of GM130 with syntaxin 5 and Rab1 are also regulated by mitotic phosphorylation, which is likely to contribute to the inhibition of ER to Golgi trafficking that occurs when mammalian cells enter mitosis.

Use of 3D printer technology to facilitate surgical correction of a complex vascular anomaly with esophageal entrapment in a dog.

PubMed

Dundie, A; Hayes, G; Scrivani, P; Campoy, L; Fletcher, D; Ash, K; Oxford, E; Moïse, N S

2017-04-01

A 10 week old female intact Staffordshire terrier was presented with a total of five congenital cardio-thoracic vascular anomalies consisting of a patent ductus arteriosus (PDA) with an aneurysmic dilation, pulmonic stenosis, persistent right aortic arch, aberrant left subclavian artery and persistent left cranial vena cava. These abnormalities were identified with a combination of echocardiogram and computed tomography angiography (CTA). The abnormalities were associated with esophageal entrapment, regurgitation, and volume overload of the left heart with left atrial and ventricular enlargement. A 2 cm diameter aneurysmic dilation at the junction of the PDA, right aortic arch and aberrant left subclavian artery presented an unusual surgical challenge and precluded simple circumferential ligation and transection of the structure. A full scale three dimensional model of the heart and vasculature was constructed from the CTA and plasma sterilized. The model was used preoperatively to facilitate surgical planning and enhance intraoperative communication and coordination between the surgical and anesthesia teams. Intraoperatively the model facilitated spatial orientation, atraumatic vascular dissection, instrument sizing and positioning. A thoracoabdominal stapler was used to close the PDA aneurysm prior to transection. At the four-month postoperative follow-up the patient was doing well. This is the first reported application of new imaging and modeling technology to enhance surgical planning when approaching correction of complex cardiovascular anomalies in a dog. Copyright © 2016 Elsevier B.V. All rights reserved.

Facilitators & Barriers to the Adoption of Ergonomic Solutions in Construction

PubMed Central

Dale, Ann Marie; Jaegers, Lisa; Welch, Laura; Barnidge, Ellen; Weaver, Nancy; Evanoff, Bradley A.

2017-01-01

Background Rates of musculoskeletal disorders in construction remain high. Few studies have described barriers and facilitators to the use of available ergonomic solutions. This paper describes these barriers and facilitators and their relationship to the level of adoption. Methods Three analysts rated 16 proposed ergonomic solutions from a participatory ergonomics study and assessed the level of adoption, six adoption characteristics, and identified the category of adoption from a theoretical model. Results Twelve solutions were always or intermittently used and were rated positively for characteristics of relative advantage, compatibility with existing work processes, and trialability. Locus of control (worker vs. contractor) was not related to adoption. Simple solutions faced fewer barriers to adoption than those rated as complex. Conclusions Specific adoption characteristics can help predict the use of new ergonomic solutions in construction. Adoption of complex solutions must involve multiple stakeholders, more time, and shifts in culture or work systems. PMID:28195660

Music Preferences, Friendship, and Externalizing Behavior in Early Adolescence: A SIENA Examination of the Music Marker Theory Using the SNARE Study.

PubMed

Franken, Aart; Keijsers, Loes; Dijkstra, Jan Kornelis; Ter Bogt, Tom

2017-08-01

Music Marker Theory posits that music is relevant for the structuring of peer groups and that rock, urban, or dance music preferences relate to externalizing behavior. The present study tested these hypotheses, by investigating the role of music preference similarity in friendship selection and the development of externalizing behavior, while taking the effects of friends' externalizing behavior into account. Data were used from the first three waves of the SNARE (Social Network Analysis of Risk behavior in Early adolescence) study (N = 1144; 50% boys; M age  = 12.7; SD = 0.47), including students who entered the first-year of secondary school. Two hypotheses were tested. First, adolescents were expected to select friends based both on a similarity in externalizing behavior and music genre preference. Second, a preference for rock, urban, or dance, music types was expected to predict the development of externalizing behavior, even when taking friends' influence on externalizing behavior into account. Stochastic Actor-Based Modeling indicated that adolescents select their friends based on both externalizing behavior and highbrow music preference. Moreover, both friends' externalizing behavior and a preference for dance music predicted the development of externalizing behavior. Intervention programs might focus on adolescents with dance music preferences.

Synaptotagmin 11 interacts with components of the RNA-induced silencing complex RISC in clonal pancreatic β-cells.

PubMed

Milochau, Alexandra; Lagrée, Valérie; Benassy, Marie-Noëlle; Chaignepain, Stéphane; Papin, Julien; Garcia-Arcos, Itsaso; Lajoix, Anne; Monterrat, Carole; Coudert, Laetitia; Schmitter, Jean-Marie; Ochoa, Begoña; Lang, Jochen

2014-06-27

Synaptotagmins are two C2 domain-containing transmembrane proteins. The function of calcium-sensitive members in the regulation of post-Golgi traffic has been well established whereas little is known about the calcium-insensitive isoforms constituting half of the protein family. Novel binding partners of synaptotagmin 11 were identified in β-cells. A number of them had been assigned previously to ER/Golgi derived-vesicles or linked to RNA synthesis, translation and processing. Whereas the C2A domain interacted with the Q-SNARE Vti1a, the C2B domain of syt11 interacted with the SND1, Ago2 and FMRP, components of the RNA-induced silencing complex (RISC). Binding to SND was direct via its N-terminal tandem repeats. Our data indicate that syt11 may provide a link between gene regulation by microRNAs and membrane traffic. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Disease resistance through impairment of α-SNAP–NSF interaction and vesicular trafficking by soybean Rhg1

PubMed Central

Bayless, Adam M.; Smith, John M.; Song, Junqi; McMinn, Patrick H.; Teillet, Alice; August, Benjamin K.

2016-01-01

α-SNAP [soluble NSF (N-ethylmaleimide–sensitive factor) attachment protein] and NSF proteins are conserved across eukaryotes and sustain cellular vesicle trafficking by mediating disassembly and reuse of SNARE protein complexes, which facilitate fusion of vesicles to target membranes. However, certain haplotypes of the Rhg1 (resistance to Heterodera glycines 1) locus of soybean possess multiple repeat copies of an α-SNAP gene (Glyma.18G022500) that encodes atypical amino acids at a highly conserved functional site. These Rhg1 loci mediate resistance to soybean cyst nematode (SCN; H. glycines), the most economically damaging pathogen of soybeans worldwide. Rhg1 is widely used in agriculture, but the mechanisms of Rhg1 disease resistance have remained unclear. In the present study, we found that the resistance-type Rhg1 α-SNAP is defective in interaction with NSF. Elevated in planta expression of resistance-type Rhg1 α-SNAPs depleted the abundance of SNARE-recycling 20S complexes, disrupted vesicle trafficking, induced elevated abundance of NSF, and caused cytotoxicity. Soybean, due to ancient genome duplication events, carries other loci that encode canonical (wild-type) α-SNAPs. Expression of these α-SNAPs counteracted the cytotoxicity of resistance-type Rhg1 α-SNAPs. For successful growth and reproduction, SCN dramatically reprograms a set of plant root cells and must sustain this sedentary feeding site for 2–4 weeks. Immunoblots and electron microscopy immunolocalization revealed that resistance-type α-SNAPs specifically hyperaccumulate relative to wild-type α-SNAPs at the nematode feeding site, promoting the demise of this biotrophic interface. The paradigm of disease resistance through a dysfunctional variant of an essential gene may be applicable to other plant–pathogen interactions. PMID:27821740

The Robustness of a Signaling Complex to Domain Rearrangements Facilitates Network Evolution

PubMed Central

Sato, Paloma M.; Yoganathan, Kogulan; Jung, Jae H.; Peisajovich, Sergio G.

2014-01-01

The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering. PMID:25490747

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

PubMed Central

MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

2015-01-01

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

PubMed Central

Gaffaney, Jon D.; Dunning, F. Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R.

2008-01-01

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B. PMID:18784080

Practice Facilitators' and Leaders' Perspectives on a Facilitated Quality Improvement Program.

PubMed

McHugh, Megan; Brown, Tiffany; Liss, David T; Walunas, Theresa L; Persell, Stephen D

2018-04-01

Practice facilitation is a promising approach to helping practices implement quality improvements. Our purpose was to describe practice facilitators' and practice leaders' perspectives on implementation of a practice facilitator-supported quality improvement program and describe where their perspectives aligned and diverged. We conducted interviews with practice leaders and practice facilitators who participated in a program that included 35 improvement strategies aimed at the ABCS of heart health (aspirin use in high-risk individuals, blood pressure control, cholesterol management, and smoking cessation). Rapid qualitative analysis was used to collect, organize, and analyze the data. We interviewed 17 of the 33 eligible practice leaders, and the 10 practice facilitators assigned to those practices. Practice leaders and practice facilitators both reported value in the program's ability to bring needed, high-quality resources to practices. Practice leaders appreciated being able to set the schedule for facilitation and select among the 35 interventions. According to practice facilitators, however, relying on practice leaders to set the pace of the intervention resulted in a lower level of program intensity than intended. Practice leaders preferred targeted assistance, particularly electronic health record documentation guidance and linkages to state smoking cessation programs. Practice facilitators reported that the easiest interventions were those that did not alter care practices. The dual perspectives of practice leaders and practice facilitators provide a more holistic picture of enablers and barriers to program implementation. There may be greater opportunities to assist small practices through simple, targeted practice facilitator-supported efforts rather than larger, comprehensive quality improvement projects. © 2018 Annals of Family Medicine, Inc.

SNAP-23 regulates phagosome formation and maturation in macrophages

PubMed Central

Sakurai, Chiye; Hashimoto, Hitoshi; Nakanishi, Hideki; Arai, Seisuke; Wada, Yoh; Sun-Wada, Ge-Hong; Wada, Ikuo; Hatsuzawa, Kiyotaka

2012-01-01

Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane–localized soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus–tagged SNAP-23 was established. These cells showed enhanced Fc receptor–mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H+-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic. PMID:23087210

Facilitating Argumentative Knowledge Construction through a Transactive Discussion Script in CSCL

ERIC Educational Resources Information Center

Noroozi, Omid; Weinberger, Armin; Biemans, Harm J. A.; Mulder, Martin; Chizari, Mohammad

2013-01-01

Learning to argue is prerequisite to solving complex problems in groups, especially when they are multidisciplinary and collaborate online. Environments for Computer-Supported Collaborative Learning (CSCL) can be designed to facilitate argumentative knowledge construction. This study investigates how argumentative knowledge construction in…

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

PubMed

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex

PubMed Central

Ganaie, Safder S.; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve

2017-01-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases. PMID:28459842

The Influence of Facilitator and Facilitation Characteristics on Participants' Ratings of Stepfamily Education

ERIC Educational Resources Information Center

Higginbotham, Brian J.; Myler, Cory

2010-01-01

We examine the relative importance of facilitator and facilitation characteristics on participant ratings of a stepfamily education program. Data from 48 facilitators and 598 participants suggest that quality facilitation is more meaningful to participants than whether facilitators have comparable demographic characteristics or life experiences.…

Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

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

Chung, Le Thi Kim, E-mail: ngocanh@nutr.med.tokushima-u.ac.jp; Hosaka, Toshio; Harada, Nagakatsu

2010-01-01

In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4more » to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.« less

Importance of stimulation paradigm in determining facilitation and effects of neuromodulation.

PubMed

Crider, M E; Cooper, R L

1999-09-25

Evoked synaptic activity within the CNS and at the neuromuscular junction in most in vivo preparations studied occurs not with single isolated stimuli, but with trains, or bursts, of stimuli. Although for ease in studying the mechanisms of vesicular synaptic transmission one often uses single discrete stimuli, the true mechanisms in the animal may be far more complex. When repetitive stimuli are present at a nerve terminal, often a heightened (i.e., facilitated) postsynaptic potential can be as a result. Facilitation is commonly used as an index of synaptic function and plasticity induced by chronic stimulation or by neuromodulation. The mechanisms that give rise to facilitation are thought to be the same that may underlie short-term learning and memory [C.H. Bailey, E.R. Kandel, Structural changes accompanying memory storage. Annu. Rev. Physiol. 55 (1993) 397-426.]. Differences in short term facilitation (STF) are seen depending on the conventional stimulation paradigm (twin pulse, train, or continuous) used to induce facilitation. Thus, a battery of paradigms should be used to characterize synaptic function to obtain a closer understanding of the possible in vivo conditions.

Removal of diminutive colorectal polyps: A prospective randomized clinical trial between cold snare polypectomy and hot forceps biopsy

PubMed Central

Komeda, Yoriaki; Kashida, Hiroshi; Sakurai, Toshiharu; Tribonias, George; Okamoto, Kazuki; Kono, Masashi; Yamada, Mitsunari; Adachi, Teppei; Mine, Hiromasa; Nagai, Tomoyuki; Asakuma, Yutaka; Hagiwara, Satoru; Matsui, Shigenaga; Watanabe, Tomohiro; Kitano, Masayuki; Chikugo, Takaaki; Chiba, Yasutaka; Kudo, Masatoshi

2017-01-01

AIM To compare the efficacy and safety of cold snare polypectomy (CSP) and hot forceps biopsy (HFB) for diminutive colorectal polyps. METHODS This prospective, randomized single-center clinical trial included consecutive patients ≥ 20 years of age with diminutive colorectal polyps 3-5 mm from December 2014 to October 2015. The primary outcome measures were en-bloc resection (endoscopic evaluation) and complete resection rates (pathological evaluation). The secondary outcome measures were the immediate bleeding or immediate perforation rate after polypectomy, delayed bleeding or delayed perforation rate after polypectomy, use of clipping for bleeding or perforation, and polyp retrieval rate. Prophylactic clipping after polyp removal wasn’t routinely performed. RESULTS Two hundred eight patients were randomized into the CSP (102), HFB (106) and 283 polyps were evaluated (CSP: 148, HFB: 135). The en-bloc resection rate was significantly higher with CSP than with HFB [99.3% (147/148) vs 80.0% (108/135), P < 0.0001]. The complete resection rate was significantly higher with CSP than with HFB [80.4% (119/148) vs 47.4% (64/135), P < 0.0001]. The immediate bleeding rate was similar between the groups [8.6% (13/148) vs 8.1% (11/135), P = 1.000], and endoscopic hemostasis with hemoclips was successful in all cases. No cases of perforation or delayed bleeding occurred. The rate of severe tissue injury to the pathological specimen was higher HFB than CSP [52.6% (71/135) vs 1.3% (2/148), P < 0.0001]. Polyp retrieval failure was encountered CSP (7), HFB (2). CONCLUSION CSP is more effective than HFB for resecting diminutive polyps. Further long-term follow-up study is required. PMID:28127206

Removal of diminutive colorectal polyps: A prospective randomized clinical trial between cold snare polypectomy and hot forceps biopsy.

PubMed

Komeda, Yoriaki; Kashida, Hiroshi; Sakurai, Toshiharu; Tribonias, George; Okamoto, Kazuki; Kono, Masashi; Yamada, Mitsunari; Adachi, Teppei; Mine, Hiromasa; Nagai, Tomoyuki; Asakuma, Yutaka; Hagiwara, Satoru; Matsui, Shigenaga; Watanabe, Tomohiro; Kitano, Masayuki; Chikugo, Takaaki; Chiba, Yasutaka; Kudo, Masatoshi

2017-01-14

To compare the efficacy and safety of cold snare polypectomy (CSP) and hot forceps biopsy (HFB) for diminutive colorectal polyps. This prospective, randomized single-center clinical trial included consecutive patients ≥ 20 years of age with diminutive colorectal polyps 3-5 mm from December 2014 to October 2015. The primary outcome measures were en-bloc resection (endoscopic evaluation) and complete resection rates (pathological evaluation). The secondary outcome measures were the immediate bleeding or immediate perforation rate after polypectomy, delayed bleeding or delayed perforation rate after polypectomy, use of clipping for bleeding or perforation, and polyp retrieval rate. Prophylactic clipping after polyp removal wasn't routinely performed. Two hundred eight patients were randomized into the CSP (102), HFB (106) and 283 polyps were evaluated (CSP: 148, HFB: 135). The en-bloc resection rate was significantly higher with CSP than with HFB [99.3% (147/148) vs 80.0% (108/135), P < 0.0001]. The complete resection rate was significantly higher with CSP than with HFB [80.4% (119/148) vs 47.4% (64/135), P < 0.0001]. The immediate bleeding rate was similar between the groups [8.6% (13/148) vs 8.1% (11/135), P = 1.000], and endoscopic hemostasis with hemoclips was successful in all cases. No cases of perforation or delayed bleeding occurred. The rate of severe tissue injury to the pathological specimen was higher HFB than CSP [52.6% (71/135) vs 1.3% (2/148), P < 0.0001]. Polyp retrieval failure was encountered CSP (7), HFB (2). CSP is more effective than HFB for resecting diminutive polyps. Further long-term follow-up study is required.

Facilitating social support: member-leader communication in a breast cancer support group.

PubMed

Beck, Stephenson J; Keyton, Joann

2014-01-01

Early detection and treatment have resulted in more women surviving breast cancer; increased survivorship has also increased the need for breast cancer support groups (BCSG). The ostensible goal of such groups is to provide support for the physical and emotional stressors that cancer survivors face, as well as provide information on coping and treatment options. Although scholars have examined the effects of support groups on their group members, the examination of group facilitator messages has been largely neglected. The goal of this study was to extend theory on group leader behavior, specifically investigating how member-leader messages create social support in support groups. The transcribed conversations of weekly meetings of a BCSG were examined using Interaction Process Analysis to discover how the member-leader facilitated the group's enactment and management of social support. Across the meetings, task talk dominated (primarily statements of orientation or information). Furthermore, analysis of interaction sequences between the support group facilitator and other members revealed 2 broad categories of task-oriented facilitation techniques (changing the focus, clarification) and 1 category of socioemotional facilitation techniques (showing support). Support group facilitators need the ability to facilitate both task and relational aspects of social support. Facilitator behaviors were highlighted as being instrumental to the creation of social support. The results from this study indicate that the ability to change the focus of interaction, to provide and require clarification on complex issues, and to show support through relational messages is needed in facilitator training.

Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion

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

Wasik, Anita A.; Dumont, Vincent; Tienari, Jukka

Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex,more » as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane. - Highlights: • Septin 7, nonmuscle myosin heavy chain IIA (NMHC-IIA) and SNAP23 form a complex. • Knockdown of septin 7 increases NM-IIA activity in the SNAP23 complex. • Insulin decreases septin 7 level and increases NM-IIA activity in the SNAP23 complex. • Septin 7 hinders GSV docking/fusion by reducing NM-IIA activity in the SNAP23 complex.« less

Using learning theory, interprofessional facilitation competencies, and behavioral indicators to evaluate facilitator training.

PubMed

LeGros, Theresa A; Amerongen, Helen M; Cooley, Janet H; Schloss, Ernest P

2015-01-01

Despite the increasing need for faculty and preceptors skilled in interprofessional facilitation (IPF), the relative novelty of the field poses a challenge to the development and evaluation of IPF programs. We use learning theory and IPF competencies with associated behavioral indicators to develop and evaluate six key messages in IPF training and experience. Our mixed methods approach included two phases: quantitative data collection with embedded qualitative data, followed by qualitative data collection in explanatory sequential fashion. This enabled triangulated analyses of both data types and of facilitation behaviors from facilitator and student perspectives. Results indicate the competency-based training was effective. Facilitators felt comfortable performing behaviors associated with IPF competencies; student observations of those behaviors supported facilitator self-reported performance. Overall, students perceived more facilitation opportunities than facilitators. Findings corroborate the importance of recruiting seasoned facilitators and establishing IPF guidelines that acknowledge variable team dynamics and help facilitators recognize teachable moments.

Managing Postsocialist Transitions: Politicized Sense Making as a Facilitator of Organizational Change

ERIC Educational Resources Information Center

Tiplic, Dijana

2011-01-01

This study explores what organizational strategies are employed to initiate and facilitate organizational change in higher education institutions in the increasingly complex and competitive postsocialist environment of Bosnia-Herzegovina. By studying organizations trapped between their inert socialist-era legacies and desired organizational…

Stimulus Equalization: Temporary Reduction of Stimulus Complexity to Facilitate Discrimination Learning.

ERIC Educational Resources Information Center

Hoko, J. Aaron; LeBlanc, Judith M.

1988-01-01

Because disabled learners may profit from procedures using gradual stimulus change, this study utilized a microcomputer to investigate the effectiveness of stimulus equalization, an error reduction procedure involving an abrupt but temporary reduction of dimensional complexity. The procedure was found to be generally effective and implications for…

Positive feelings facilitate working memory and complex decision making among older adults.

PubMed

Carpenter, Stephanie M; Peters, Ellen; Västfjäll, Daniel; Isen, Alice M

2013-01-01

The impact of induced mild positive feelings on working memory and complex decision making among older adults (aged 63-85) was examined. Participants completed a computer administered card task in which participants could win money if they chose from "gain" decks and lose money if they chose from "loss" decks. Individuals in the positive-feeling condition chose better than neutral-feeling participants and earned more money overall. Participants in the positive-feeling condition also demonstrated improved working-memory capacity. These effects of positive-feeling induction have implications for affect theory, as well as, potentially, practical implications for people of all ages dealing with complex decisions.

The informativity of sound modulates crossmodal facilitation of visual discrimination: a fMRI study.

PubMed

Li, Qi; Yu, Hongtao; Li, Xiujun; Sun, Hongzan; Yang, Jingjing; Li, Chunlin

2017-01-18

Many studies have investigated behavioral crossmodal facilitation when a visual stimulus is accompanied by a concurrent task-irrelevant sound. Lippert and colleagues reported that a concurrent task-irrelevant sound reduced the uncertainty of the timing of the visual display and improved perceptional responses (informative sound). However, the neural mechanism by which the informativity of sound affected crossmodal facilitation of visual discrimination remained unclear. In this study, we used event-related functional MRI to investigate the neural mechanisms underlying the role of informativity of sound in crossmodal facilitation of visual discrimination. Significantly faster reaction times were observed when there was an informative relationship between auditory and visual stimuli. The functional MRI results showed sound informativity-induced activation enhancement including the left fusiform gyrus and the right lateral occipital complex. Further correlation analysis showed that the right lateral occipital complex was significantly correlated with the behavioral benefit in reaction times. This suggests that this region was modulated by the informative relationship within audiovisual stimuli that was learnt during the experiment, resulting in late-stage multisensory integration and enhanced behavioral responses.

Genetic analysis of the role of amyloplasts in shoot gravisensing

NASA Astrophysics Data System (ADS)

Tasaka, M.; Morita, M.

Plant can change the growth direction after sensing the gravity orientation This response calls gravitropism and the initial step is the gravisensing We have isolated many Arabidopsis mutants shoot gravitropism sgr with reduced or no gravitropic response in inflorescence stems The analysis of sgr1 and sgr7 revealed that endoderm cells in the inflorescence stems were gravisensing sites zig zigzag sgr4 and sgr3 showed no or reduced gravitropism in shoot respectively and their amyloplasts thought to be statoliths did not sedimented to the orientation of gravity in the endoderm cells ZIG encoded a SNARE AtVTI11 and SGR3 encoded other SNARE AtVAM3 These two SNAREs made a complex in the shoot endoderm cells suggesting that the vesicle transport from trans-Golgi network TGN to prevacuolar compartment PVC and or vacuole was involved in the amyloplasts localization and movement The analysis to visualize amyloplasts and vacuolar membrane in living endoderm cells supported that the vacuole function was important for the amyloplasts movement Recently we have isolated many suppressor mutants of zig One of them named zig suppressor zip 1 had a point mutation in the gene encoded other SNARE of AtVTI12 This protein is a homologous to ZIG AtVTI11 and these two proteins have partially redundant functions Although wild type At VTI 12 could not rescued zig mutated AtVTI12 protein ZIP1 could almost completely play the part of ZIG In zigzip1 amyloplasts in endoderm cells sedimented normally and the shoots showed normal gravitropic response The other

Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

PubMed

MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

2015-04-24

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Algorithms for treatment of complex hand injuries].

PubMed

Pillukat, T; Prommersberger, K-J

2011-07-01

The primary treatment strongly influences the course and prognosis of hand injuries. Complex injuries which compromise functional recovery are especially challenging. Despite an apparently unlimited number of injury patterns it is possible to develop strategies which facilitate a standardized approach to operative treatment. In this situation algorithms can be important guidelines for a rational approach. The following algorithms have been proven in the treatment of complex injuries of the hand by our own experience. They were modified according to the current literature and refer to prehospital care, emergency room management, basic strategy in general and reconstruction of bone and joints, vessels, nerves, tendons and soft tissue coverage in detail. Algorithms facilitate the treatment of severe hand injuries. Applying simple yes/no decisions complex injury patterns are split into distinct partial problems which can be managed step by step.

Homeless youth: Barriers and facilitators for service referrals.

PubMed

Black, Emma B; Fedyszyn, Izabela E; Mildred, Helen; Perkin, Rhianna; Lough, Richard; Brann, Peter; Ritter, Cheryl

2018-06-01

Young people who are homeless and experiencing mental health issues are reluctant to use relevant services for numerous reasons. Youth are also at risk of disengaging from services at times of referral to additional or alternative services. This study aimed to identify barriers and facilitators for inter-service referrals for homeless youth with mental health issues who have already engaged with a service. Qualitative, semi-structured interviews were conducted with homeless youth (n = 10), homelessness support workers (n = 10), and mental health clinicians (n = 10). Barriers included: resource shortages; programs or services having inflexible entry criteria; complexity of service systems; homeless youth feeling devalued; and a lack of communication between services, for example, abrupt referrals with no follow up. Referral facilitators included: services providers offering friendly and client-centred support; supported referrals; awareness of other services; and collaboration between services. Relationships with service providers and inter-service collaboration appeared essential for successful referrals for homeless youth. These facilitating factors may be undermined by sector separation and siloing, as well as resource shortages in both the homelessness and mental health sectors. Service transitions may be conceptualised as a genuine service outcome for homeless youth, and as a basis for successful future service provision. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pretreatment with β-adrenergic receptor agonists facilitates induction of LTP and sharp wave ripple complexes in rodent hippocampus.

PubMed

Ul Haq, Rizwan; Anderson, Marlene; Liotta, Agustin; Shafiq, Maria; Sherkheli, Muhammad Azhar; Heinemann, Uwe

2016-12-01

Norepinephrine, is involved in the enhancement of learning and memory formation by regulating synaptic mechanisms through its ability to activate pre- and post-synaptic adrenergic receptors. Here we show that β-agonists of norepinephrine facilitate the induction of both associational LTP and sharp wave ripples (SPW-Rs) in acute slices of rat hippocampus in area CA3. Surprisingly, this facilitating effect persists when slices are only pretreated with β-receptor agonists followed by wash out and application of the unspecific β-adrenoreceptor (βAR) antagonist propranolol. During application of βAR agonists repeated stimulation resulted in facilitated induction of SPW-Rs. Since SPW-Rs are thought to be involved in memory replay we studied the effects of βAR-agonists on spontaneous SPW-Rs in murine hippocampus and found that amplitude and incidence of SPW-Rs increased. These effects involve cyclic-AMP and the activation of protein kinase A and suggest a supportive role in memory consolidation. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing.

PubMed

Yu, Simei; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Jain, Shruti; Rolicka, Anna; Östlund Farrants, Ann-Kristin; Visa, Neus

2018-05-31

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

Experiences of practice facilitators working on the Improved Delivery of Cardiovascular Care project: Retrospective case study.

PubMed

Liddy, Clare; Rowan, Margo; Valiquette-Tessier, Sophie-Claire; Drosinis, Paul; Crowe, Lois; Hogg, William

2018-01-01

To examine the barriers to and facilitators of practice facilitation experienced by participants in the Improving Delivery of Cardiovascular Care (IDOCC) project. Case studies of practice facilitators' narrative reports. Eastern Ontario. Primary care practices that participated in the IDOCC project. Cases were identified by calculating sum scores in order to determine practices' performance relative to their peers. Two case exemplars were selected that scored within ± 1 SD of the total mean score, and a qualitative analysis of practice facilitators' narrative reports was conducted using a 5-factor implementation framework to identify barriers and facilitators. Narratives were divided into 3 phases: planning, implementation, and sustainability. Barriers and facilitators fluctuated over the intervention's 3 phases. Site A reported more barriers (n = 47) than facilitators (n = 38), while site B reported a roughly equal number of barriers (n = 144) and facilitators (n = 136). In both sites, the most common barriers involved organizational and provider factors and the most common facilitators were associated with innovation and structural factors. Both practices encountered various barriers and facilitators throughout the IDOCC's 3 phases. The case studies reveal the complex interactions of these factors over time, and provide insight into the implementation of practice facilitation programs. Copyright© the College of Family Physicians of Canada.

Understanding Facilitation: Theory and Principles.

ERIC Educational Resources Information Center

Hogan, Christine

This book introduces newcomers to the concept of facilitation, and it presents a critical analysis of established and current theory on facilitation for existing practitioners. The following are among the topics discussed: (1) emergence of the field of facilitation; (2) development of facilitation in management; (3) development of facilitation in…

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

PubMed Central

Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves

2016-01-01

ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. PMID:27803188

Interactions between Depression and Facilitation within Neural Networks: Updating the Dual-Process Theory of Plasticity

PubMed Central

Prescott, Steven A.

1998-01-01

Repetitive stimulation often results in habituation of the elicited response. However, if the stimulus is sufficiently strong, habituation may be preceded by transient sensitization or even replaced by enduring sensitization. In 1970, Groves and Thompson formulated the dual-process theory of plasticity to explain these characteristic behavioral changes on the basis of competition between decremental plasticity (depression) and incremental plasticity (facilitation) occurring within the neural network. Data from both vertebrate and invertebrate systems are reviewed and indicate that the effects of depression and facilitation are not exclusively additive but, rather, that those processes interact in a complex manner. Serial ordering of induction of learning, in which a depressing locus precedes the modulatory system responsible for inducing facilitation, causes the facilitation to wane. The parallel and/or serial expression of depression and waning facilitation within the stimulus–response pathway culminates in the behavioral changes that characterize dual-process learning. A mathematical model is presented to formally express and extend understanding of the interactions between depression and facilitation. PMID:10489261

Cold snare polypectomy reduced delayed postpolypectomy bleeding compared with conventional hot polypectomy: a propensity score-matching analysis

PubMed Central

Yamashina, Takeshi; Fukuhara, Manabu; Maruo, Takanori; Tanke, Gensho; Marui, Saiko; Sada, Ryota; Taki, Mio; Ohara, Yoshiaki; Sakamoto, Azusa; Henmi, Shinichiro; Sawai, Yugo; Saito, Sumio; Nishijima, Norihiro; Nasu, Akihiro; Komekado, Hideyuki; Sekikawa, Akira; Asada, Masanori; Tumura, Takehiko; Kita, Ryuichi; Kimura, Toru; Osaki, Yukio

2017-01-01

Background and study aims  Cold snare polypectomy (CSP) for small colorectal polyps has lower incidence of adverse events, especially delayed postpolypectomy bleeding (DPPB). However, few data are available on comparisons of the incidence of DPPB of CSP and hot polypectomy (HP). The aim of this study was to evaluate the incidence of DPPB after CSP and compare it with that of HP. A propensity score model was used as a secondary analysis. Patients and methods  This was a retrospective cohort study conducted in a single municipal hospital. We identified 539 patients with colorectal polyps from 2 mm to 11 mm in size who underwent CSP (804 polyps in 330 patients) or HP (530 polyps in 209 patients) between July 2013 and June 2015. Results  There were no cases of DPPB in the CSP group. Conversely, DPPB occurred in 4 patients (1.9 %) after HP, resulting in a significant difference between the CSP and HP groups (0.008 % vs 0 %, P  = 0.02). Propensity score-matching analysis created 402 matched pairs, yielding a significantly higher DPPB rate in the HP group than CSP group (0.02 % vs 0 %, P  = 0.04). However, significantly more patients in the CSP group had unclear horizontal margins that precluded assessment (83 vs 38 cases, P  < 0.001). The retrieval failure rate was significantly higher in the CSP group than in the HP group (3 % vs 0.7 %, P  = 0.01). Conclusions  DPPB was less frequent with CSP than HP, as selected by the propensity score-matching model. Our findings indicate that CSP is recommended polypectomy in daily clinical setting. However, special care should be taken during polyp retrieval and horizontal margin assessment, and these issues could be taken into account in follow-up after CSP. PMID:28670615

Design Research as a Mechanism for Consultants to Facilitate and Evaluate Educational Innovations

ERIC Educational Resources Information Center

Castillo, Jose M.; Dorman, Clark; Gaunt, Brian; Hardcastle, Beth; Justice, Kelly; March, Amanda L.

2016-01-01

Schools across the nation are implementing innovative practices; however, questions remain regarding how to facilitate quality implementation. Research designs that emphasize high degrees of control over independent variables result in findings with internal validity, but that may not generalize to complex, dynamic educational systems. The purpose…

Endovascular Retrieval of Entrapped Elephant Trunk Graft During Complex Hybrid Aortic Arch Repair

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

Damodharan, Karthikeyan, E-mail: drdkarthik@hotmail.com; Chao, Victor T. T., E-mail: victor.chao.t.t@singhealth.com.sg; Tay, Kiang Hiong, E-mail: tay.kiang.hiong@singhealth.com.sg

Entrapment of the elephant trunk graft within the false lumen is a rare complication of surgical repair of an aortic dissection. This is normally retrieved by emergent open surgery. We describe a technique of endovascular retrieval of the dislodged graft, during hybrid aortic arch repair. The elephant trunk was cannulated through and through from a femoral access and the free end of the wire was snared and retrieved from a brachial access. The wire was externalised from both accesses and was used to reposition the graft into the true lumen using a body flossing technique.

Facilitated versus Non-Facilitated Online Case Discussions: Comparing Differences in Problem Space Coverage

ERIC Educational Resources Information Center

Ertmer, Peggy A.; Koehler, Adrie A.

2015-01-01

The facilitator plays a key role in guiding students' efforts during case discussions. However, few studies have compared differences in learning outcomes for students participating in facilitated versus non-facilitated discussions. In this research, we used "problem space coverage" as a learning measure to compare outcomes between…

Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms.

PubMed

Bhalla, Akhil; Chicka, Michael C; Chapman, Edwin R

2008-08-01

Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca(2+)-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca(2+), while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca(2+). Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca(2+)-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.

Unpacking the Complexity of Planning with Persons with Cognitive Disability and Complex Support Needs

ERIC Educational Resources Information Center

Collings, Susan; Dew, Angela; Dowse, Leanne

2018-01-01

Background: Planners will engage with people with cognitive disability and complex support needs in the Australian National Disability Insurance Scheme, but the specific skills needed to build sustainable plans with this group are not yet known. Method: A qualitative study was conducted to explore the barriers and facilitators to planning with…

Induced vibrations facilitate traversal of cluttered obstacles

NASA Astrophysics Data System (ADS)

Thoms, George; Yu, Siyuan; Kang, Yucheng; Li, Chen

When negotiating cluttered terrains such as grass-like beams, cockroaches and legged robots with rounded body shapes most often rolled their bodies to traverse narrow gaps between beams. Recent locomotion energy landscape modeling suggests that this locomotor pathway overcomes the lowest potential energy barriers. Here, we tested the hypothesis that body vibrations induced by intermittent leg-ground contact facilitate obstacle traversal by allowing exploration of locomotion energy landscape to find this lowest barrier pathway. To mimic a cockroach / legged robot pushing against two adjacent blades of grass, we developed an automated robotic system to move an ellipsoidal body into two adjacent beams, and varied body vibrations by controlling an oscillation actuator. A novel gyroscope mechanism allowed the body to freely rotate in response to interaction with the beams, and an IMU and cameras recorded the motion of the body and beams. We discovered that body vibrations facilitated body rolling, significantly increasing traversal probability and reducing traversal time (P <0.0001, ANOVA). Traversal probability increased with and traversal time decreased with beam separation. These results confirmed our hypothesis and support the plausibility of locomotion energy landscapes for understanding the formation of locomotor pathways in complex 3-D terrains.

Citizen social science: a methodology to facilitate and evaluate workplace learning in continuing interprofessional education.

PubMed

Dadich, Ann

2014-05-01

Workplace learning in continuing interprofessional education (CIPE) can be difficult to facilitate and evaluate, which can create a number of challenges for this type of learning. This article presents an innovative method to foster and investigate workplace learning in CIPE - citizen social science. Citizen social science involves clinicians as co-researchers in the systematic examination of social phenomena. When facilitated by an open-source online social networking platform, clinicians can participate via computer, smartphone, or tablet in ways that suit their needs and preferences. Furthermore, as co-researchers they can help to reveal the dynamic interplay that facilitates workplace learning in CIPE. Although yet to be tested, citizen social science offers four potential benefits: it recognises and accommodates the complexity of workplace learning in CIPE; it has the capacity to both foster and evaluate the phenomena; it can be used in situ, capturing and having direct relevance to the complexity of the workplace; and by advancing both theoretical and methodological debates on CIPE, it may reveal opportunities to improve and sustain workplace learning. By describing an example situated in the youth health sector, this article demonstrates how these benefits might be realised.

Self-recognition in corals facilitates deep-sea habitat engineering

USGS Publications Warehouse

Hennige, Sebastian J; Morrison, Cheryl L.; Form, Armin U.; Buscher, Janina; Kamenos, Nicholas A.; Roberts, J. Murray

2014-01-01

The ability of coral reefs to engineer complex three-dimensional habitats is central to their success and the rich biodiversity they support. In tropical reefs, encrusting coralline algae bind together substrates and dead coral framework to make continuous reef structures, but beyond the photic zone, the cold-water coral Lophelia pertusa also forms large biogenic reefs, facilitated by skeletal fusion. Skeletal fusion in tropical corals can occur in closely related or juvenile individuals as a result of non-aggressive skeletal overgrowth or allogeneic tissue fusion, but contact reactions in many species result in mortality if there is no ‘self-recognition’ on a broad species level. This study reveals areas of ‘flawless’ skeletal fusion in Lophelia pertusa, potentially facilitated by allogeneic tissue fusion, are identified as having small aragonitic crystals or low levels of crystal organisation, and strong molecular bonding. Regardless of the mechanism, the recognition of ‘self’ between adjacent L. pertusa colonies leads to no observable mortality, facilitates ecosystem engineering and reduces aggression-related energetic expenditure in an environment where energy conservation is crucial. The potential for self-recognition at a species level, and subsequent skeletal fusion in framework-forming cold-water corals is an important first step in understanding their significance as ecological engineers in deep-seas worldwide.

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

PubMed

Blaikley, Elizabeth J; Tinline-Purvis, Helen; Kasparek, Torben R; Marguerat, Samuel; Sarkar, Sovan; Hulme, Lydia; Hussey, Sharon; Wee, Boon-Yu; Deegan, Rachel S; Walker, Carol A; Pai, Chen-Chun; Bähler, Jürg; Nakagawa, Takuro; Humphrey, Timothy C

2014-05-01

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability. © The Author(s) 2014. Published by Oxford University Press.

Economic Feasibility of Staffing the Intensive Care Unit with a Communication Facilitator.

PubMed

Khandelwal, Nita; Benkeser, David; Coe, Norma B; Engelberg, Ruth A; Curtis, J Randall

2016-12-01

In the intensive care unit (ICU), complex decision making by clinicians and families requires good communication to ensure that care is consistent with the patients' values and goals. To assess the economic feasibility of staffing ICUs with a communication facilitator. Data were from a randomized trial of an "ICU communication facilitator" linked to hospital financial records; eligible patients (n = 135) were admitted to the ICU at a single hospital with predicted mortality ≥30% and a surrogate decision maker. Adjusted regression analyses assessed differences in ICU total and direct variable costs between intervention and control patients. A bootstrap-based simulation assessed the cost efficiency of a facilitator while varying the full-time equivalent of the facilitator and the ICU mortality risk. Total ICU costs (mean 22.8k; 95% CI, -42.0k to -3.6k; P = 0.02) and average daily ICU costs (mean, -0.38k; 95% CI, -0.65k to -0.11k; P = 0.006)] were reduced significantly with the intervention. Despite more contacts, families of survivors spent less time per encounter with facilitators than did families of decedents (mean, 25 [SD, 11] min vs. 36 [SD, 14] min). Simulation demonstrated maximal weekly savings with a 1.0 full-time equivalent facilitator and a predicted ICU mortality of 15% (total weekly ICU cost savings, $58.4k [95% CI, $57.7k-59.2k]; weekly direct variable savings, $5.7k [95% CI, $5.5k-5.8k]) after incorporating facilitator costs. Adding a full-time trained communication facilitator in the ICU may improve the quality of care while simultaneously reducing short-term (direct variable) and long-term (total) health care costs. This intervention is likely to be more cost effective in a lower-mortality population.

Endovascular rescue method for undesirably stretched coil.

PubMed

Cho, Jae Hoon

2014-10-01

Undesirable detachment or stretching of coils within the parent artery during aneurysm embolization can be related with thrombus formation, which can be caused occlusion of parent artery or embolic event(s). To escape from this situation, several rescue methods have been reported. A case with undesirably stretched coil in which another rescue method was used, is presented. When the stretched coil is still located in the coil delivery microcatheter, the stretched coil can be removed safely using a snare and a handmade monorail microcatheter. After a snare is lodged in the handmade monorail microcatheter, the snare is introduced over the coil delivery micorcatheter and located in the distal part of the stretched coil. After then, the handmade monorail microcatheter captures the stretched coil and the snare as one unit. This technique using a handmade monorail microcatheter and a snare can be a good rescue modality for the undesirably stretched coil, still remained within the coil delivery microcatheter.

Endovascular Rescue Method for Undesirably Stretched Coil

PubMed Central

2014-01-01

Undesirable detachment or stretching of coils within the parent artery during aneurysm embolization can be related with thrombus formation, which can be caused occlusion of parent artery or embolic event(s). To escape from this situation, several rescue methods have been reported. A case with undesirably stretched coil in which another rescue method was used, is presented. When the stretched coil is still located in the coil delivery microcatheter, the stretched coil can be removed safely using a snare and a handmade monorail microcatheter. After a snare is lodged in the handmade monorail microcatheter, the snare is introduced over the coil delivery micorcatheter and located in the distal part of the stretched coil. After then, the handmade monorail microcatheter captures the stretched coil and the snare as one unit. This technique using a handmade monorail microcatheter and a snare can be a good rescue modality for the undesirably stretched coil, still remained within the coil delivery microcatheter. PMID:25371791

PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

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

Lim, So-Hee; Moon, Jeonghee; Lee, Myungkyu

2013-09-13

Highlights: •PTPRT is a brain-specific, expressed, protein tyrosine phosphatase. •PTPRT regulated the interaction of Syntaxin-binding protein 1 with Syntaxin 1. •PTPRT dephosphorylated the specific tyrosine residue of Syntaxin-binding protein 1. •Dephosphorylation of Syntaxin-binding protein 1 enhanced the interaction with Syntaxin 1. •PTPRT appears to regulate the fusion of synaptic vesicle through dephosphorylation. -- Abstract: PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified asmore » a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.« less

Leucine facilitates insulin signaling through a Gαi protein-dependent signaling pathway in hepatocytes.

PubMed

Yang, Xuefeng; Mei, Shuang; Wang, Xiaolei; Li, Xiang; Liu, Rui; Ma, Yan; Hao, Liping; Yao, Ping; Liu, Liegang; Sun, Xiufa; Gu, Haihua; Liu, Zhenqi; Cao, Wenhong

2013-03-29

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt(473) and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K (LY294002), Gαi protein (pertussis toxin or siRNA against Gαi1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gαi1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gαi protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly.

Leucine Facilitates Insulin Signaling through a Gαi Protein-dependent Signaling Pathway in Hepatocytes*

PubMed Central

Yang, Xuefeng; Mei, Shuang; Wang, Xiaolei; Li, Xiang; Liu, Rui; Ma, Yan; Hao, Liping; Yao, Ping; Liu, Liegang; Sun, Xiufa; Gu, Haihua; Liu, Zhenqi; Cao, Wenhong

2013-01-01

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt473 and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K (LY294002), Gαi protein (pertussis toxin or siRNA against Gαi1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gαi1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gαi protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly. PMID:23404499

Barriers and Facilitators to Implementing a Change Initiative in Long-Term Care Using the INTERACT® Quality Improvement Program.

PubMed

Tappen, Ruth M; Wolf, David G; Rahemi, Zahra; Engstrom, Gabriella; Rojido, Carolina; Shutes, Jill M; Ouslander, Joseph G

Implementation of major organizational change initiatives presents a challenge for long-term care leadership. Implementation of the INTERACT® (Interventions to Reduce Acute Care Transfers) quality improvement program, designed to improve the management of acute changes in condition and reduce unnecessary emergency department visits and hospitalizations of nursing home residents, serves as an example to illustrate the facilitators and barriers to major change in long-term care. As part of a larger study of the impact of INTERACT® on rates of emergency department visits and hospitalizations, staff of 71 nursing homes were called monthly to follow-up on their progress and discuss successful facilitating strategies and any challenges and barriers they encountered during the yearlong implementation period. Themes related to barriers and facilitators were identified. Six major barriers to implementation were identified: the magnitude and complexity of the change (35%), instability of facility leadership (27%), competing demands (40%), stakeholder resistance (49%), scarce resources (86%), and technical problems (31%). Six facilitating strategies were also reported: organization-wide involvement (68%), leadership support (41%), use of administrative authority (14%), adequate training (66%), persistence and oversight on the part of the champion (73%), and unfolding positive results (14%). Successful introduction of a complex change such as the INTERACT® quality improvement program in a long-term care facility requires attention to the facilitators and barriers identified in this report from those at the frontline.

From Vesalius to Virtual Reality: How Embodied Cognition Facilitates the Visualization of Anatomy

ERIC Educational Resources Information Center

Jang, Susan

2010-01-01

This study examines the facilitative effects of embodiment of a complex internal anatomical structure through three-dimensional ("3-D") interactivity in a virtual reality ("VR") program. Since Shepard and Metzler's influential 1971 study, it has been known that 3-D objects (e.g., multiple-armed cube or external body parts) are visually and…

Separation of Olefin/Paraffin Mixtures with Carrier Facilitated Membrane Final Report

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

Merkel, T.C.; Blanc, R.; Zeid, J.

2007-03-12

This document describes the results of a DOE funded joint effort of Membrane Technology and Research Inc. (MTR), SRI International (SRI), and ABB Lummus (ABB) to develop facilitated transport membranes for olefin/paraffin separations. Currently, olefin/paraffin separation is done by distillation—an extremely energy-intensive process because of the low relative volatilities of olefins and paraffins. If facilitated transport membranes could be successfully commercialized, the potential energy savings achievable with this membrane technology are estimated to be 48 trillion Btu per year by the year 2020. We discovered in this work that silver salt-based facilitated transport membranes are not stable even in themore » presence of ideal olefin/paraffin mixtures. This decline in membrane performance appears to be caused by a previously unrecognized phenomenon that we have named olefin conditioning. As the name implies, this mechanism of performance degradation becomes operative once a membrane starts permeating olefins. This project is the first study to identify olefin conditioning as a significant factor impacting the performance of facilitated olefin transport membranes. To date, we have not identified an effective strategy to mitigate the impact of olefin conditioning. other than running at low temperatures or with low olefin feed pressures. In our opinion, this issue must be addressed before further development of facilitated olefin transport membranes can proceed. In addition to olefin conditioning, traditional carrier poisoning challenges must also be overcome. Light, hydrogen, hydrogen sulfide, and acetylene exposure adversely affect membrane performance through unwanted reaction with silver ions. Harsh poisoning tests with these species showed useful membrane lifetimes of only one week. These tests demonstrate a need to improve the stability of the olefin complexing agent to develop membranes with lifetimes satisfactory for commercial application. A

Transition from strike-slip faulting to oblique subduction: active tectonics at the Puysegur Margin, South New Zealand

NASA Astrophysics Data System (ADS)

Lamarche, Geoffroy; Lebrun, Jean-Frédéric

2000-01-01

South of New Zealand the Pacific-Australia (PAC-AUS) plate boundary runs along the intracontinental Alpine Fault, the Puysegur subduction front and the intraoceanic Puysegur Fault. The Puysegur Fault is located along Puysegur Ridge, which terminates at ca. 47°S against the continental Puysegur Bank in a complex zone of deformation called the Snares Zone. At Puysegur Trench, the Australian Plate subducts beneath Puysegur Bank and the Fiordland Massif. East of Fiordland and Puysegur Bank, the Moonlight Fault System (MFS) represents the Eocene strike-slip plate boundary. Interpretation of seafloor morphology and seismic reflection profiles acquired over Puysegur Bank and the Snares Zone allows study of the transition from intraoceanic strike-slip faulting along the Puysegur Ridge to oblique subduction at the Puysegur Trench and to better understand the genetic link between the Puysegur Fault and the MFS. Seafloor morphology is interpreted from a bathymetric dataset compiled from swath bathymetry data acquired during the 1993 Geodynz survey, and single beam echo soundings acquired by the NZ Royal Navy. The Snares Zone is the key transition zone from strike-slip faulting to subduction. It divides into three sectors, namely East, NW and SW sectors. A conspicuous 3600 m-deep trough (the Snares Trough) separates the NW and East sectors. The East sector is characterised by the NE termination of Puysegur Ridge into right-stepping en echelon ridges that accommodate a change of strike from the Puysegur Fault to the MFS. Between 48°S and 47°S, in the NW sector and the Snares Trough, a series of transpressional faults splay northwards from the Puysegur Fault. Between 49°50'S and 48°S, thrusts develop progressively at Puysegur Trench into a decollement. North of 48°S the Snares Trough develops between two splays of the Puysegur Fault, indicating superficial extension associated with the subsidence of Puysegur Ridge. Seismic reflection profiles and bathymetric maps show a

Engineering botulinum neurotoxin domains for activation by toxin light chain.

PubMed

Stancombe, Patrick R; Masuyer, Geoffrey; Birch-Machin, Ian; Beard, Matthew; Foster, Keith A; Chaddock, John A; Acharya, K Ravi

2012-02-01

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions. © 2011 Syntaxin Ltd. Journal compilation © 2011 FEBS.

Surgical treatment for hypopharyngeal cysts with a side-opened direct laryngoscope.

PubMed

Kawaida, M; Fukuda, H; Shiotani, A; Kohno, N

1994-01-01

Two cases of hypopharyngeal cyst are reported. Both cysts occurred in the piriform sinus of the hypopharynx. Histopathological examination indicated that both were retention cysts. These cysts were removed by laryngomicrosurgical technique using a side-opened direct laryngoscope. In the cyst with a distinct base, a laryngomicrosurgical snare was used for removal. In the wide-based cyst, the mucous membrane around the cyst was incised with an electrosurgical instrument and then detached to facilitate removal. In this paper, we describe our surgical procedure for removing hypopharyngeal cysts and discuss the causes of such cysts.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

PubMed

Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

2017-06-01

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.

Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion.

PubMed

Sitarska, Ewa; Xu, Junjie; Park, Seungmee; Liu, Xiaoxia; Quade, Bradley; Stepien, Karolina; Sugita, Kyoko; Brautigam, Chad A; Sugita, Shuzo; Rizo, Josep

2017-05-06

Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release. Munc18-1 binds to synaptobrevin, but the relevance of this interaction and its relation to Munc13 function are unclear. NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin. Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the 'furled conformation' of a Munc18-1 loop. Correspondingly, the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation. Moreover, the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in Caenorhabditis elegans unc-18 nulls. Together with previous studies, our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly, and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1.

The Fas/Fap-1/Cav-1 complex regulates IL-1RA secretion in mesenchymal stem cells to accelerate wound healing.

PubMed

Kou, Xiaoxing; Xu, Xingtian; Chen, Chider; Sanmillan, Maria Laura; Cai, Tao; Zhou, Yanheng; Giraudo, Claudio; Le, Anh; Shi, Songtao

2018-03-14

Mesenchymal stem cells (MSCs) are capable of secreting exosomes, extracellular vesicles, and cytokines to regulate cell and tissue homeostasis. However, it is unknown whether MSCs use a specific exocytotic fusion mechanism to secrete exosomes and cytokines. We show that Fas binds with Fas-associated phosphatase-1 (Fap-1) and caveolin-1 (Cav-1) to activate a common soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated membrane fusion mechanism to release small extracellular vesicles (sEVs) in MSCs. Moreover, we reveal that MSCs produce and secrete interleukin-1 receptor antagonist (IL-1RA) associated with sEVs to maintain rapid wound healing in the gingiva via the Fas/Fap-1/Cav-1 cascade. Tumor necrosis factor-α (TNF-α) serves as an activator to up-regulate Fas and Fap-1 expression via the nuclear factor κB pathway to promote IL-1RA release. This study identifies a previously unknown Fas/Fap-1/Cav-1 axis that regulates SNARE-mediated sEV and IL-1RA secretion in stem cells, which contributes to accelerated wound healing. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Selective expression of a sec1/munc18 member in sea urchin eggs and embryos.

PubMed

Leguia, Mariana; Wessel, Gary M

2004-10-01

Regulated secretion is mediated by SNAREs (soluble NSF attachment receptors) and their regulators and effectors, which include the SM (sec1/munc18) family of proteins. Homologs of the SNAREs have been identified in sea urchins, associated with cortical granule exocytosis at fertilization, with membranes of the cleavage furrow, and in secretory cells later in development. To contribute to the understanding of regulated secretion in sea urchins we have cloned the single SM protein homolog from two species of sea urchin, Lytechinus variegatus and Strongylocentrotus purpuratus. In oocytes and eggs, we find that it localizes to the plasma membrane and the cortical region of the egg, consistent with a role in one of the steps leading to cortical granule exocytosis. The protein is also expressed throughout development, enriched in membranes of the cleavage furrow in early embryos, and in cells of the gut in advanced embryos. Furthermore, we find that sec1/munc18 co-localizes with its cognate binding partner syntaxin. Finally, our biochemical analysis shows that the protein associates with rab3 in high molecular weight complexes, suggesting that the exocytotic machinery functions as a multi-protein subunit to mediate regulated secretion in sea urchins. These results will be instrumental in the future to functionally test the SNARE regulators associated with multiple membrane fusion events.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

PubMed Central

Woo, Sang Su; James, Declan J.; Martin, Thomas F. J.

2017-01-01

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. PMID:28100639

Facilitating Facilitators to Facilitate, in Problem or Enquiry Based Learning Sessions

ERIC Educational Resources Information Center

Coelho, Catherine

2014-01-01

Problem based learning (PBL) has been used in dental education over the past 20 years and uses a patient case scenario to stimulate learning in a small group setting, where a trained facilitator does not teach but guides the group to bring about deep contextualized learning, to be empathetic to each other and to encourage fair and equitable…

Happy creativity: Listening to happy music facilitates divergent thinking.

PubMed

Ritter, Simone M; Ferguson, Sam

2017-01-01

Creativity can be considered one of the key competencies for the twenty-first century. It provides us with the capacity to deal with the opportunities and challenges that are part of our complex and fast-changing world. The question as to what facilitates creative cognition-the ability to come up with creative ideas, problem solutions and products-is as old as the human sciences, and various means to enhance creative cognition have been studied. Despite earlier scientific studies demonstrating a beneficial effect of music on cognition, the effect of music listening on creative cognition has remained largely unexplored. The current study experimentally tests whether listening to specific types of music (four classical music excerpts systematically varying on valance and arousal), as compared to a silence control condition, facilitates divergent and convergent creativity. Creativity was higher for participants who listened to 'happy music' (i.e., classical music high on arousal and positive mood) while performing the divergent creativity task, than for participants who performed the task in silence. No effect of music was found for convergent creativity. In addition to the scientific contribution, the current findings may have important practical implications. Music listening can be easily integrated into daily life and may provide an innovative means to facilitate creative cognition in an efficient way in various scientific, educational and organizational settings when creative thinking is needed.

Happy creativity: Listening to happy music facilitates divergent thinking

PubMed Central

Ferguson, Sam

2017-01-01

Creativity can be considered one of the key competencies for the twenty-first century. It provides us with the capacity to deal with the opportunities and challenges that are part of our complex and fast-changing world. The question as to what facilitates creative cognition—the ability to come up with creative ideas, problem solutions and products—is as old as the human sciences, and various means to enhance creative cognition have been studied. Despite earlier scientific studies demonstrating a beneficial effect of music on cognition, the effect of music listening on creative cognition has remained largely unexplored. The current study experimentally tests whether listening to specific types of music (four classical music excerpts systematically varying on valance and arousal), as compared to a silence control condition, facilitates divergent and convergent creativity. Creativity was higher for participants who listened to ‘happy music’ (i.e., classical music high on arousal and positive mood) while performing the divergent creativity task, than for participants who performed the task in silence. No effect of music was found for convergent creativity. In addition to the scientific contribution, the current findings may have important practical implications. Music listening can be easily integrated into daily life and may provide an innovative means to facilitate creative cognition in an efficient way in various scientific, educational and organizational settings when creative thinking is needed. PMID:28877176

Facilitation of the arterial baroreflex by the preoptic area in anaesthetized rats.

PubMed Central

Inui, K; Nomura, J; Murase, S; Nosaka, S

1995-01-01

1. Activation of cell bodies in the ventrolateral part of the midbrain periaqueductal grey matter (PAG) facilitates the arterial baroreflex via the nucleus raphe magnus. The facilitatory effects of stimulation within the hypothalamus on the arterial baroreflex and their relation to the PAG and nucleus raphe magnus were studied in urethane- and chloralose-anaesthetized rats. 2. Systematic mapping experiments revealed that the preoptic area (POA) is the principal location in the hypothalamus of neuronal cell bodies that are responsible for the potentiation of the baroreflex. In addition to provoking hypotension and vagal bradycardia, both electrical and chemical stimulation of the POA produced facilitation of baroreflex vagal bradycardia (BVB) that was evoked by electrical stimulation of the aortic depressor nerve. Baroreflex hypotension was slightly augmented during activation of the POA in vagotomized rats. 3. Selective destruction of cell bodies either in the ventrolateral PAG or in the nucleus raphe magnus reduced facilitation of BVB by the POA. Hypotension and bradycardia due to POA stimulation were also markedly attenuated after such selective destruction. 4. In conclusion, the POA, the ventrolateral PAG and the nucleus raphe magnus constitute a functional complex that produces cardiovascular trophotropic effects including hypotension, vagal bradycardia and baroreflex facilitation. PMID:8568691

Improving the organization of palliative care: identification of barriers and facilitators in five European countries.

PubMed

van Riet Paap, Jasper; Vernooij-Dassen, Myrra; Brouwer, Frederike; Meiland, Franka; Iliffe, Steve; Davies, Nathan; Leppert, Wojciech; Jaspers, Birgit; Mariani, Elena; Sommerbakk, Ragni; Vissers, Kris; Engels, Yvonne

2014-10-16

Interventions to improve palliative care encounter challenges beyond the usual implementation problems because of palliative care's complex and changing character. In this study, we explored barriers and facilitators faced by health-care professionals in five European countries (England, Germany, Italy, Norway and the Netherlands) with regard to improving the organization of their palliative care service. Semi-structured individual and focus group interviews were conducted with purposefully selected health-care professionals. The constant comparative method was used to analyse the data. Professionals working in hospitals, hospices, nursing homes and primary care facilities who provide palliative care to adult patients were interviewed (n =40) or participated in ten focus group interviews (n =59). Barriers and facilitators were inductively grouped into 16 categories and arranged into five themes: innovation, individual professional level, group dynamics, organizational context and local political-economic context. Although the barriers and facilitators identified differed in scope, context, strength and provenance, they were shared by professionals from different European countries. This study identified barriers and facilitators to organizational change in palliative care. Some of these barriers and facilitators were experienced by professionals in almost all countries and are therefore prerequisites to change. Understanding the barriers to and facilitators of change will help tailor organizational improvements to the needs of individuals and organizations.

Using a meta-ethnographic approach to explore the nature of facilitation and teaching approaches employed in interprofessional education.

PubMed

Reeves, Scott; Pelone, Ferruccio; Hendry, Julie; Lock, Nicholas; Marshall, Jayne; Pillay, Leontia; Wood, Ruth

2016-12-01

Interprofessional facilitators and teachers are regarded as central to the effective delivery of interprofessional education (IPE). As the IPE literature continues to expand, most studies have focused on reporting learner outcomes, with little attention paid to IPE facilitation. However, a number of studies have recently emerged reporting on this phenomenon. To present a synthesis of qualitative evidence on the facilitation of IPE, using a meta-ethnographic approach. Electronic databases and journals were searched for the past 10 years. Of the 2164 abstracts initially found, 94 full papers were reviewed and subsequently 12 papers were included. Teams of two reviewers independently completed each step in the review process. The quality of these papers was assessed using a modified critical appraisal checklist. Seven key concepts embedded in the included studies were synthesized into three main factors which provided an insight into the nature of IPE facilitation. Specifically, the synthesis found that IPE facilitation is influenced by "contextual characteristics"; "facilitator experiences"; and the "use of different facilitation strategies". IPE facilitation is a complex activity affected by contextual, experiential and pedagogical factors. Further research is needed to explore the effects of these factors on the delivery of IPE.

Paper SERS chromatography for detection of trace analytes in complex samples

NASA Astrophysics Data System (ADS)

Yu, Wei W.; White, Ian M.

2013-05-01

We report the application of paper SERS substrates for the detection of trace quantities of multiple analytes in a complex sample in the form of paper chromatography. Paper chromatography facilitates the separation of different analytes from a complex sample into distinct sections in the chromatogram, which can then be uniquely identified using SERS. As an example, the separation and quantitative detection of heroin in a highly fluorescent mixture is demonstrated. Paper SERS chromatography has obvious applications, including law enforcement, food safety, and border protection, and facilitates the rapid detection of chemical and biological threats at the point of sample.

Rational Design of Therapeutic and Diagnostic Against Botulinum Neurotoxin

DTIC Science & Technology

2006-12-01

serotypes A, C, and E cleave SNAP-25; and serotype C cleaves syntaxin. Without acetylcholine release, the muscle is unable to contract . (after [24...binds to acetylcholine receptors on muscle cells for normal nerve impulse and muscle contraction . Cleavage of the neuronal SNARE complex proteins...A., Williams, T.A., Chauvet, M., and Corvol, P . (1997). Substrate dependence of angiotensin I – converting enzyme inhibition: captopril displays a

Perceptions of Barriers and Facilitators During Implementation of a Complex Model of Group Prenatal Care in Six Urban Sites

PubMed Central

Novick, Gina; Womack, Julie A.; Lewis, Jessica; Stasko, Emily C.; Rising, Sharon S.; Sadler, Lois S.; Cunningham, Shayna C.; Tobin, Jonathan N.; Ickovics, Jeannette R.

2016-01-01

Group prenatal care improves perinatal outcomes, but implementing this complex model places substantial demands on settings designed for individual care. To describe perceived barriers and facilitators to implementing and sustaining Centering Pregnancy Plus (CP+) group prenatal care, 24 in-depth interviews were conducted with 22 clinicians, staff, administrators, and study personnel in six of the 14 sites of a randomized trial of the model. All sites served low-income, minority women. Sites for the present evaluation were selected for variation in location, study arm, and initial implementation response. Implementing CP+ was challenging in all sites, requiring substantial adaptations of clinical systems. All sites had barriers to meeting the model’s demands, but how sites responded to these barriers affected whether implementation thrived or struggled. Thriving sites had organizational cultures that supported innovation, champions who advocated for CP+, and staff who viewed logistical demands as manageable hurdles. Struggling sites had bureaucratic organizational structures and lacked buy-in and financial resources, and staff were overwhelmed by the model’s challenges. Findings suggested that implementing and sustaining health care innovation requires new practices and different ways of thinking, and health systems may not fully recognize the magnitude of change required. Consequently, evidence-based practices are modified or discontinued, and outcomes may differ from those in the original controlled studies. Before implementing new models of care, clinical settings should anticipate model demands and assess capacity for adapting to the disruptions of innovation. PMID:26340483

Hope for recovery - how clinicians may facilitate this in their work.

PubMed

Hobbs, Mia; Baker, Martyn

2012-04-01

The importance of having hope for recovery has been highlighted in numerous qualitative studies of recovery. It is identified as a vital part of this process, and guidelines suggest that service providers should therefore facilitate hope in their clinical work; however, they do not indicate how this guidance can be operationalised. To identify the sources of hope for recovery based on the accounts of people with experience of recovery; to ascertain how these accounts show service providers can facilitate such hope in their therapeutic work. Semi-structured interviews were conducted with eight people with experience of recovering from mental health problems. A grounded theory analysis was undertaken. A model conceptualising the role of hope in recovery was developed with three categories: "influence of others on hope", "personal hope" and "doing recovery". The model indicates a complex interaction between hope and recovery with an important role for social context and interpersonal relationships, including those with clinicians. Mental health service providers occupy a powerful position in relation to service users' hope, and must carefully consider how they communicate their own hopefulness about clients' recovery. Suggestions are made about facilitating hope for recovery.

[Health: an adaptive complex system].

PubMed

Toro-Palacio, Luis Fernando; Ochoa-Jaramillo, Francisco Luis

2012-02-01

This article points out the enormous gap that exists between complex thinking of an intellectual nature currently present in our environment, and complex experimental thinking that has facilitated the scientific and technological advances that have radically changed the world. The article suggests that life, human beings, global society, and all that constitutes health be considered as adaptive complex systems. This idea, in turn, prioritizes the adoption of a different approach that seeks to expand understanding. When this rationale is recognized, the principal characteristics and emerging properties of health as an adaptive complex system are sustained, following a care and services delivery model. Finally, some pertinent questions from this perspective are put forward in terms of research, and a series of appraisals are expressed that will hopefully serve to help us understand all that we have become as individuals and as a species. The article proposes that the delivery of health care services be regarded as an adaptive complex system.

Developing facilitation skills--a narrative.

PubMed

Newton, Jennifer M

2003-07-01

Effective facilitation has been identified in the literature as one of three elements, along with context and evidence, that have a dynamic and coexisting relationship to enable the successful uptake of evidence into practice. This paper presents an overview of the concept of facilitation within the context of practice development, ahead of a personal and professional reflective account of a 'developing facilitator'. In the summer of 2001, the author was instrumental in organising the first Practice Development School in Melbourne. Thrown in at the deep end, she found herself co-facilitating with an experienced practice developer from the United Kingdom. Having never facilitated in the arena of an action learning group, nor worked in the field of practice development, there was initially a sense of impending overload and drowning in the new knowledge and skills that needed to be acquired. Drawing upon the work of narrative inquiry the author shares her experiences in the anticipation that in telling her story it will assist others in their journey of becoming a facilitator.

Professor as Facilitator: Shaping an Emerging, Living System of Shared Leadership in the Classroom

ERIC Educational Resources Information Center

Bright, David S.; Turesky, Elizabeth Fisher; Putzel, Roger; Stang, Thomas

2012-01-01

From the perspective of emergence, professors can facilitate and shape a class as a complex, adaptive, and living system. A case study illustrates phases of emergence in the classroom by tracing how a professor may use this perspective to empower students to share in the leadership of the classroom. Instead of presenting lessons, the professor…

Visual Complexity and Pictorial Memory: A Fifteen Year Research Perspective.

ERIC Educational Resources Information Center

Berry, Louis H.

For 15 years an ongoing research project at the University of Pittsburgh has focused on the effects of variations in visual complexity and color on the storage and retrieval of visual information by learners. Research has shown that visual materials facilitate instruction, but has not fully delineated the interactions of visual complexity and…

The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

PubMed

Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

2015-03-01

RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

PubMed Central

Prasanth, K. Reddisiva; Barajas, Daniel

2014-01-01

ABSTRACT RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCE RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA

Provider perspectives on barriers and facilitators to adjuvant endocrine therapy-related symptom management.

PubMed

Samuel, Cleo A; Turner, Kea; Donovan, Heidi A S; Beckjord, Ellen; Cardy, Alexandra; Dew, Mary Amanda; van Londen, G J

2017-12-01

Adjuvant endocrine therapy (AET) utilization is linked to improved clinical outcomes among breast cancer survivors (BCS); yet, AET adherence rates remain suboptimal. Little is known about provider perspectives regarding barriers and facilitators to AET-related symptom management (SM). In this study, we examined provider perspectives on the barriers and facilitators to AET-related SM among BCS and opportunities for improvement. We conducted three focus groups (FGs) with a multidisciplinary group of healthcare providers (n = 13) experienced in caring for BCS undergoing AET. We utilized semi-structured discussion guides to elicit provider perspectives on AET-related SM. FGs were audiotaped, transcribed, and analyzed using qualitative software to identify key themes. Providers described patient-, provider-, and system-level barriers and facilitators to AET-related SM. At the patient-level, barriers included competing demands, limited time/resources, and possible misattribution of some symptoms to AET, while family/social relationships and insurance emerged as important facilitators. Discomfort with SM, limited time, and challenges distinguishing AET-related symptoms from other conditions were key provider-level barriers. Provider-level facilitators included routine symptom documentation and strong provider relationships. Care fragmentation and complexity of the cancer care delivery system were described as system-level barriers; however, survivor clinics were endorsed by providers. Provider perspectives on AET-related SM can shed light on SM barriers and facilitators spanning multiple levels of the cancer care delivery system. Strategies for improving AET-related SM in BCS include increasing patients' knowledge and engagement in SM, equipping providers with efficient SM strategies, and improving coordination of symptom-related services through survivorship programs.

Medical students' and facilitators' experiences of an Early Professional Contact course: active and motivated students, strained facilitators.

PubMed

von Below, Bernhard; Hellquist, Gunilla; Rödjer, Stig; Gunnarsson, Ronny; Björkelund, Cecilia; Wahlqvist, Mats

2008-12-02

Today, medical students are introduced to patient contact, communication skills, and clinical examination in the preclinical years of the curriculum with the purpose of gaining clinical experience. These courses are often evaluated from the student perspective. Reports with an additional emphasis on the facilitator perspective are scarce. According to constructive alignment, an influential concept from research in higher education, the learning climate between students and teachers is also of great importance. In this paper, we approach the learning climate by studying both students' and facilitators' course experiences.In 2001, a new "Early Professional Contact" longitudinal strand through term 1-4, was introduced at the Sahlgrenska Academy, University of Gothenburg, Sweden. General practitioners and hospital specialists were facilitators.The aim of this study was to assess and analyse students' and clinical facilitators' experiences of the Early Professional Contact course and to illuminate facilitators' working conditions. Inspired by a Swedish adaptation of the Course Experience Questionnaire, an Early Professional Contact Questionnaire was constructed. In 2003, on the completion of the first longitudinal strand, a student and facilitator version was distributed to 86 students and 21 facilitators. In the analysis, both Chi-square and the Mann-Whitney tests were used. Sixty students (70%) and 15 facilitators (71%) completed the questionnaire. Both students and facilitators were satisfied with the course. Students reported gaining iiration for their future work as doctors along with increased confidence in meeting patients. They also reported increased motivation for biomedical studies. Differences in attitudes between facilitators and students were found. Facilitators experienced a greater workload, less reasonable demands and less support, than students. In this project, a new Early Professional Contact course was analysed from both student and facilitator

The Effect of Intensity and Age on the Perception of Accent in Isochronous Sequences of a Snare Drum Timbre

NASA Astrophysics Data System (ADS)

Walls, Kimberly Kyle Curley

1992-01-01

Musical expression is largely dependent upon accentuation, yet there have been few attempts to study the perception of dynamic accent in music or to relate the results of psychoacoustical research in intensity to realistic musical situations. The purpose of the experiment was to estimate the relationships among (a) the intensity increment in dB(A) required to meet an 80% correct criterion in the perception of one accented tone embedded within a seven -tone isochronous series of identical 87 dB(A) snare drum timbre stimuli of 333 ms onsets (accent level, or AL), (b) the different limen (DL) for intensity increase to meet a 75% correct criterion in a 2AFC task for pairs for the stimuli, and (c) the age of the subjects, all of whom have normal audiograms. The 51 subjects (N = 51) were female nonmusicians ranging in age from 9 to 33 years (M = 17.98, SD = 5.21). The response tasks involved saying whether the second tone of each pair was louder or softer and circling the accented note in notated quarter notes. The stimuli production, the headphone calibration process, and their rationales were detailed. The global regression model was significant (F(2, 48) = 5.505, p =.007, R^2 =.187), and the relationship between AL and DL was not significant (F(1, 48) = 5.505, p =.197, R^2 change =.029), the relationship between AL and age was significant (F(1, 48) = 5.732, p =.021, R ^2 change =.098) at an alpha level of.05 and power calculated at.66 for a medium ES. It was concluded that accented sounds are easier to perceive in tone pairs than they are in a musical setting and that subject maturation improves performance of intensity judgement tasks. Suggestions for further research include shortening the length of the experimental session for younger subjects and increasing the number of intensity increments as well as using smaller increments to accommodate individual differences in perception.

Impairment of autophagosome-lysosome fusion in the buff mutant mice with the VPS33AD251E mutation

PubMed Central

Zhen, Yuanli; Li, Wei

2015-01-01

The HOPS (homotypic fusion and protein sorting) complex functions in endocytic and autophagic pathways in both lower eukaryotes and mammalian cells through its involvement in fusion events between endosomes and lysosomes or autophagosomes and lysosomes. However, the differential molecular mechanisms underlying these fusion processes are largely unknown. Buff (bf) is a mouse mutant that carries an Asp251-to-Glu point mutation (D251E) in the VPS33A protein, a tethering protein and a core subunit of the HOPS complex. Bf mice showed impaired spontaneous locomotor activity, motor learning, and autophagic activity. Although the gross anatomy of the brain was apparently normal, the number of Purkinje cells was significantly reduced. Furthermore, we found that fusion between autophagosomes and lysosomes was defective in bf cells without compromising the endocytic pathway. The direct association of mutant VPS33AD251E with the autophagic SNARE complex, STX17 (syntaxin 17)-VAMP8-SNAP29, was enhanced. In addition, the VPS33AD251E mutation enhanced interactions with other HOPS subunits, namely VPS41, VPS39, VPS18, and VPS11, except for VPS16. Reduction of the interactions between VPS33AY440D and several other HOPS subunits led to decreased association with STX17. These results suggest that the VPS33AD251E mutation plays dual roles by increasing the HOPS complex assembly and its association with the autophagic SNARE complex, which selectively affects the autophagosome-lysosome fusion that impairs basal autophagic activity and induces Purkinje cell loss. PMID:26259518

Golgi-to-Endoplasmic Reticulum (ER) Retrograde Traffic in Yeast Requires Dsl1p, a Component of the ER Target Site that Interacts with a COPI Coat Subunit

PubMed Central

Reilly, Barbara A.; Kraynack, Bryan A.; VanRheenen, Susan M.; Waters, M. Gerard

2001-01-01

DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic. Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature. Here, we show that dsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident. These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic. Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex. Immunoisolation of the complex yielded Dsl1p and proteins of ∼80 and ∼55 kDa. The ∼80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is ∼50 kDa. Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p. These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex. Last, we show that Dsl1p interacts with the δ-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction. PMID:11739780

Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes*

PubMed Central

Lolis, Alexandra A.; Londhe, Priya; Beggs, Benjamin C.; Byrum, Stephanie D.; Tackett, Alan J.; Davie, Judith K.

2013-01-01

Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell. PMID:23364797

Photodissociation of Non-Covalent Peptide-Crown Ether Complexes

PubMed Central

Wilson, Jeffrey J.; Kirkovits, Gregory J.; Sessler, Jonathan L.; Brodbelt, Jennifer S.

2008-01-01

Highly chromogenic 18-crown-6-dipyrrolylquinoxaline coordinates primary amines of peptides, forming non-covalent complexes that can be transferred to the gas phase by electrospray ionization. The appended chromogenic crown ether facilitates efficient energy transfer to the peptide upon ultraviolet irradiation in the gas phase, resulting in diagnostic peptide fragmentation. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) of these non-covalent complexes results only in their disassembly with the charge retained on either the peptide or crown ether, yielding no sequence ions. Upon UV photon absorption the intermolecular energy transfer is facilitated by the fast activation time scale of UVPD (< 10 ns) and by the collectively strong hydrogen bonding between the crown ether and peptide, thus allowing effective transfer of energy to the peptide moiety prior to disruption of the intermolecular hydrogen bonds. PMID:18077179

Modeling complex tone perception: grouping harmonics with combination-sensitive neurons.

PubMed

Medvedev, Andrei V; Chiao, Faye; Kanwal, Jagmeet S

2002-06-01

Perception of complex communication sounds is a major function of the auditory system. To create a coherent precept of these sounds the auditory system may instantaneously group or bind multiple harmonics within complex sounds. This perception strategy simplifies further processing of complex sounds and facilitates their meaningful integration with other sensory inputs. Based on experimental data and a realistic model, we propose that associative learning of combinations of harmonic frequencies and nonlinear facilitation of responses to those combinations, also referred to as "combination-sensitivity," are important for spectral grouping. For our model, we simulated combination sensitivity using Hebbian and associative types of synaptic plasticity in auditory neurons. We also provided a parallel tonotopic input that converges and diverges within the network. Neurons in higher-order layers of the network exhibited an emergent property of multifrequency tuning that is consistent with experimental findings. Furthermore, this network had the capacity to "recognize" the pitch or fundamental frequency of a harmonic tone complex even when the fundamental frequency itself was missing.

Practice change in community pharmacy: quantification of facilitators.

PubMed

Roberts, Alison S; Benrimoj, Shalom I; Chen, Timothy F; Williams, Kylie A; Aslani, Parisa

2008-06-01

There has been an increasing international trend toward the delivery of cognitive pharmaceutical services (CPS) in community pharmacy. CPS have been developed and disseminated individually, without a framework underpinning their implementation and with limited knowledge of factors that might assist practice change. The implementation process is complex, involving a range of internal and external factors. To quantify facilitators of practice change in Australian community pharmacies. We employed a literature review and qualitative study to facilitate the design of a 43-item "facilitators of practice change" scale as part of a quantitative survey instrument, using a framework of organizational theory. The questionnaire was pilot-tested (n = 100), then mailed to a random sample of 2000 community pharmacies, with a copy each for the pharmacy owner, employed pharmacist, and pharmacy assistant. The construct validity and reliability of the scale were established using exploratory factor analysis and Cronbach's alpha, respectively. A total of 735 (37%) pharmacies responded, with 1303 individual questionnaires. Factor analysis of the scale yielded 7 factors, explaining 48.8% of the total variance. The factors were: relationship with physicians (item loading range 0.59-0.85; Cronbach's alpha 0.90), remuneration (0.52-0.74; 0.82), pharmacy layout (0.52-0.79; 0.81), patient expectation (0.52-0.85; 0.82), manpower/staff (0.49-0.66; 0.80), communication and teamwork (0.37-0.65; 0.77), and external support/assistance (0.47-0.69; 0.74). All of the factors demonstrated good reliability and construct validity and explained approximately half of the variance. Implementing CPS requires support not only with the clinical aspects of service delivery, but also for the process of implementation itself, and remuneration models must reflect this. The identified facilitators should be used in a multilevel strategy to integrate professional services into the community pharmacy business

In Vitro Reconstitution of Autophagosome-Lysosome Fusion.

PubMed

Diao, J; Li, L; Lai, Y; Zhong, Q

2017-01-01

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins are a highly regulated class of membrane proteins lying in the center of membrane fusion. In conjunction with accessory proteins, SNAREs drive efficient merger of two distinct lipid bilayers into one interconnected structure. This chapter describes our fluorescence resonance energy transfer (FRET)-based proteoliposome fusion assays for the roles of various SNARE proteins, accessory proteins, and effects of different lipid compositions on membrane fusion involved in autophagy. © 2017 Elsevier Inc. All rights reserved.

Chansporter complexes in cell signaling.

PubMed

Abbott, Geoffrey W

2017-09-01

Ion channels facilitate diffusion of ions across cell membranes for such diverse purposes as neuronal signaling, muscular contraction, and fluid homeostasis. Solute transporters often utilize ionic gradients to move aqueous solutes up their concentration gradient, also fulfilling a wide variety of tasks. Recently, an increasing number of ion channel-transporter ('chansporter') complexes have been discovered. Chansporter complex formation may overcome what could otherwise be considerable spatial barriers to rapid signal integration and feedback between channels and transporters, the ions and other substrates they transport, and environmental factors to which they must respond. Here, current knowledge in this field is summarized, covering both heterologous expression structure/function findings and potential mechanisms by which chansporter complexes fulfill contrasting roles in cell signaling in vivo. © 2017 Federation of European Biochemical Societies.

Online interprofessional education facilitation: A scoping review.

PubMed

Evans, Sherryn Maree; Ward, Catherine; Reeves, Scott

2018-04-22

The use of online media to deliver interprofessional education (IPE) is becoming more prevalent across health professions education settings. Facilitation of IPE activities is known to be critical to the effective delivery of IPE, however, specifics about the nature of online IPE facilitation remains unclear. To explore the health professions education literature to understand the extent, range and nature of research on online IPE facilitation. Scoping review methodology was used to guide a search of four electronic databases for relevant papers. Of the 2095 abstracts initially identified, after screening of both abstracts and full-text papers, 10 studies were selected for inclusion in this review. Following abstraction of key information from each study, a thematic analysis was undertaken. Three key themes emerged to describe the nature of the IPE facilitation literature: (1) types of online IPE facilitation contributions, (2) the experience of online IPE facilitation and (3) personal outcomes of online IPE facilitation. These IPE facilitation themes were particularly focused on facilitation of interprofessional student teams on an asynchronous basis. While the included studies provide some insight into the nature of online IPE facilitation, future research is needed to better understand facilitator contributions, and the facilitation experience and associated outcomes, both relating to synchronous and asynchronous online environments.

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

PubMed

Iki, Taichiro; Yoshikawa, Manabu; Meshi, Tetsuo; Ishikawa, Masayuki

2012-01-18

Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

Elaboration over a Discourse Facilitates Retrieval in Sentence Processing

PubMed Central

Troyer, Melissa; Hofmeister, Philip; Kutas, Marta

2016-01-01

Language comprehension requires access to stored knowledge and the ability to combine knowledge in new, meaningful ways. Previous work has shown that processing linguistically more complex expressions (‘Texas cattle rancher’ vs. ‘rancher’) leads to slow-downs in reading during initial processing, possibly reflecting effort in combining information. Conversely, when this information must subsequently be retrieved (as in filler-gap constructions), processing is facilitated for more complex expressions, possibly because more semantic cues are available during retrieval. To follow up on this hypothesis, we tested whether information distributed across a short discourse can similarly provide effective cues for retrieval. Participants read texts introducing two referents (e.g., two senators), one of whom was described in greater detail than the other (e.g., ‘The Democrat had voted for one of the senators, and the Republican had voted for the other, a man from Ohio who was running for president’). The final sentence (e.g., ‘The senator who the {Republican/Democrat}had voted for…’) contained a relative clause picking out either the Many-Cue referent (with ‘Republican’) or the One-Cue referent (with ‘Democrat’). We predicted facilitated retrieval (faster reading times) for the Many-Cue condition at the verb region (‘had voted for’), where readers could understand that ‘The senator’ is the object of the verb. As predicted, this pattern was observed at the retrieval region and continued throughout the rest of the sentence. Participants also completed the Author/Magazine Recognition Tests (ART/MRT; Stanovich and West, 1989), providing a proxy for world knowledge. Since higher ART/MRT scores may index (a) greater experience accessing relevant knowledge and/or (b) richer/more highly structured representations in semantic memory, we predicted it would be positively associated with effects of elaboration on retrieval. We did not observe the

Elaboration over a Discourse Facilitates Retrieval in Sentence Processing.

PubMed

Troyer, Melissa; Hofmeister, Philip; Kutas, Marta

2016-01-01

Language comprehension requires access to stored knowledge and the ability to combine knowledge in new, meaningful ways. Previous work has shown that processing linguistically more complex expressions ('Texas cattle rancher' vs. 'rancher') leads to slow-downs in reading during initial processing, possibly reflecting effort in combining information. Conversely, when this information must subsequently be retrieved (as in filler-gap constructions), processing is facilitated for more complex expressions, possibly because more semantic cues are available during retrieval. To follow up on this hypothesis, we tested whether information distributed across a short discourse can similarly provide effective cues for retrieval. Participants read texts introducing two referents (e.g., two senators), one of whom was described in greater detail than the other (e.g., 'The Democrat had voted for one of the senators, and the Republican had voted for the other, a man from Ohio who was running for president'). The final sentence (e.g., 'The senator who the {Republican/Democrat}had voted for…') contained a relative clause picking out either the Many-Cue referent (with 'Republican') or the One-Cue referent (with 'Democrat'). We predicted facilitated retrieval (faster reading times) for the Many-Cue condition at the verb region ('had voted for'), where readers could understand that 'The senator' is the object of the verb. As predicted, this pattern was observed at the retrieval region and continued throughout the rest of the sentence. Participants also completed the Author/Magazine Recognition Tests (ART/MRT; Stanovich and West, 1989), providing a proxy for world knowledge. Since higher ART/MRT scores may index (a) greater experience accessing relevant knowledge and/or (b) richer/more highly structured representations in semantic memory, we predicted it would be positively associated with effects of elaboration on retrieval. We did not observe the predicted interaction between ART

A symbiosis-dedicated SYNTAXIN OF PLANTS 13II isoform controls the formation of a stable host-microbe interface in symbiosis.

PubMed

Huisman, Rik; Hontelez, Jan; Mysore, Kirankumar S; Wen, Jiangqi; Bisseling, Ton; Limpens, Erik

2016-09-01

Arbuscular mycorrhizal (AM) fungi and rhizobium bacteria are accommodated in specialized membrane compartments that form a host-microbe interface. To better understand how these interfaces are made, we studied the regulation of exocytosis during interface formation. We used a phylogenetic approach to identify target soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) that are dedicated to symbiosis and used cell-specific expression analysis together with protein localization to identify t-SNAREs that are present on the host-microbe interface in Medicago truncatula. We investigated the role of these t-SNAREs during the formation of a host-microbe interface. We showed that multiple syntaxins are present on the peri-arbuscular membrane. From these, we identified SYNTAXIN OF PLANTS 13II (SYP13II) as a t-SNARE that is essential for the formation of a stable symbiotic interface in both AM and rhizobium symbiosis. In most dicot plants, the SYP13II transcript is alternatively spliced, resulting in two isoforms, SYP13IIα and SYP13IIβ. These splice-forms differentially mark functional and degrading arbuscule branches. Our results show that vesicle traffic to the symbiotic interface is specialized and required for its maintenance. Alternative splicing of SYP13II allows plants to replace a t-SNARE involved in traffic to the plasma membrane with a t-SNARE that is more stringent in its localization to functional arbuscules. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

From parent to 'peer facilitator': a qualitative study of a peer-led parenting programme.

PubMed

Thomson, S; Michelson, D; Day, C

2015-01-01

Peer-led interventions are increasingly common in community health settings. Although peer-led approaches have proven benefits for service users, relatively little is known about the process and outcomes of participation for peer leaders. This study investigated experiences of parents who had participated as 'peer facilitators' in Empowering Parents, Empowering Communities (EPEC), a peer-led programme designed to improve access to evidence-based parenting support in socially disadvantaged communities. A qualitative cross-sectional design was used. Semi-structured interviews were conducted with 14 peer facilitators and scrutinized using thematic analysis. Peer facilitators developed their knowledge and skills through personal experience of receiving parenting support, participation in formal training and supervised practice, access to an intervention manual, and peer modelling. Peer facilitators described positive changes in their own families, confidence and social status. Transformative personal gains reinforced peer facilitators' role commitment and contributed to a cohesive 'family' identity among EPEC staff and service users. Peer facilitators' enthusiasm, openness and mutual identification with families were seen as critical to EPEC's effectiveness and sustainability. Peer facilitators also found the training emotionally and intellectually demanding. There were particular difficulties around logistical issues (e.g. finding convenient supervision times), managing psychosocial complexity and child safeguarding. The successful delivery and sustained implementation of peer-led interventions requires careful attention to the personal qualities and support of peer leaders. Based on the findings of this study, support should include training, access to intervention manuals, regular and responsive supervision, and logistical/administrative assistance. Further research is required to elaborate and extend these findings to other peer-led programmes. © 2014 John Wiley

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila

PubMed Central

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P.

2017-01-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. PMID:28381425

Complexity in language learning and treatment.

PubMed

Thompson, Cynthia K

2007-02-01

To introduce a Clinical Forum focused on the Complexity Account of Treatment Efficacy (C. K. Thompson, L. P. Shapiro, S. Kiran, & J. Sobecks, 2003), a counterintuitive but effective approach for treating language disorders. This approach espouses training complex structures to promote generalized improvement of simpler, linguistically related structures. Three articles are included, addressing complexity in treatment of phonology, lexical-semantics, and syntax. Complexity hierarchies based on models of normal language representation and processing are discussed in each language domain. In addition, each article presents single-subject controlled experimental studies examining the complexity effect. By counterbalancing treatment of complex and simple structures across participants, acquisition and generalization patterns are examined as they emerge. In all language domains, cascading generalization occurs from more to less complex structures; however, the opposite pattern is rarely seen. The results are robust, with replication within and across participants. The construct of complexity appears to be a general principle that is relevant to treating a range of language disorders in both children and adults. While challenging the long-standing clinical notion that treatment should begin with simple structures, mounting evidence points toward the facilitative effects of using more complex structures as a starting point for treatment.

Evolution of an RNP assembly system: A minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster

PubMed Central

Kroiss, Matthias; Schultz, Jörg; Wiesner, Julia; Chari, Ashwin; Sickmann, Albert; Fischer, Utz

2008-01-01

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2–8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. Our data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction. PMID:18621711

The Resourceful Facilitator: Teacher Leaders Constructing Identities as Facilitators of Teacher Peer Groups

ERIC Educational Resources Information Center

Allen, David

2016-01-01

The use of teacher peer groups is a prevalent strategy for school-based professional development and instructional improvement. Facilitation of such groups is an increasingly vital dimension of teacher leadership as a component of school improvement efforts. Drawing on a qualitative study of facilitation of teacher peer groups, the article…

Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry.

PubMed

Karalewitz, Andrew P-A; Fu, Zhuji; Baldwin, Michael R; Kim, Jung-Ja P; Barbieri, Joseph T

2012-11-23

How botulinum neurotoxin serotype C (BoNT/C) enters neurons is unclear. BoNT/C utilizes dual gangliosides as host cell receptors. BoNT/C accesses gangliosides on the plasma membrane. Plasma membrane accessibility of the dual ganglioside receptors suggests synaptic vesicle exocytosis may not be necessary to expose BoNT/C receptors. Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A-G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.

Facilitating the implementation of clinical technology in healthcare: what role does a national agency play?

PubMed

Harvey, Gill; Llewellyn, Sue; Maniatopoulos, Greg; Boyd, Alan; Procter, Rob

2018-05-10

Accelerating the implementation of new technology in healthcare is typically complex and multi-faceted. One strategy is to charge a national agency with the responsibility for facilitating implementation. This study examines the role of such an agency in the English National Health Service. In particular, it compares two different facilitation strategies employed by the agency to support the implementation of insulin pump therapy. The research involved an empirical case study of four healthcare organisations receiving different levels of facilitation from the national agency: two received active hands-on facilitation; one was the intended recipient of a more passive, web-based facilitation strategy; the other implemented the technology without any external facilitation. The primary method of data collection was semi-structured qualitative interviews with key individuals involved in implementation. The integrated-PARIHS framework was applied as a conceptual lens to analyse the data. The two sites that received active facilitation from an Implementation Manager in the national agency made positive progress in implementing the technology. In both sites there was a high level of initial receptiveness to implementation. This was similar to a site that had successfully introduced insulin pump therapy without facilitation support from the national agency. By contrast, a site that did not have direct contact with the national agency made little progress with implementation, despite the availability of a web-based implementation resource. Clinicians expressed differences of opinion around the value and effectiveness of the technology and contextual barriers related to funding for implementation persisted. The national agency's intended roll out strategy using passive web-based facilitation appeared to have little impact. When favourable conditions exist, in terms of agreement around the value of the technology, clinician receptiveness and motivation to change, active

The potential of sarcospan in adhesion complex replacement therapeutics for the treatment of muscular dystrophy

PubMed Central

Marshall, Jamie L.; Kwok, Yukwah; McMorran, Brian; Baum, Linda G.; Crosbie-Watson, Rachelle H.

2013-01-01

Three adhesion complexes span the sarcolemma and facilitate critical connections between the extracellular matrix and the actin cytoskeleton: the dystrophin- and utrophin-glycoprotein complexes and α7β1 integrin. Loss of individual protein components results in a loss of the entire protein complex and muscular dystrophy. Muscular dystrophy is a progressive, lethal wasting disease characterized by repetitive cycles of myofiber degeneration and regeneration. Protein replacement therapy offers a promising approach for the treatment of muscular dystrophy. Recently, we demonstrated that sarcospan facilitates protein-protein interactions amongst the adhesion complexes and is an important therapeutic target. Here, we review current protein replacement strategies, discuss the potential benefits of sarcospan expression, and identify important experiments that must be addressed for sarcospan to move to the clinic. PMID:23601082

Competition and facilitation between a native and a domestic herbivore: trade-offs between forage quantity and quality.

PubMed

Augustine, David J; Springer, Tim L

2013-06-01

Potential competition between native and domestic herbivores is a major consideration influencing the management and conservation of native herbivores in rangeland ecosystems. In grasslands of the North American Great Plains, black-tailed prairie dogs (Cynomys ludovicianus) are widely viewed as competitors with cattle but are also important for biodiversity conservation due to their role in creating habitat for other native species. We examined spatiotemporal variation in prairie dog effects on growing-season forage quality and quantity using measurements from three colony complexes in Colorado and South Dakota and from a previous study of a fourth complex in Montana. At two complexes experiencing below-average precipitation, forage availability both on and off colonies was so low (12-54 g/m2) that daily forage intake rates of cattle were likely constrained by instantaneous intake rates and daily foraging time. Under these dry conditions, prairie dogs (1) substantially reduced forage availability, thus further limiting cattle daily intake rates, and (2) had either no or a small positive effect on forage digestibility. Under such conditions, prairie dogs are likely to compete with cattle in direct proportion to their abundance. For two complexes experiencing above-average precipitation, forage quantity on and off colonies (77-208 g/m2) was sufficient for daily forage intake of cattle to be limited by digestion rather than instantaneous forage intake. At one complex where prairie dogs enhanced forage digestibility and [N] while having no effect on forage quantity, prairie dogs are predicted to facilitate cattle mass gains regardless of prairie dog abundance. At the second complex where prairie dogs enhanced digestibility and [N] but reduced forage quantity, effects on cattle can vary from competition to facilitation depending on prairie dog abundance. Our findings show that the high spatiotemporal variation in vegetation dynamics characteristic of semiarid grasslands

The Multiple Origins of Complex Multicellularity

NASA Astrophysics Data System (ADS)

Knoll, Andrew H.

2011-05-01

Simple multicellularity has evolved numerous times within the Eukarya, but complex multicellular organisms belong to only six clades: animals, embryophytic land plants, florideophyte red algae, laminarialean brown algae, and two groups of fungi. Phylogeny and genomics suggest a generalized trajectory for the evolution of complex multicellularity, beginning with the co-optation of existing genes for adhesion. Molecular channels to facilitate cell-cell transfer of nutrients and signaling molecules appear to be critical, as this trait occurs in all complex multicellular organisms but few others. Proliferation of gene families for transcription factors and cell signals accompany the key functional innovation of complex multicellular clades: differentiated cells and tissues for the bulk transport of oxygen, nutrients, and molecular signals that enable organisms to circumvent the physical limitations of diffusion. The fossil records of animals and plants document key stages of this trajectory.

Navigating Complex Trade-Offs in Conservation and Development: An Integrative Framework

ERIC Educational Resources Information Center

Hirsch, Paul D.; Brosius, J. Peter

2013-01-01

We present a framework that makes space for multiple perspectives and ways of thinking about complex trade-off problems in conservation and development. At the core of the framework are three "integrative lenses" designed to facilitate lines of inquiry according to three unique ways of perceiving complexity. The aim of the framework is…

Evolution of complex adaptations in molecular systems

PubMed Central

Pál, Csaba; Papp, Balázs

2017-01-01

A central challenge in evolutionary biology concerns the mechanisms by which complex adaptations arise. Such adaptations depend on the fixation of multiple, highly specific mutations, where intermediate stages of evolution seemingly provide little or no benefit. It is generally assumed that the establishment of complex adaptations is very slow in nature, as evolution of such traits demands special population genetic or environmental circumstances. However, blueprints of complex adaptations in molecular systems are pervasive, indicating that they can readily evolve. We discuss the prospects and limitations of non-adaptive scenarios, which assume multiple neutral or deleterious steps in the evolution of complex adaptations. Next, we examine how complex adaptations can evolve by natural selection in changing environment. Finally, we argue that molecular ’springboards’, such as phenotypic heterogeneity and promiscuous interactions facilitate this process by providing access to new adaptive paths. PMID:28782044

Using electrical and optical tweezers to facilitate studies of molecular motors.

PubMed

Arsenault, Mark E; Sun, Yujie; Bau, Haim H; Goldman, Yale E

2009-06-28

Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells.

Using electrical and optical tweezers to facilitate studies of molecular motors†

PubMed Central

Arsenault, Mark E.; Sun, Yujie; Bau, Haim H.; Goldman, Yale E.

2013-01-01

Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells. PMID:19506758

3D printing of an aortic aneurysm to facilitate decision making and device selection for endovascular aneurysm repair in complex neck anatomy.

PubMed

Tam, Matthew D B S; Laycock, Stephen D; Brown, James R I; Jakeways, Matthew

2013-12-01

To describe rapid prototyping or 3-dimensional (3D) printing of aneurysms with complex neck anatomy to facilitate endovascular aneurysm repair (EVAR). A 75-year-old man had a 6.6-cm infrarenal aortic aneurysm that appeared on computed tomographic angiography to have a sharp neck angulation of ~90°. However, although the computed tomography (CT) data were analyzed using centerline of flow, the true neck length and relations of the ostial origins were difficult to determine. No multidisciplinary consensus could be reached as to which stent-graft to use owing to these borderline features of the neck anatomy. Based on past experience with rapid prototyping technology, a decision was taken to print a model of the aneurysm to aid in visualization of the neck anatomy. The CT data were segmented, processed, and converted into a stereolithographic format representing the lumen as a 3D volume, from which a full-sized replica was printed within 24 hours. The model demonstrated that the neck was adequate for stent-graft repair using the Aorfix device. Rapid prototyping of aortic aneurysms is feasible and can aid decision making and device delivery. Further work is required to test the value of 3D replicas in planning procedures and their impact on procedure time, radiation dose, and procedure cost.

Graphic facilitation as a novel approach to practice development.

PubMed

Leonard, Angela; Bonaconsa, Candice; Ssenyonga, Lydia; Coetzee, Minette

2017-10-10

The active participation of staff from the outset of any health service or practice improvement process ensures they are more likely to become engaged in the implementation phases that follow initial service analyses. Graphic facilitation is a way of getting participants to develop an understanding of complex systems and articulate solutions from within them. This article describes how a graphic facilitation process enabled the members of a multidisciplinary team at a specialist paediatric neurosurgery hospital in Uganda to understand how their system worked. The large graphic representation the team created helped each team member to visualise their day-to-day practice, understand each person's contribution, celebrate their triumphs and highlight opportunities for service improvement. The process highlighted three features of their practice: an authentic interdisciplinary team approach to care, admission of the primary carer with the child, and previously unrecognised delays in patient flow through the outpatients department. The team's active participation and ownership of the process resulted in sustainable improvements to clinical practice. ©2012 RCN Publishing Company Ltd. All rights reserved. Not to be copied, transmitted or recorded in any way, in whole or part, without prior permission of the publishers.

Addressing Complex Challenges through Adaptive Leadership: A Promising Approach to Collaborative Problem Solving

ERIC Educational Resources Information Center

Nelson, Tenneisha; Squires, Vicki

2017-01-01

Organizations are faced with solving increasingly complex problems. Addressing these issues requires effective leadership that can facilitate a collaborative problem solving approach where multiple perspectives are leveraged. In this conceptual paper, we critique the effectiveness of earlier leadership models in tackling complex organizational…

Facilitation as Attenuating of Environmental Stress among Structured Microbial Populations.

PubMed

Martins, Suzana Cláudia Silveira; Santaella, Sandra Tédde; Martins, Claudia Miranda; Martins, Rogério Parentoni

2016-01-01

There is currently an intense debate in microbial societies on whether evolution in complex communities is driven by competition or cooperation. Since Darwin, competition for scarce food resources has been considered the main ecological interaction shaping population dynamics and community structure both in vivo and in vitro. However, facilitation may be widespread across several animal and plant species. This could also be true in microbial strains growing under environmental stress. Pure and mixed strains of Serratia marcescens and Candida rugosa were grown in mineral culture media containing phenol. Growth rates were estimated as the angular coefficients computed from linearized growth curves. Fitness index was estimated as the quotient between growth rates computed for lineages grown in isolation and in mixed cultures. The growth rates were significantly higher in associated cultures than in pure cultures and fitness index was greater than 1 for both microbial species showing that the interaction between Serratia marcescens and Candida rugosa yielded more efficient phenol utilization by both lineages. This result corroborates the hypothesis that facilitation between microbial strains can increase their fitness and performance in environmental bioremediation.

Computer-aided identification, synthesis, and biological evaluation of novel inhibitors for botulinum neurotoxin serotype A

DOE PAGES

Teng, Y. G.; Berger, W. T.; Nesbitt, N. M.; ...

2015-07-27

Botulinum neurotoxins (BoNTs) are among the most potent biological toxin known to humans, and are classified as Category A bioterrorism agents by the Centers for Disease Control and prevention (CDC). There are seven known BoNT serotypes (A-G) which have been thus far identified in literature. BoNTs have been shown to block neurotransmitter release by cleaving proteins of the soluble NSF attachment protein receptor (SNARE) complex. Disruption of the SNARE complex precludes motor neuron failure which ultimately results in flaccid paralysis in humans and animals. Currently, there are no effective therapeutic treatments against the neurotoxin light chain (LC) after translocation intomore » the cytosols of motor neurons. In this work, high-throughput virtual screening was employed to screen a library of commercially available compounds from ZINC database against BoNT/A-LC. Among the hit compounds from the in-silico screening, two lead compounds were identified and found to have potent inhibitory activity against BoNT/A-LC in vitro, as well as in Neuro-2a cells. A few analogues of the lead compounds were synthesized and their potency examined. One of these analogues showed an enhanced activity than the lead compounds« less

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

PubMed Central

Kabachinski, Greg; Kielar-Grevstad, D. Michelle; Zhang, Xingmin; James, Declan J.; Martin, Thomas F. J.

2016-01-01

The Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly. PMID:26700319

Evaluation of the Facilitated Communication Pilot

ERIC Educational Resources Information Center

Cooper-Martin, Elizabeth

2014-01-01

The Office of Special Education and Student Services asked the Office of Shared Accountability to evaluate the "Facilitated Communication Pilot." In facilitated communication (FC), people with communication impairments express themselves by typing with the aid of a communication partner, called a facilitator, who provides physical (and…

An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity

PubMed Central

Choudhary, Vineet

2017-01-01

Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess ceramide is toxic, causing growth arrest and apoptosis. In this study, we identify a novel mechanism by which cells prevent the toxic accumulation of ceramides; they facilitate nonvesicular ceramide transfer from the endoplasmic reticulum (ER) to the Golgi complex, where ceramides are converted to complex sphingolipids. We find that the yeast protein Nvj2p promotes the nonvesicular transfer of ceramides from the ER to the Golgi complex. The protein is a tether that generates close contacts between these compartments and may directly transport ceramide. Nvj2p normally resides at contacts between the ER and other organelles, but during ER stress, it relocalizes to and increases ER–Golgi contacts. ER–Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER–Golgi contacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the buildup of toxic amounts of ceramides. PMID:28011845

Bat echolocation calls facilitate social communication

PubMed Central

Knörnschild, Mirjam; Jung, Kirsten; Nagy, Martina; Metz, Markus; Kalko, Elisabeth

2012-01-01

Bat echolocation is primarily used for orientation and foraging but also holds great potential for social communication. The communicative function of echolocation calls is still largely unstudied, especially in the wild. Eavesdropping on vocal signatures encoding social information in echolocation calls has not, to our knowledge, been studied in free-living bats so far. We analysed echolocation calls of the polygynous bat Saccopteryx bilineata and found pronounced vocal signatures encoding sex and individual identity. We showed experimentally that free-living males discriminate approaching male and female conspecifics solely based on their echolocation calls. Males always produced aggressive vocalizations when hearing male echolocation calls and courtship vocalizations when hearing female echolocation calls; hence, they responded with complex social vocalizations in the appropriate social context. Our study demonstrates that social information encoded in bat echolocation calls plays a crucial and hitherto underestimated role for eavesdropping conspecifics and thus facilitates social communication in a highly mobile nocturnal mammal. PMID:23034703

Bat echolocation calls facilitate social communication.

PubMed

Knörnschild, Mirjam; Jung, Kirsten; Nagy, Martina; Metz, Markus; Kalko, Elisabeth

2012-12-07

Bat echolocation is primarily used for orientation and foraging but also holds great potential for social communication. The communicative function of echolocation calls is still largely unstudied, especially in the wild. Eavesdropping on vocal signatures encoding social information in echolocation calls has not, to our knowledge, been studied in free-living bats so far. We analysed echolocation calls of the polygynous bat Saccopteryx bilineata and found pronounced vocal signatures encoding sex and individual identity. We showed experimentally that free-living males discriminate approaching male and female conspecifics solely based on their echolocation calls. Males always produced aggressive vocalizations when hearing male echolocation calls and courtship vocalizations when hearing female echolocation calls; hence, they responded with complex social vocalizations in the appropriate social context. Our study demonstrates that social information encoded in bat echolocation calls plays a crucial and hitherto underestimated role for eavesdropping conspecifics and thus facilitates social communication in a highly mobile nocturnal mammal.

Dissociative tendencies and facilitated emotional processing.

PubMed

Oathes, Desmond J; Ray, William J

2008-10-01

Dissociation is a process linked to lapses of attention, history of abuse or trauma, compromised emotional memory, and a disintegrated sense of self. It is theorized that dissociation stems from avoiding emotional information, especially negative emotion, to protect a fragile psyche. The present study tested whether or not dissociaters do actually avoid processing emotion by asking groups scoring high or low on the Dissociative Experiences Scale to judge the affective valence of several types of emotional stimuli. Manipulations of valence, modality (pictures or words), task complexity, and personal relevance lead to results suggesting that dissociation is linked to facilitated rather than deficient emotional processing. Our results are consistent with a theory that sensitivity to emotional material may be a contributing factor in subsequent dissociation to avoid further elaboration of upsetting emotion in these individuals. The findings for dissociation further exemplify the influence of individual differences in the link between cognition and emotion. (c) 2008 APA, all rights reserved

LINCing complex functions at the nuclear envelope

PubMed Central

Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

2013-01-01

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

The potential of sarcospan in adhesion complex replacement therapeutics for the treatment of muscular dystrophy.

PubMed

Marshall, Jamie L; Kwok, Yukwah; McMorran, Brian J; Baum, Linda G; Crosbie-Watson, Rachelle H

2013-09-01

Three adhesion complexes span the sarcolemma and facilitate critical connections between the extracellular matrix and the actin cytoskeleton: the dystrophin- and utrophin-glycoprotein complexes and α7β1 integrin. Loss of individual protein components results in a loss of the entire protein complex and muscular dystrophy. Muscular dystrophy is a progressive, lethal wasting disease characterized by repetitive cycles of myofiber degeneration and regeneration. Protein-replacement therapy offers a promising approach for the treatment of muscular dystrophy. Recently, we demonstrated that sarcospan facilitates protein-protein interactions amongst the adhesion complexes and is an important potential therapeutic target. Here, we review current protein-replacement strategies, discuss the potential benefits of sarcospan expression, and identify important experiments that must be addressed for sarcospan to move to the clinic. © 2013 FEBS.

Respiratory-aspirated 35-mm hairpin successfully retrieved with a Teflon® snare system under fluoroscopic guidance via a split endotracheal tube: a useful technique in cases of failed extraction by bronchoscopy and avoiding the need for a thoracotomy.

PubMed

Gill, S S; Pease, R A; Ashwin, C J; Gill, S S; Tait, N P

2012-09-01

Respiratory foreign body aspiration (FBA) is a common global health problem requiring prompt recognition and early treatment to prevent potentially fatal complications. The majority of FBAs are due to organic objects and treatment is usually via either endoscopic or surgical extraction. FBA of a straight hairpin has been described as a unique entity in the literature, occurring most commonly in females, particularly during adolescence. In the process of inserting hairpins, the pins will typically be between the teeth with the head tilted backwards, while tying their hair with both hands. This position increases the risk of aspiration, particularly if there is any sudden coughing or laughing. To our knowledge, this is the first case report of a 35-mm straight metallic hairpin foreign body that has been successfully retrieved by a radiological snare system under fluoroscopic guidance. This was achieved with the use of a split endotracheal tube, and therefore avoided the need for a thoracotomy in an adolescent female patient.

Barriers and facilitators to participation of minorities in clinical trials.

PubMed

Schmotzer, Geri L

2012-01-01

Historically, researchers have experienced difficulties with the recruitment of underrepresented populations, especially for women and minorities to cancer clinical trials. This has lead to marked health disparities among these groups. The purpose of this literature review is to investigate barriers and facilitators that provide explanations for the low participation rate of women and minorities in clinical trials. A search was conducted for published work in medical and social research from 1995 to 2008 using computerized databases: PubMed, CINAHL, and PsyclNFO. The following MeSH terms were used; clinical trials, minorities, minority groups, participation, recruitment, research subjects, and neoplasm. This netted a total of 43 articles, 22 of which were deemed appropriate for this article. Most striking throughout the literature was that barriers to trial participation were reported at an appreciably higher rate than facilitators. Health care provider barriers were captured by two themes: physician triage and physician knowledge. Patient barriers to trial participation emerged as reports of fear, mistrust of the medical community and the burden associated with trial participation. Facilitators to trial participation included physician enthusiasm and good communication skills, a good provider-patient relationship, having a perceived benefit, and feelings of altruism. This review provides a background into women and minorities' participation in clinical research. Patient recruitment into clinical trials is a complex process and there is limited research exploring the optimization of study recruitment. More information is needed to understand the issues surrounding the decision making process of the potential trial participant.

Group Facilitation: Functions and Skills.

ERIC Educational Resources Information Center

Anderson, L. Frances; Robertson, Sharon E.

1985-01-01

Discusses a model based on a specific set of assumptions about causality and effectiveness in interactional groups. Discusses personal qualities of group facilitators and proposes five major functions and seven skill clusters central to effective group facilitation. (Author/BH)

Exocyst and autophagy-related membrane trafficking in plants.

PubMed

Pecenková, Tamara; Markovic, Vedrana; Sabol, Peter; Kulich, Ivan; Žárský, Viktor

2017-12-18

Endomembrane traffic in eukaryotic cells functions partially as a means of communication; delivery of membrane in one direction has to be balanced with a reduction at the other end. This effect is typically the case during the defence against pathogens. To combat pathogens, cellular growth and differentiation are suppressed, while endomembrane traffic is poised towards limiting the pathogen attack. The octameric exocyst vesicle-tethering complex was originally discovered as a factor facilitating vesicle-targeting and vesicle-plasma membrane (PM) fusion during exocytosis prior to and possibly during SNARE complex formation. Interestingly, it was recently implicated both in animals and plants in autophagy membrane traffic. In animal cells, the exocyst is integrated into the mTOR-regulated energy metabolism stress/starvation pathway, participating in the formation and especially initiation of an autophagosome. In plants, the first functional link was to autophagy-related anthocyanin import to the vacuole and to starvation. In this concise review, we summarize the current knowledge of exocyst functions in autophagy and defence in plants that might involve unconventional secretion and compare it with animal conditions. Formation of different exocyst complexes during undisturbed cell growth, as opposed to periods of cellular stress reactions involving autophagy, might contribute to the coordination of endomembrane trafficking pathways. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Effects of Student-Facilitated Learning on Instructional Facilitators

ERIC Educational Resources Information Center

Bonner, Sarah M.; Somers, Jennifer A.; Rivera, Gwendelyn J.; Keiler, Leslie S.

2017-01-01

We investigated perceptions about learning strategy use and instructional roles among a sample of high needs adolescents (n = 230) who acted as near-peer instructional facilitators. The sample was drawn from science and mathematics classes in nonselective public secondary schools in New York City. Students participated in an inschool intervention…

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

PubMed

Tani, Motohiro; Kuge, Osamu

2012-12-01

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. © 2012 Blackwell Publishing Ltd.

Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment.

PubMed

Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves; Einav, Shirit

2016-11-01

Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether

Blm10 facilitates nuclear import of proteasome core particles

PubMed Central

Weberruss, Marion H; Savulescu, Anca F; Jando, Julia; Bissinger, Thomas; Harel, Amnon; Glickman, Michael H; Enenkel, Cordula

2013-01-01

Short-lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT-like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP-CP assemblies are primarily nuclear and promote cell division. During quiescence, RP-CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10-bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG-rich nucleoporins and is dissociated from the CP by Ran-GTP. Thus, Blm10 represents the first CP-dedicated nuclear import receptor in yeast. PMID:23982732

Identifying Facilitating Factors and Barriers to Improving Student Retention Rates in Tertiary Teaching Courses: A Systematic Review

ERIC Educational Resources Information Center

Bowles, Terence V.; Brindle, Kimberley A.

2017-01-01

Factors which impact student retention in tertiary level teaching courses are complex. This study examined facilitating factors and barriers to student retention for students studying education. Due to a limited number of studies, the search was extended to factors impacting students undertaking tertiary education. A systematic review was…

Minorities with lupus nephritis and medications: a study of facilitators to medication decision-making.

PubMed

Singh, Jasvinder A; Qu, Haiyan; Yazdany, Jinoos; Chatham, Winn; Shewchuk, Richard

2015-12-17

Medication decision-making poses a challenge for a significant proportion of patients. This is an even more challenging for patients who have complex, rare, immune conditions that affect them at a young age and are associated with the use of life-long treatment, perceived by some as having significant risk of side effects and toxicity. The aim of our study was to examine the perspectives of women with lupus nephritis on facilitators to medication decision-making. We used the nominal group technique (NGT), a structured formative process to elicit patient perspectives. An NGT expert moderated eight patient group meetings. Participants (n = 52) responded to the question "What sorts of things make it easier for people to decide to take the medicines that doctors prescribe for treating their lupus kidney disease?" Patients nominated, discussed, and prioritized facilitators to medication decisional processes. Fifty-two women with lupus nephritis participated in eight NGT meetings (27 African-American, 13 Hispanic, and 12 Caucasian). Average age was 40.6 years (standard deviation (SD) = 13.3), and disease duration was 11.8 years (SD = 8.3); 36.5 % obtained at least a college education, and 55.8 % had difficulty in reading health materials. Patients generated 280 decision-making facilitators (range of 26 to 42 per panel). Of these, 102 (36 %) facilitators were perceived by patients as having relatively more influence in decision-making processes than others. Prioritized facilitators included effective patient-physician communication regarding benefits/harms, patient desire to live a normal life and improve quality of life, concern for their dependents, experiencing benefits and few/infrequent/no harms with lupus medications, and their affordability. Relative to African-Americans, Caucasian and Hispanic patients endorsed a smaller percentage of facilitators as influential. Level of agreement with which patients within panels independently agreed in their

Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

ERIC Educational Resources Information Center

Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

2005-01-01

In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

Results of a feasibility study: barriers and facilitators in implementing the Sherbrooke model in France.

PubMed

Fassier, Jean-Baptiste; Durand, Marie-José; Caillard, Jean-François; Roquelaure, Yves; Loisel, Patrick

2015-05-01

Return-to-work interventions associated with the workplace environment are often more effective than conventional care. The Sherbrooke model is an integrated intervention that has proved successful in preventing work disability due to low-back pain. Implementation, however, runs up against many obstacles, and failure has been reported in many countries. The present study sought to identify barriers to and facilitators of the implementation of the Sherbrooke model within the French health system. A multiple case study with nested levels of analysis was performed in two regions of France. A conceptual framework was designed and refined to identify barriers and facilitators at the individual, organizational and contextual levels. Qualitative data were collected via semi-structured interview (N=22), focus groups (N=7), and observation and from the gray literature. Participants (N=61) belonged to three fields: healthcare, social insurance, and the workplace. Numerous barriers and facilitators were identified in each field and at each level, some specific and others common to workers in all fields. Individual and organizational barriers comprised lack of time and resources, discordant professional values, and perceived risk. Legal barriers comprised medical confidentiality, legal complexity, and priority given to primary prevention. Individual-level facilitators comprised needs and perceived benefits. Some organizations had concordant values and practices. Legal facilitators comprised possibilities of collaboration and gradual return to work. The present feasibility analysis of implementing the Sherbrooke model revealed numerous barriers and facilitators suggesting a new implementation strategy be drawn up if failure is to be avoided.

Competing Distractors Facilitate Visual Search in Heterogeneous Displays.

PubMed

Kong, Garry; Alais, David; Van der Burg, Erik

2016-01-01

In the present study, we examine how observers search among complex displays. Participants were asked to search for a big red horizontal line among 119 distractor lines of various sizes, orientations and colours, leading to 36 different feature combinations. To understand how people search in such a heterogeneous display, we evolved the search display by using a genetic algorithm (Experiment 1). The best displays (i.e., displays corresponding to the fastest reaction times) were selected and combined to create new, evolved displays. Search times declined over generations. Results show that items sharing the same colour and orientation as the target disappeared over generations, implying they interfered with search, but items sharing the same colour and were 12.5° different in orientation only interfered if they were also the same size. Furthermore, and inconsistent with most dominant visual search theories, we found that non-red horizontal distractors increased over generations, indicating that these distractors facilitated visual search while participants were searching for a big red horizontally oriented target. In Experiments 2 and 3, we replicated these results using conventional, factorial experiments. Interestingly, in Experiment 4, we found that this facilitation effect was only present when the displays were very heterogeneous. While current models of visual search are able to successfully describe search in homogeneous displays, our results challenge the ability of these models to describe visual search in heterogeneous environments.

Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction

NASA Astrophysics Data System (ADS)

Zhang, Ying; Cui, Weidong; Wecksler, Aaron T.; Zhang, Hao; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

2016-07-01

Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis.

Structures of Ca(V) Ca**2+/CaM-IQ Domain Complexes Reveal Binding Modes That Underlie Calcium-Dependent Inactivation And Facilitation

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

Kim, E.Y.; Rumpf, C.H.; Fujiwara, Y.

2009-05-20

Calcium influx drives two opposing voltage-activated calcium channel (Ca{sub V}) self-modulatory processes: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Specific Ca{sup 2+}/calmodulin (Ca{sup 2+}/CaM) lobes produce CDI and CDF through interactions with the Ca{sub V}{alpha}{sub 1} subunit IQ domain. Curiously, Ca{sup 2+}/CaM lobe modulation polarity appears inverted between Ca{sub V}1s and Ca{sub V}2s. Here, we present crystal structures of Ca{sub V}2.1, Ca{sub V}2.2, and Ca{sub V}2.3 Ca{sup 2+}/CaM-IQ domain complexes. All display binding orientations opposite to Ca{sub V}1.2 with a physical reversal of the CaM lobe positions relative to the IQ {alpha}-helix. Titration calorimetry reveals lobe competition for a high-affinitymore » site common to Ca{sub V}1 and Ca{sub V}2 IQ domains that is occupied by the CDI lobe in the structures. Electrophysiological experiments demonstrate that the N-terminal Ca{sub V}2 Ca{sup 2+}/C-lobe anchors affect CDF. Together, the data unveil the remarkable structural plasticity at the heart of Ca{sub V} feedback modulation and indicate that Ca{sub V}1 and Ca{sub V}2 IQ domains bear a dedicated CDF site that exchanges Ca{sup 2+}/CaM lobe occupants.« less

Facilitated Communication: An Experimental Evaluation.

ERIC Educational Resources Information Center

Regal, Robert A.; And Others

1994-01-01

Nineteen adults with developmental disabilities, judged competent in facilitated communication, participated in a validation study using an information passing design requiring short-term recall of stimulus cards with shapes, colors, and numbers. Results failed to validate facilitated communication for the group as a whole, any individual…

Virtual OD: Facilitating Groups Online

ERIC Educational Resources Information Center

Milton, Judy; Watkins, Karen E.; Daley, Barbara J.

2005-01-01

This study examined the role of facilitators in nine virtual action learning groups. A qualitative analysis of the facilitators' interventions across all groups resulted in a typology that included group management, group process, and support interventions. A model showing the relationship among these categories proposes that effective…

An Investigation of the Relationship between Cohesion and Syntactic Complexity of Take-Away Writing and Advanced Content Complexity and Depth

ERIC Educational Resources Information Center

Gardner, Matthew Thomas

2017-01-01

In secondary and post-secondary content courses, the use of writing to facilitate complex learning in advanced content areas, which is called writing to learn content, can help students to evaluate their understanding, higher order cognition, and thinking about the content to learned (Carifio, 2005; Hayes, 2006; Carifio, 2015). The primary focus…

Cross-Linking and Mass Spectrometry Methodologies to Facilitate Structural Biology: Finding a Path through the Maze

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

Merkley, Eric D.; Cort, John R.; Adkins, Joshua N.

2013-09-01

Multiprotein complexes, rather than individual proteins, make up a large part of the biological macromolecular machinery of a cell. Understanding the structure and organization of these complexes is critical to understanding cellular function. Chemical cross-linking coupled with mass spectrometry is emerging as a complementary technique to traditional structural biology methods and can provide low-resolution structural information for a multitude of purposes, such as distance constraints in computational modeling of protein complexes. In this review, we discuss the experimental considerations for successful application of chemical cross-linking-mass spectrometry in biological studies and highlight three examples of such studies from the recent literature.more » These examples (as well as many others) illustrate the utility of a chemical cross-linking-mass spectrometry approach in facilitating structural analysis of large and challenging complexes.« less

Facilitating Dialogues about Racial Realities

ERIC Educational Resources Information Center

Quaye, Stephen John

2014-01-01

Background/Context: Facilitating dialogues about racial issues in higher education classroom settings continues to be a vexing problem facing postsecondary educators. In order for students to discuss race with their peers, they need skilled facilitators who are knowledgeable about racial issues and able to support students in these difficult…

The Essential Elements of Facilitation.

ERIC Educational Resources Information Center

Priest, Simon; Gass, Michael; Gillis, Lee

Most organizations find it difficult to implement change, and only about 10 percent of learning from training and development experiences is actually applied in the workplace. This book advocates facilitation as a means of enhancing change and increasing productivity. Facilitation engages employees by enhancing the processes associated with their…

Leucine facilitates the insulin-stimulated glucose uptake and insulin signaling in skeletal muscle cells: involving mTORC1 and mTORC2.

PubMed

Liu, Hui; Liu, Rui; Xiong, Yufang; Li, Xiang; Wang, Xiaolei; Ma, Yan; Guo, Huailan; Hao, Liping; Yao, Ping; Liu, Liegang; Wang, Di; Yang, Xuefeng

2014-08-01

Leucine, a branched-chain amino acid, has been shown to promote glucose uptake and increase insulin sensitivity in skeletal muscle, but the exact mechanism remains unestablished. We addressed this issue in cultured skeletal muscle cells in this study. Our results showed that leucine alone did not have an effect on glucose uptake or phosphorylation of protein kinase B (AKT), but facilitated the insulin-induced glucose uptake and AKT phosphorylation. The insulin-stimulated glucose uptake and AKT phosphorylation were inhibited by the phosphatidylinositol 3-kinase inhibitor, wortmannin, but the inhibition was partially reversed by leucine. The inhibitor of mammalian target of rapamycin complex 1 (mTORC1), rapamycin, had no effect on the insulin-stimulated glucose uptake, but eliminated the facilitating effect of leucine in the insulin-stimulated glucose uptake and AKT phosphorylation. In addition, leucine facilitation of the insulin-induced AKT phosphorylation was neutralized by knocking down the core component of the mammalian target of rapamycin complex 2 (mTORC2) with specific siRNA. Together, these findings show that leucine can facilitate the insulin-induced insulin signaling and glucose uptake in skeletal muscle cells through both mTORC1 and mTORC2, implicating the potential importance of this amino acid in glucose homeostasis and providing new mechanistic insights.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

PubMed

Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

2009-07-01

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Presynaptic kainate receptor-mediated facilitation of glutamate release involves Ca2+ -calmodulin at mossy fiber-CA3 synapses.

PubMed

Andrade-Talavera, Yuniesky; Duque-Feria, Paloma; Negrete-Díaz, José Vicente; Sihra, Talvinder S; Flores, Gonzalo; Rodríguez-Moreno, Antonio

2012-09-01

Presynaptic kainate receptors (KARs) modulate the release of glutamate at synapses established between mossy fibers (MF) and CA3 pyramidal cells in the hippocampus. The activation of KAR by low, nanomolar, kainate concentrations facilitates glutamate release. KAR-mediated facilitation of glutamate release involves the activation of an adenylate cyclase/cyclic adenosine monophosphate/protein kinase A cascade at MF-CA3 synapses. Here, we studied the mechanisms by which KAR activation produces this facilitation of glutamate release in slices and synaptosomes. We find that the facilitation of glutamate release mediated by KAR activation requires an increase in Ca(2+) levels in the cytosol and the formation of a Ca(2+) -calmodulin complex to activate adenylate cyclase. The increase in cytosolic Ca(2+) underpinning this modulation is achieved, both, by Ca(2+) entering via Ca(2+) -permeable KARs and, by the mobilization of intraterminal Ca(2+) stores. Finally, we find that, congruent with the Ca(2+) -calmodulin support of KAR-mediated facilitation of glutamate release, induction of long-term potentiation at MF-CA3 synapses has an obligate requirement for Ca(2+) -calmodulin activity. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Clinical education facilitators: a literature review.

PubMed

Lambert, Veronica; Glacken, Michèle

2005-07-01

The aim of this literature review, set within an Irish context, is to present a broad overview of former and existing clinical support personnel, explore the concept of facilitation and examine what is known about the role of the clinical education facilitator. The importance of providing a supportive clinical environment to enhance clinical teaching and learning is strongly portrayed in the literature. While the past two decades have borne witness to various clinical support personnel, the literature identifies conflicting demands that these personnel face. No suggestions are advanced as to how to overcome these difficulties, which inevitably influence the quality and quantity of their clinical teaching role. An identifiable gap exists over who has prime responsibility for clinical teaching. It is timely that alternative possibilities for organizing clinical teaching are investigated. A new post emerging in practice settings is that of the clinical education facilitator who is meant to be the key linchpin in clinical areas for reducing the theory-practice gap. Relevant literature for this review was sourced using the computerized databases CINAHL, Medline and Synergy. Manual searching of relevant nursing journals and sourcing of secondary references extended the search. Government reports and other relevant documents were obtained through pertinent websites. Papers that explicitly examined the concept of facilitation and explored the posts of clinical education facilitators were included; six research papers were accessed and reviewed. In addition seven non-empirical papers were included. It is clear that considerable lack of role clarity resides over what constitutes clinical facilitation and the role of the clinical facilitator. Thus, it is paramount to strengthen this support role with Irish empirical evidence. A major advantage in having a ward-based clinical education facilitator is the benefit of having access to someone who can concentrate solely on

Facilitation as Attenuating of Environmental Stress among Structured Microbial Populations

PubMed Central

Santaella, Sandra Tédde; Martins, Claudia Miranda; Martins, Rogério Parentoni

2016-01-01

There is currently an intense debate in microbial societies on whether evolution in complex communities is driven by competition or cooperation. Since Darwin, competition for scarce food resources has been considered the main ecological interaction shaping population dynamics and community structure both in vivo and in vitro. However, facilitation may be widespread across several animal and plant species. This could also be true in microbial strains growing under environmental stress. Pure and mixed strains of Serratia marcescens and Candida rugosa were grown in mineral culture media containing phenol. Growth rates were estimated as the angular coefficients computed from linearized growth curves. Fitness index was estimated as the quotient between growth rates computed for lineages grown in isolation and in mixed cultures. The growth rates were significantly higher in associated cultures than in pure cultures and fitness index was greater than 1 for both microbial species showing that the interaction between Serratia marcescens and Candida rugosa yielded more efficient phenol utilization by both lineages. This result corroborates the hypothesis that facilitation between microbial strains can increase their fitness and performance in environmental bioremediation. PMID:26904719

Selective isolation of gold facilitated by second-sphere coordination with α-cyclodextrin.

PubMed

Liu, Zhichang; Frasconi, Marco; Lei, Juying; Brown, Zachary J; Zhu, Zhixue; Cao, Dennis; Iehl, Julien; Liu, Guoliang; Fahrenbach, Albert C; Botros, Youssry Y; Farha, Omar K; Hupp, Joseph T; Mirkin, Chad A; Fraser Stoddart, J

2013-01-01

Gold recovery using environmentally benign chemistry is imperative from an environmental perspective. Here we report the spontaneous assembly of a one-dimensional supramolecular complex with an extended {[K(OH₂)₆][AuBr₄](α-cyclodextrin)₂}n chain superstructure formed during the rapid co-precipitation of α-cyclodextrin and KAuBr₄ in water. This phase change is selective for this gold salt, even in the presence of other square-planar palladium and platinum complexes. From single-crystal X-ray analyses of six inclusion complexes between α-, β- and γ-cyclodextrins with KAuBr₄ and KAuCl₄, we hypothesize that a perfect match in molecular recognition between α-cyclodextrin and [AuBr₄](-) leads to a near-axial orientation of the ion with respect to the α-cyclodextrin channel, which facilitates a highly specific second-sphere coordination involving [AuBr₄](-) and [K(OH₂)₆](+) and drives the co-precipitation of the 1:2 adduct. This discovery heralds a green host-guest procedure for gold recovery from gold-bearing raw materials making use of α-cyclodextrin-an inexpensive and environmentally benign carbohydrate.

Evaluation of the tratment of metal-EDTA complexes using Ti0{sub 2} photocatalysis

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

Madden, T.; Datyte, A.K.; Prairie, M.R.

1996-03-01

This study has demonstrated the feasibility of TiO{sub 2} photocatalysis to treat EDTA and several metal-EDTA complexes that can be found in industrial wastewaters. For the EDTA complexes of metals capable of photodeposition, such as Cu and Pb, certain reaction conditions were shown to facilitate the simultaneous complex degradation and photodeposition of these metals onto the catalyst. With metals that do not easily photodeposit, such as Ni and Cd, it is shown that the complex degradation is still facilitated, and can enhance other metals removal processes after photocatalytic treatment. Because the treatment of these metal-EDTA complexes typically requires special measures,more » there may exist situations where TiO{sub 2} photocatalysis could actually be the preferred method of treatment. However, its use should be compared economically to other more established advanced oxidation technologies. This necessity is demonstrated in the economic comparison to ozone treatment for EDTA degradation alone, where ozone treatment appears to be the clear choice in this application.« less

Barriers and facilitators influencing ethical evaluation in health technology assessment.

PubMed

Assasi, Nazila; Schwartz, Lisa; Tarride, Jean-Eric; O'Reilly, Daria; Goeree, Ron

2015-01-01

The objective of this study was to explore barriers and facilitators influencing the integration of ethical considerations in health technology assessment (HTA). The study consisted of two complementary approaches: (a) a systematic review of the literature; and (b) an eighteen-item online survey that was distributed to fifty-six HTA agencies affiliated with the International Network of Agencies for Health Technology Assessment. The review identified twenty-six relevant articles. The most often cited barriers in the literature were: scarcity, heterogeneity and complexity of ethical analysis methods; challenges in translating ethical analysis results into knowledge that is useful for decision makers; and lack of organizational support in terms of required expertise, time and financial resources. The most frequently cited facilitators included: usage of value-based appraisal methods, stakeholder and public engagement, enhancement of practice guidelines, ethical expertise, and educational interventions. Representatives of twenty-six (46.5 percent) agencies from nineteen countries completed the survey. A median of 10 percent (interquartile range, 5 percent to 50 percent) of the HTA products produced by the agencies was reported to include an assessment of ethical aspects. The most commonly perceived barriers were: limited ethical knowledge and expertise, insufficient time and resources, and difficulties in finding ethical evidence or using ethical guidelines. Educational interventions, demand by policy makers, and involvement of ethicists in HTA were the most commonly perceived facilitators. Our results emphasize the importance of simplification of ethics methodology and development of good practice guidelines in HTA, as well as capacity building for engaging HTA practitioners in ethical analyses.

Childbirth Supporters' Experiences in a Built Hospital Birth Environment: Exploring Inhibiting and Facilitating Factors in Negotiating the Supporter Role.

PubMed

Harte, J Davis; Sheehan, Athena; Stewart, Susan C; Foureur, Maralyn

2016-04-01

To explore inhibiting and facilitating design factors influencing childbirth supporters' experiences. Birthing women benefit from the continuous, cooperative presence of supporters. However, little research has investigated how birth room design facilitates or inhibits supporters' role navigation. We conducted an exploratory video ethnographic single case study of childbirth supporters' experiences, within an Australian hospital birth environment. Video, field notes, and video-cued reflexive interviews with the woman, her midwives, and supporters were thematically analyzed using ethnographic/symbolic interactionist perspectives to frame supporters' understandings. Findings suggest supporters' experiences are complex, made more complicated by sparse understanding or accommodation of their needs in the built environment. Supporters' presence and roles are not facilitated by the physical space; they experience "an unbelonging paradox" of being needed, yet uncertain and "in the way" during "tenuous nest-building" activities. Suggested design guidelines to facilitate supporters' well-being and their roles in designed hospital birth spaces are provided. © The Author(s) 2016.

SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

PubMed Central

Nogueira, Cristina; Erlmann, Patrik; Villeneuve, Julien; Santos, António JM; Martínez-Alonso, Emma; Martínez-Menárguez, José Ángel; Malhotra, Vivek

2014-01-01

TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18. DOI: http://dx.doi.org/10.7554/eLife.02784.001 PMID:24842878

SCScore: Synthetic Complexity Learned from a Reaction Corpus.

PubMed

Coley, Connor W; Rogers, Luke; Green, William H; Jensen, Klavs F

2018-02-26

Several definitions of molecular complexity exist to facilitate prioritization of lead compounds, to identify diversity-inducing and complexifying reactions, and to guide retrosynthetic searches. In this work, we focus on synthetic complexity and reformalize its definition to correlate with the expected number of reaction steps required to produce a target molecule, with implicit knowledge about what compounds are reasonable starting materials. We train a neural network model on 12 million reactions from the Reaxys database to impose a pairwise inequality constraint enforcing the premise of this definition: that on average, the products of published chemical reactions should be more synthetically complex than their corresponding reactants. The learned metric (SCScore) exhibits highly desirable nonlinear behavior, particularly in recognizing increases in synthetic complexity throughout a number of linear synthetic routes.

Structural plasticity in hippocampal cells related to the facilitative effect of intracranial self-stimulation on a spatial memory task.

PubMed

Chamorro-López, Jacobo; Miguéns, Miguel; Morgado-Bernal, Ignacio; Kastanauskaite, Asta; Selvas, Abraham; Cabané-Cucurella, Alberto; Aldavert-Vera, Laura; DeFelipe, Javier; Segura-Torres, Pilar

2015-12-01

Posttraining intracranial self-stimulation (SS) in the lateral hypothalamus facilitates the acquisition and retention of several implicit and explicit memory tasks. Here, intracellular injections of Lucifer yellow were used to assess morphological changes in hippocampal neurons that might be specifically related to the facilitative posttraining SS effect upon the acquisition and retention of a distributed spatial task in the Morris water maze. We examined the structure, size and branching complexity of cornus ammonis 1 (CA1) cells, and the spine density of CA1 pyramidal neurons and granular cells of the dentate gyrus (DG). Animals that received SS after each acquisition session performed faster and better than Sham ones--an improvement that was also evident in a probe trial 3 days after the last training session. The neuromorphological analysis revealed an increment in the size and branching complexity in apical CA1 dendritic arborization in SS-treated subjects as compared with Sham animals. Furthermore, increased spine density was observed in the CA1 field in SS animals, whereas no effects were observed in DG cells. Our results support the hypothesis that the facilitating effect of SS on the acquisition and retention of a spatial memory task could be related to structural plasticity in CA1 hippocampal cells. (c) 2015 APA, all rights reserved).

A care improvement program acting as a powerful learning environment to support nursing students learning facilitation competencies.

PubMed

Jukema, Jan S; Harps-Timmerman, Annelies; Stoopendaal, Annemiek; Smits, Carolien H M

2015-11-01

Change management is an important area of training in undergraduate nursing education. Successful change management in healthcare aimed at improving practices requires facilitation skills that support teams in attaining the desired change. Developing facilitation skills in nursing students requires formal educational support. A Dutch Regional Care Improvement Program based on a nationwide format of change management in healthcare was designed to act as a Powerful Learning Environment for nursing students developing competencies in facilitating change. This article has two aims: to provide comprehensive insight into the program components and to describe students' learning experiences in developing their facilitation skills. This Dutch Regional Care Improvement Program considers three aspects of a Powerful Learning Environment: self-regulated learning; problem-based learning; and complex, realistic and challenging learning tasks. These three aspects were operationalised in five distinct areas of facilitation: increasing awareness of the need for change; leadership and project management; relationship building and communication; importance of the local context; and ongoing monitoring and evaluation. Over a period of 18 months, 42 nursing students, supported by trained lecturer-coaches, took part in nine improvement teams in our Regional Care Improvement Program, executing activities in all five areas of facilitation. Based on the students' experiences, we propose refinements to various components of this program, aimed at strengthenin the learning environment. There is a need for further detailed empirical research to study the impact this kind of learning environment has on students developing facilitation competencies in healthcare improvement. Copyright © 2015 Elsevier Ltd. All rights reserved.

Productive and Non-productive Pathways for Synaptotagmin 1 to Support Ca2+-Triggered Fast Exocytosis.

PubMed

Kim, Jaewook; Shin, Yeon-Kyun

2017-01-01

Ca 2+ -triggered SNARE-mediated membrane fusion is essential for neuronal communication. The speed of this process is of particular importance because it sets a time limit to cognitive and physical activities. In this work, we expand the proteoliposome-to-supported bilayer (SBL) fusion assay by successfully incorporating synaptotagmin 1 (Syt1), a major Ca 2+ sensor. We report that Syt1 and Ca 2+ together can elicit more than a 50-fold increase in the number of membrane fusion events when compared with membrane fusion mediated by SNAREs only. What is remarkable is that ~55% of all vesicle fusion events occurs within 20 ms upon vesicle docking. Furthermore, pre-binding of Syt1 to SNAREs prior to Ca 2+ inhibits spontaneous fusion, but intriguingly, this leads to a complete loss of the Ca 2+ responsiveness. Thus, our results suggest that there is a productive and a non-productive pathway for Syt1, depending on whether there is a premature interaction between Syt1 and SNAREs. Our results show that Ca 2+ binding to Syt1 prior to Syt1's binding to SNAREs may be a prerequisite for the productive pathway. The successful reconstitution of Syt1 activities in the physiological time scale provides new opportunities to test the current mechanistic models for Ca 2+ -triggered exocytosis.

Constructing Videocases to Help Novices Learn to Facilitate Discussions in Science and English: How Does Subject Matter Matter?

ERIC Educational Resources Information Center

Rosaen, Cheryl L.; Lundeberg, Mary; Terpstra, Marjorie; Cooper, Marjorie; Niu, Rui; Fu, Jing

2010-01-01

Learning to conduct interactive classroom discussions is a high priority for becoming an effective teacher, and most teachers view conducting productive classroom discussions as a complex undertaking. Because the dynamics of facilitating classroom discussions are multifaceted and hard to analyze in real time, there is a growing interest in how…

Competition and cooperation among similar representations: toward a unified account of facilitative and inhibitory effects of lexical neighbors.

PubMed

Chen, Qi; Mirman, Daniel

2012-04-01

One of the core principles of how the mind works is the graded, parallel activation of multiple related or similar representations. Parallel activation of multiple representations has been particularly important in the development of theories and models of language processing, where coactivated representations (neighbors) have been shown to exhibit both facilitative and inhibitory effects on word recognition and production. Researchers generally ascribe these effects to interactive activation and competition, but there is no unified explanation for why the effects are facilitative in some cases and inhibitory in others. We present a series of simulations of a simple domain-general interactive activation and competition model that is broadly consistent with more specialized domain-specific models of lexical processing. The results showed that interactive activation and competition can indeed account for the complex pattern of reversals. Critically, the simulations revealed a core computational principle that determines whether neighbor effects are facilitative or inhibitory: strongly active neighbors exert a net inhibitory effect, and weakly active neighbors exert a net facilitative effect.

Facilitation of contrast detection in near-peripheral vision.

PubMed

Giorgi, Robert G; Soong, Grace P; Woods, Russell L; Peli, Eli

2004-12-01

Foveal detection of a Gabor patch (target) is facilitated by collinear, displaced high-contrast flankers. Polat and Sagi reported that the same phenomenon occurred in the periphery, but no data were presented [Proc. Natl. Acad. Sci. 91 (1994) 1206]. Others have found no facilitation in a limited number of conditions tested. To resolve this apparent conflict, we measured lateral facilitation in the near-periphery using a range of stimulus parameters. We found facilitation for a range of target-flanker distances for peripheral eccentricities up to 6 degrees , but the magnitude of the effect was less than found in central vision. Facilitation varied across subjects and with spatial frequency. Flanker contrast had no effect over the range evaluated (10-80%). Equal facilitation was found for two global arrangements of the stimulus pattern. Facilitation was found using a temporal, but not a spatial two-alternative forced-choice paradigm, accounting for the different results among previous studies. This finding supports previous indications of the role of attention in altering such facilitation. The value of facilitation from lateral interactions for persons with central vision impairment, who have to shift their attention to a peripheral locus constantly, needs to be examined.

Chronic stress may facilitate the recruitment of habit- and addiction-related neurocircuitries through neuronal restructuring of the striatum.

PubMed

Taylor, S B; Anglin, J M; Paode, P R; Riggert, A G; Olive, M F; Conrad, C D

2014-11-07

Chronic stress is an established risk factor in the development of addiction. Addiction is characterized by a progressive transition from casual drug use to habitual and compulsive drug use. The ability of chronic stress to facilitate the transition to addiction may be mediated by increased engagement of the neurocircuitries underlying habitual behavior and addiction. In the present study, striatal morphology was evaluated after 2 weeks of chronic variable stress in male Sprague-Dawley rats. Dendritic complexity of medium spiny neurons was visualized and quantified with Golgi staining in the dorsolateral and dorsomedial striatum, as well as in the nucleus accumbens core and shell. In separate cohorts, the effects of chronic stress on habitual behavior and the acute locomotor response to methamphetamine were also assessed. Chronic stress resulted in increased dendritic complexity in the dorsolateral striatum and nucleus accumbens core, regions implicated in habitual behavior and addiction, while decreased complexity was found in the nucleus accumbens shell, a region critical for the initial rewarding effects of drugs of abuse. Chronic stress did not affect dendritic complexity in the dorsomedial striatum. A parallel shift toward habitual learning strategies following chronic stress was also identified. There was an initial reduction in acute locomotor response to methamphetamine, but no lasting effect as a result of chronic stress exposure. These findings suggest that chronic stress may facilitate the recruitment of habit- and addiction-related neurocircuitries through neuronal restructuring in the striatum. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Chronic stress may facilitate the recruitment of habit- and addiction-related neurocircuitries through neuronal restructuring of the striatum

PubMed Central

Taylor, S.B.; Anglin, J.M.; Paode, P.R.; Riggert, A.G.; Olive, M.F.; Conrad, C.D.

2014-01-01

Chronic stress is an established risk factor in the development of addiction. Addiction is characterized by a progressive transition from casual drug use to habitual and compulsive drug use. The ability of chronic stress to facilitate the transition to addiction may be mediated by increased engagement of the neurocircuitries underlying habitual behavior and addiction. In the present study, striatal morphology was evaluated after two weeks of chronic variable stress in male Sprague-Dawley rats. Dendritic complexity of medium spiny neurons was visualized and quantified with Golgi staining in the dorsolateral and dorsomedial striatum, as well as in the nucleus accumbens core and shell. In separate cohorts, the effects of chronic stress on habitual behavior and the acute locomotor response to methamphetamine were also assessed. Chronic stress resulted in increased dendritic complexity in the dorsolateral striatum and nucleus accumbens core, regions implicated in habitual behavior and addiction, while decreased complexity was found in the nucleus accumbens shell, a region critical for the initial rewarding effects of drugs of abuse. Chronic stress did not affect dendritic complexity in the dorsomedial striatum. A parallel shift toward habitual learning strategies following chronic stress was also identified. There was an initial reduction in acute locomotor response to methamphetamine, but no lasting effect as a result of chronic stress exposure. These findings suggest that chronic stress may facilitate the recruitment of habit- and addiction-related neurocircuitries through neuronal restructuring in the striatum. PMID:25242641

Molecular Speciation of Trace Metal Organic Complexes in the Pacific Ocean

NASA Astrophysics Data System (ADS)

Repeta, D.; Boiteau, R. M.; Bundy, R. M.; Babcock-Adams, L.

2017-12-01

Microbial production across approximately one third of the surface ocean is limited by extraordinarily low (picomolar) concentrations of dissolved iron, essentially all of which is complexed to strong organic ligands of unknown composition. Other biologically important trace metals (cobalt, copper, zinc, nickel) are also complexed to strong organic ligands, which again have not been extensively characterized. Nevertheless, organic ligands exert a strong influence on metal bioavailability and toxicity. For example, amendment experiments using commercially available siderophores, organic compounds synthesized by microbes to facilitate iron uptake, show these ligands can both facilitate or impede iron uptake depending on the siderophore composition and available uptake pathways. Over the past few years we have developed analytical techniques using high pressure liquid chromatography interfaced with inductively coupled plasma and electrospray ionization mass spectrometry to identify and quantify trace metal organic complexes in laboratory cultures of marine microbes and in seawater. We found siderophores to be widely distributed in the ocean, particularly in regions characterized by low iron concentrations. We also find chemically distinct complexes of copper, zinc, colbalt and nickel that we have yet to fully characterize. We will discuss some of our recent work on trace metal organic speciation in seawater and laboratory cultures, and outline future efforts to better understand the microbial cycling of trace metal organic complexes in the sea.

μ2-Dependent endocytosis of N-cadherin is regulated by β-catenin to facilitate neurite outgrowth.

PubMed

Chen, Yi-Ting; Tai, Chin-Yin

2017-05-01

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the μ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, β-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of β-catenin facilitates μ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through β-catenin-regulated μ2 binding to modulate neurite outgrowth. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Knockdown Brm and Baf170, components of chromatin remodeling complex, facilitates reprogramming of somatic cells

USDA-ARS?s Scientific Manuscript database

The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells (ESCs) esBAF contains Brg1 and Baf...

Malleable machines in transcription regulation: the mediator complex.

PubMed

Tóth-Petróczy, Agnes; Oldfield, Christopher J; Simon, István; Takagi, Yuichiro; Dunker, A Keith; Uversky, Vladimir N; Fuxreiter, Monika

2008-12-01

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the

Complementarity of stability patches at the interfaces of protein complexes: Implication for the structural organization of energetic hot spots.

PubMed

Kuttner, Yosef Y; Engel, Stanislav

2018-02-01

A rational design of protein complexes with defined functionalities and of drugs aimed at disrupting protein-protein interactions requires fundamental understanding of the mechanisms underlying the formation of specific protein complexes. Efforts to develop efficient small-molecule or protein-based binders often exploit energetic hot spots on protein surfaces, namely, the interfacial residues that provide most of the binding free energy in the complex. The molecular basis underlying the unusually high energy contribution of the hot spots remains obscure, and its elucidation would facilitate the design of interface-targeted drugs. To study the nature of the energetic hot spots, we analyzed the backbone dynamic properties of contact surfaces in several protein complexes. We demonstrate that, in most complexes, the backbone dynamic landscapes of interacting surfaces form complementary "stability patches," in which static areas from the opposing surfaces superimpose, and that these areas are predominantly located near the geometric center of the interface. We propose that a diminished enthalpy-entropy compensation effect augments the degree to which residues positioned within the complementary stability patches contribute to complex affinity, thereby giving rise to the energetic hot spots. These findings offer new insights into the nature of energetic hot spots and the role that backbone dynamics play in facilitating intermolecular recognition. Mapping the interfacial stability patches may provide guidance for protein engineering approaches aimed at improving the stability of protein complexes and could facilitate the design of ligands that target complex interfaces. © 2017 Wiley Periodicals, Inc.

Language production is facilitated by semantic richness but inhibited by semantic density: Evidence from picture naming.

PubMed

Rabovsky, Milena; Schad, Daniel J; Abdel Rahman, Rasha

2016-01-01

Communicating meaningful messages is the ultimate goal of language production. Yet, verbal messages can differ widely in the complexity and richness of their semantic content, and such differences should strongly modulate conceptual and lexical encoding processes during speech planning. However, despite the crucial role of semantic content in language production, the influence of this variability is currently unclear. Here, we investigate influences of the number of associated semantic features and intercorrelational feature density on language production during picture naming. While the number of semantic features facilitated naming, intercorrelational feature density inhibited naming. Both effects follow naturally from the assumption of conceptual facilitation and simultaneous lexical competition. They are difficult to accommodate with language production theories dismissing lexical competition. Copyright © 2015 Elsevier B.V. All rights reserved.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

NASA Astrophysics Data System (ADS)

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

PubMed

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

Patterns of task and network actions performed by navigators to facilitate cancer care.

PubMed

Clark, Jack A; Parker, Victoria A; Battaglia, Tracy A; Freund, Karen M

2014-01-01

Patient navigation is a widely implemented intervention to facilitate access to care and reduce disparities in cancer care, but the activities of navigators are not well characterized. The aim of this study is to describe what patient navigators actually do and explore patterns of activity that clarify the roles they perform in facilitating cancer care. We conducted field observations of nine patient navigation programs operating in diverse health settings of the national patient navigation research program, including 34 patient navigators, each observed an average of four times. Trained observers used a structured observation protocol to code as they recorded navigator actions and write qualitative field notes capturing all activities in 15-minute intervals during observations ranging from 2 to 7 hours; yielding a total of 133 observations. Rates of coded activity were analyzed using numerical cluster analysis of identified patterns, informed by qualitative analysis of field notes. Six distinct patterns of navigator activity were identified, which differed most relative to how much time navigators spent directly interacting with patients and how much time they spent dealing with medical records and documentation tasks. Navigator actions reveal a complex set of roles in which navigators both provide the direct help to patients denoted by their title and also carry out a variety of actions that function to keep the health system operating smoothly. Working to navigate patients through complex health services entails working to repair the persistent challenges of health services that can render them inhospitable to patients. The organizations that deploy navigators might learn from navigators' efforts and explore alternative approaches, structures, or systems of care in addressing both the barriers patients face and the complex solutions navigators create in helping patients.

Integrating Human Factors Engineering and Information Processing Approaches to Facilitate Evaluations in Criminal Justice Technology Research.

PubMed

Salvemini, Anthony V; Piza, Eric L; Carter, Jeremy G; Grommon, Eric L; Merritt, Nancy

2015-06-01

Evaluations are routinely conducted by government agencies and research organizations to assess the effectiveness of technology in criminal justice. Interdisciplinary research methods are salient to this effort. Technology evaluations are faced with a number of challenges including (1) the need to facilitate effective communication between social science researchers, technology specialists, and practitioners, (2) the need to better understand procedural and contextual aspects of a given technology, and (3) the need to generate findings that can be readily used for decision making and policy recommendations. Process and outcome evaluations of technology can be enhanced by integrating concepts from human factors engineering and information processing. This systemic approach, which focuses on the interaction between humans, technology, and information, enables researchers to better assess how a given technology is used in practice. Examples are drawn from complex technologies currently deployed within the criminal justice system where traditional evaluations have primarily focused on outcome metrics. Although this evidence-based approach has significant value, it is vulnerable to fully account for human and structural complexities that compose technology operations. Guiding principles for technology evaluations are described for identifying and defining key study metrics, facilitating communication within an interdisciplinary research team, and for understanding the interaction between users, technology, and information. The approach posited here can also enable researchers to better assess factors that may facilitate or degrade the operational impact of the technology and answer fundamental questions concerning whether the technology works as intended, at what level, and cost. © The Author(s) 2015.

Isolation of integrin-based adhesion complexes.

PubMed

Jones, Matthew C; Humphries, Jonathan D; Byron, Adam; Millon-Frémillon, Angélique; Robertson, Joseph; Paul, Nikki R; Ng, Daniel H J; Askari, Janet A; Humphries, Martin J

2015-03-02

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry. Copyright © 2015 John Wiley & Sons, Inc.

TANGO1 recruits ERGIC membranes to the endoplasmic reticulum for procollagen export

PubMed Central

Santos, António JM; Raote, Ishier; Scarpa, Margherita; Brouwers, Nathalie; Malhotra, Vivek

2015-01-01

Previously we showed that membrane fusion is required for TANGO1-dependent export of procollagen VII from the endoplasmic reticulum (ER) (Nogueira, et al., 2014). Along with the t-SNARE Syntaxin 18, we now reveal the complete complement of SNAREs required in this process, t-SNAREs BNIP1 and USE1, and v-SNARE YKT6. TANGO1 recruits YKT6-containing ER Golgi Intermediate Compartment (ERGIC) membranes to procollagen VII-enriched patches on the ER. Moreover residues 1214-1396, that include the first coiled coil of TANGO1, specifically recruit ERGIC membranes even when targeted to mitochondria. TANGO1 is thus pivotal in concentrating procollagen VII in the lumen and recruiting ERGIC membranes on the cytoplasmic surface of the ER. Our data reveal that growth of a mega transport carrier for collagen export from the ER is not by acquisition of a larger patch of ER membrane, but instead by addition of ERGIC membranes to procollagen-enriched domains of the ER by a TANGO1-mediated process. DOI: http://dx.doi.org/10.7554/eLife.10982.001 PMID:26568311

Technologies and Techniques for Supporting Facilitated Video

ERIC Educational Resources Information Center

Linnell, Natalie

2011-01-01

Worldwide, demand for education of all kinds is increasing beyond the capacity to provide it. One approach that shows potential for addressing this demand is facilitated video. In facilitated video, an educator is recorded teaching, and that video is sent to a remote site where it is shown to students by a facilitator who creates interaction…

Lipid and cationic polymer based transduction of botulinum holotoxin, or toxin protease alone, extends the target cell range and improves the efficiency of intoxication.

PubMed

Kuo, Chueh-Ling; Oyler, George; Shoemaker, Charles B

2010-01-01

Botulinum neurotoxin (BoNT) heavy chain (Hc) facilitates receptor-mediated endocytosis into neuronal cells and transport of the light chain (Lc) protease to the cytosol where neurotransmission is inhibited as a result of SNARE protein cleavage. Here we show that the role of BoNT Hc in cell intoxication can be replaced by commercial lipid-based and polycationic polymer DNA transfection reagents. BoNT "transduction" by these reagents permits efficient intoxication of neuronal cells as well as some non-neuronal cell lines normally refractory to BoNT. Surprisingly, the reagents facilitate delivery of recombinant BoNT Lc protease to the cytosol of both neuronal and non-neuronal cells in the absence of BoNT Hc, and with sensitivities approaching that of BoNT holotoxin. Transduction of BoNT, as with natural intoxication, is inhibited by bafilomycin A1, methylamine and ammonium chloride indicating that both pathways require endosome acidification. DNA transfection reagents facilitate intoxication by holotoxins, or isolated Lc proteases, of all three BoNT serotypes tested (A, B, E). These results suggest that lipid and cationic polymer transfection reagents facilitate cytosolic delivery of BoNT holotoxins and isolated Lc proteases by an endosomal uptake pathway.

Lipid and cationic polymer based transduction of botulinum holotoxin, or toxin protease alone, extends the target cell range and improves the efficiency of intoxication

PubMed Central

Kuo, Chueh-Ling; Oyler, George; Shoemaker, Charles B.

2009-01-01

Botulinum neurotoxin (BoNT) heavy chain (Hc) facilitates receptor-mediated endocytosis into neuronal cells and transport of the light chain (Lc) protease to the cytosol where neurotransmission is inhibited as a result of SNARE protein cleavage. Here we show that the role of BoNT Hc in cell intoxication can be replaced by commercial lipid-based and polycationic polymer DNA transfection reagents. BoNT “transduction” by these reagents permits efficient intoxication of neuronal cells as well as some non-neuronal cell lines normally refractory to BoNT. Surprisingly, the reagents facilitate delivery of recombinant BoNT Lc protease to the cytosol of both neuronal and non-neuronal cells in the absence of BoNT Hc, and with sensitivities approaching that of BoNT holotoxin. Transduction of BoNT, as with natural intoxication, is inhibited by bafilomycin A1, methylamine and ammonium chloride indicating that both pathways require endosome acidification. DNA transfection reagents facilitate intoxication by holotoxins, or isolated Lc proteases, of all three BoNT serotypes tested (A, B, E). These results suggest that lipid and cationic polymer transfection reagents facilitate cytosolic delivery of BoNT holotoxins and isolated Lc proteases by an endosomal uptake pathway. PMID:19852976

Transducer model produces facilitation from opposite-sign flanks

NASA Technical Reports Server (NTRS)

Solomon, J. A.; Watson, A. B.; Morgan, M. J.

1999-01-01

Small spots, lines and Gabor patterns can be easier to detect when they are superimposed upon similar spots, lines and Gabor patterns. Traditionally, such facilitation has been understood to be a consequence of nonlinear contrast transduction. Facilitation has also been reported to arise from non-overlapping patterns with opposite sign. We point out that this result does not preclude the traditional explanation for superimposed targets. Moreover, we find that facilitation from opposite-sign flanks is weaker than facilitation from same-sign flanks. Simulations with a transducer model produce opposite-sign facilitation.

Body shape helps legged robots climb and turn in complex 3-D terrains

NASA Astrophysics Data System (ADS)

Han, Yuanfeng; Wang, Zheliang; Li, Chen

Analogous to streamlined shapes that reduce drag in fluids, insects' ellipsoid-like rounded body shapes were recently discovered to be ``terradynamically streamlined'' and enhance locomotion in cluttered terrain by facilitating body rolling. Here, we hypothesize that there exist more terradynamic shapes that facilitate other modes of locomotion like climbing and turning in complex 3-D terrains by facilitating body pitching and yawing. To test our hypothesis, we modified the body shape of a legged robot by adding an elliptical and a rectangular shell and tested how it negotiated with circular and square vertical pillars. With a rectangular shell the robot always pitched against square pillars in an attempt to climb, whereas with an elliptical shell it always yawed and turned away from circular pillars given a small initial lateral displacement. Square / circular pillars facilitated pitching / yawing, respectively. To begin to reveal the contact physics, we developed a locomotion energy landscape model. Our model revealed that potential energy barriers to transition from pitching to yawing are high for angular locomotor and obstacle shapes (rectangular / square) but vanish for rounded shapes (elliptical / circular). Our study supports the plausibility of locomotion energy landscapes for understanding the rich locomotor transitions in complex 3-D terrains.

What can we learn from facilitator and student perceptions of facilitation skills and roles in the first year of a problem-based learning curriculum?

PubMed Central

McLean, Michelle

2003-01-01

Background The small group tutorial is a cornerstone of problem-based learning. By implication, the role of the facilitator is of pivotal importance. The present investigation canvassed perceptions of facilitators with differing levels of experience regarding their roles and duties in the tutorial. Methods In January 2002, one year after problem-based learning implementation at the Nelson R. Mandela School of Medicine, facilitators with the following experience were canvassed: trained and about to facilitate, facilitated once only and facilitated more than one six-week theme. Student comments regarding facilitator skills were obtained from a 2001 course survey. Results While facilitators generally agreed that the three-day training workshop provided sufficient insight into the facilitation process, they become more comfortable with increasing experience. Many facilitators experienced difficulty not providing content expertise. Again, this improved with increasing experience. Most facilitators saw students as colleagues. They agreed that they should be role models, but were less enthusiastic about being mentors. Students were critical of facilitators who were not up to date with curriculum implementation or who appeared disinterested. While facilitator responses suggest that there was considerable intrinsic motivation, this might in fact not be the case. Conclusions Even if they had facilitated on all six themes, facilitators could still be considered as novices. Faculty support is therefore critical for the first few years of problem-based learning, particularly for those who had facilitated once only. Since student and facilitator expectations in the small group tutorial may differ, roles and duties of facilitators must be explicit for both parties from the outset. PMID:14585108

What can we learn from facilitator and student perceptions of facilitation skills and roles in the first year of a problem-based learning curriculum?

PubMed

McLean, Michelle

2003-10-30

The small group tutorial is a cornerstone of problem-based learning. By implication, the role of the facilitator is of pivotal importance. The present investigation canvassed perceptions of facilitators with differing levels of experience regarding their roles and duties in the tutorial. In January 2002, one year after problem-based learning implementation at the Nelson R. Mandela School of Medicine, facilitators with the following experience were canvassed: trained and about to facilitate, facilitated once only and facilitated more than one six-week theme. Student comments regarding facilitator skills were obtained from a 2001 course survey. While facilitators generally agreed that the three-day training workshop provided sufficient insight into the facilitation process, they become more comfortable with increasing experience. Many facilitators experienced difficulty not providing content expertise. Again, this improved with increasing experience. Most facilitators saw students as colleagues. They agreed that they should be role models, but were less enthusiastic about being mentors. Students were critical of facilitators who were not up to date with curriculum implementation or who appeared disinterested. While facilitator responses suggest that there was considerable intrinsic motivation, this might in fact not be the case. Even if they had facilitated on all six themes, facilitators could still be considered as novices. Faculty support is therefore critical for the first few years of problem-based learning, particularly for those who had facilitated once only. Since student and facilitator expectations in the small group tutorial may differ, roles and duties of facilitators must be explicit for both parties from the outset.

Facilitator training program: The Université Laval Interprofessional Initiative.

PubMed

Milot, Élise; Museux, Anne-Claire; Careau, Emmanuelle

2017-03-01

A facilitator training program (FTP) for interprofessional learning (IPL) facilitators has been developed at Université Laval. This article describes the impacts of this program as perceived by the 22 IPL facilitators involved and outlines recommendations. Two qualitative data collection strategies were used to document the facilitators' pedagogical needs and views of the program's impacts. Results suggest that the FTP's pedagogical approach was effective. The IPL facilitators became more aware of their challenges and identified concrete strategies to use. Training initiatives should equip IPL facilitators to cope with uncertainty, create a climate supporting active learning, and facilitate positive interactions between students.

49 CFR 38.2 - Equivalent facilitation.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 1 2010-10-01 2010-10-01 false Equivalent facilitation. 38.2 Section 38.2 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES General § 38.2 Equivalent facilitation. Departures from particular...

Selective isolation of gold facilitated by second-sphere coordination with α-cyclodextrin

PubMed Central

Liu, Zhichang; Frasconi, Marco; Lei, Juying; Brown, Zachary J.; Zhu, Zhixue; Cao, Dennis; Iehl, Julien; Liu, Guoliang; Fahrenbach, Albert C.; Botros, Youssry Y.; Farha, Omar K.; Hupp, Joseph T.; Mirkin, Chad A.; Fraser Stoddart, J.

2013-01-01

Gold recovery using environmentally benign chemistry is imperative from an environmental perspective. Here we report the spontaneous assembly of a one-dimensional supramolecular complex with an extended {[K(OH2)6][AuBr4](α-cyclodextrin)2}n chain superstructure formed during the rapid co-precipitation of α-cyclodextrin and KAuBr4 in water. This phase change is selective for this gold salt, even in the presence of other square-planar palladium and platinum complexes. From single-crystal X-ray analyses of six inclusion complexes between α-, β- and γ-cyclodextrins with KAuBr4 and KAuCl4, we hypothesize that a perfect match in molecular recognition between α-cyclodextrin and [AuBr4]− leads to a near-axial orientation of the ion with respect to the α-cyclodextrin channel, which facilitates a highly specific second-sphere coordination involving [AuBr4]− and [K(OH2)6]+ and drives the co-precipitation of the 1:2 adduct. This discovery heralds a green host–guest procedure for gold recovery from gold-bearing raw materials making use of α-cyclodextrin—an inexpensive and environmentally benign carbohydrate. PMID:23673640

An exploration of tutors' experiences of facilitating problem-based learning. Part 2--implications for the facilitation of problem based learning.

PubMed

Haith-Cooper, Melanie

2003-01-01

This paper is the second of two parts exploring a study that was undertaken to investigate the role of the tutor in facilitating problem-based learning (PBL). The first part focussed on the methodological underpinnings of the study. This paper aims to focus on the findings of the study and their implications for the facilitation of PBL. Six essential themes emerged from the findings that described the facilitation role. The tutors believed that their facilitation role was essentially structured around the decision of when to intervene and how to intervene in the PBL process. Modelling and non-verbal communication were seen as essential strategies for the facilitator. Underpinning these decisions was the need to trust in the philosophy of PBL. However, within many of the themes, there was a divergence of opinion as to how the role should actually be undertaken. Despite this, these findings have implications for the future role of PBL facilitators in Health Professional Education.

Familiarity facilitates feature-based face processing.

PubMed

Visconti di Oleggio Castello, Matteo; Wheeler, Kelsey G; Cipolli, Carlo; Gobbini, M Ida

2017-01-01

Recognition of personally familiar faces is remarkably efficient, effortless and robust. We asked if feature-based face processing facilitates detection of familiar faces by testing the effect of face inversion on a visual search task for familiar and unfamiliar faces. Because face inversion disrupts configural and holistic face processing, we hypothesized that inversion would diminish the familiarity advantage to the extent that it is mediated by such processing. Subjects detected personally familiar and stranger target faces in arrays of two, four, or six face images. Subjects showed significant facilitation of personally familiar face detection for both upright and inverted faces. The effect of familiarity on target absent trials, which involved only rejection of unfamiliar face distractors, suggests that familiarity facilitates rejection of unfamiliar distractors as well as detection of familiar targets. The preserved familiarity effect for inverted faces suggests that facilitation of face detection afforded by familiarity reflects mostly feature-based processes.

Facilitating LOS Debriefings: A Training Manual

NASA Technical Reports Server (NTRS)

McDonnell, Lori K.; Jobe, Kimberly K.; Dismukes, R. Key

1997-01-01

This manual is a practical guide to help airline instructors effectively facilitate debriefings of Line Oriented Simulations (LOS). It is based on a recently completed study of Line Oriented Flight Training (LOFT) debriefings at several U.S. airlines. This manual presents specific facilitation tools instructors can use to achieve debriefing objectives. The approach of the manual is to be flexible so it can be tailored to the individual needs of each airline. Part One clarifies the purpose and objectives of facilitation in the LOS setting. Part Two provides recommendations for clarifying roles and expectations and presents a model for organizing discussion. Part Tree suggests techniques for eliciting active crew participation and in-depth analysis and evaluation. Finally, in Part Four, these techniques are organized according to the facilitation model. Examples of how to effectively use the techniques are provided throughout, including strategies to try when the debriefing objectives are not being fully achieved.

Understanding Implementation of Complex Interventions in Primary Care Teams.

PubMed

Luig, Thea; Asselin, Jodie; Sharma, Arya M; Campbell-Scherer, Denise L

2018-01-01

The implementation of interventions to support practice change in primary care settings is complex. Pragmatic strategies, grounded in empiric data, are needed to navigate real-world challenges and unanticipated interactions with context that can impact implementation and outcomes. This article uses the example of the "5As Team" randomized control trial to explore implementation strategies to promote knowledge transfer, capacity building, and practice integration, and their interaction within the context of an interdisciplinary primary care team. We performed a qualitative evaluation of the implementation process of the 5As Team intervention study, a randomized control trial of a complex intervention in primary care. We conducted thematic analysis of field notes of intervention sessions, log books of the practice facilitation team members, and semistructured interviews with 29 interdisciplinary clinician participants. We used and further developed the Interactive Systems Framework for dissemination and implementation to interpret and structure findings. Three themes emerged that illuminate interactions between implementation processes, context, and outcomes: (1) facilitating team communication supported collective and individual sense-making and adoption of the innovation, (2) iterative evaluation of the implementation process and real-time feedback-driven adaptions of the intervention proved crucial for sustainable, context-appropriate intervention impact, (3) stakeholder engagement led to both knowledge exchange that contributes to local problem solving and to shaping a clinical context that is supportive to practice change. Our findings contribute pragmatic strategies that can help practitioners and researchers to navigate interactions between context, intervention, and implementation factors to increase implementation success. We further developed an implementation framework that includes sustained engagement with stakeholders, facilitation of team sense

TP53INP2/DOR, a mediator of cell autophagy, promotes rDNA transcription via facilitating the assembly of the POLR1/RNA polymerase I preinitiation complex at rDNA promoters.

PubMed

Xu, Yinfeng; Wan, Wei; Shou, Xin; Huang, Rui; You, Zhiyuan; Shou, Yanhong; Wang, Lingling; Zhou, Tianhua; Liu, Wei

2016-07-02

Cells control their metabolism through modulating the anabolic and catabolic pathways. TP53INP2/DOR (tumor protein p53 inducible nuclear protein 2), participates in cell catabolism by serving as a promoter of autophagy. Here we uncover a novel function of TP53INP2 in protein synthesis, a major biosynthetic and energy-consuming anabolic process. TP53INP2 localizes to the nucleolus through its nucleolar localization signal (NoLS) located at the C-terminal domain. Chromatin immunoprecipitation (ChIP) assays detected an association of TP53INP2 with the ribosomal DNA (rDNA), when exclusion of TP53INP2 from the nucleolus repressed rDNA promoter activity and the production of ribosomal RNA (rRNA) and proteins. The removal of TP53INP2 also impaired the association of the POLR1/RNA polymerase I preinitiation complex (PIC) with rDNA. Further, TP53INP2 interacts directly with POLR1 PIC, and is required for the assembly of the complex. These data indicate that TP53INP2 promotes ribosome biogenesis through facilitating rRNA synthesis at the nucleolus, suggesting a dual role of TP53INP2 in cell metabolism, assisting anabolism on the nucleolus, and stimulating catabolism off the nucleolus.

Task Complexity, Learning Opportunities, and Korean EFL Learners' Question Development

ERIC Educational Resources Information Center

Kim, YouJin

2012-01-01

Building on the cognitive and interactive perspectives of task research, the cognition hypothesis states that increasing task complexity promotes greater interaction and feedback and thus facilitates second language (L2) development (Robinson, 2001b, 2007a). To date, very little research has explored this claim during learner-learner interactions…

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

PubMed

Eyboulet, Fanny; Cibot, Camille; Eychenne, Thomas; Neil, Helen; Alibert, Olivier; Werner, Michel; Soutourina, Julie

2013-12-01

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

Student Facilitators' Habits of Mind and Their Influences on Higher-Level Knowledge Construction Occurrences in Online Discussions: A Case Study

ERIC Educational Resources Information Center

Hew, Khe Foon; Cheung, Wing Sum

2011-01-01

Previous research studies on factors influencing student higher-level knowledge construction in asynchronous online discussions have largely focused on the instructors' role, student learning style, and the complexity of the discussion task. This study explores the issue from a different angle--that of student facilitators' habits of mind.…

Facilitators and Barriers to Adopting Robotic-Assisted Surgery: Contextualizing the Unified Theory of Acceptance and Use of Technology

PubMed Central

BenMessaoud, Christine; Kharrazi, Hadi; MacDorman, Karl F.

2011-01-01

Robotic-assisted surgical techniques are not yet well established among surgeon practice groups beyond a few surgical subspecialties. To help identify the facilitators and barriers to their adoption, this belief-elicitation study contextualized and supplemented constructs of the unified theory of acceptance and use of technology (UTAUT) in robotic-assisted surgery. Semi-structured individual interviews were conducted with 21 surgeons comprising two groups: users and nonusers. The main facilitators to adoption were Perceived Usefulness and Facilitating Conditions among both users and nonusers, followed by Attitude Toward Using Technology among users and Extrinsic Motivation among nonusers. The three main barriers to adoption for both users and nonusers were Perceived Ease of Use and Complexity, Perceived Usefulness, and Perceived Behavioral Control. This study's findings can assist surgeons, hospital and medical school administrators, and other policy makers on the proper adoption of robotic-assisted surgery and can guide future research on the development of theories and framing of hypotheses. PMID:21283719

Parallel workflow tools to facilitate human brain MRI post-processing

PubMed Central

Cui, Zaixu; Zhao, Chenxi; Gong, Gaolang

2015-01-01

Multi-modal magnetic resonance imaging (MRI) techniques are widely applied in human brain studies. To obtain specific brain measures of interest from MRI datasets, a number of complex image post-processing steps are typically required. Parallel workflow tools have recently been developed, concatenating individual processing steps and enabling fully automated processing of raw MRI data to obtain the final results. These workflow tools are also designed to make optimal use of available computational resources and to support the parallel processing of different subjects or of independent processing steps for a single subject. Automated, parallel MRI post-processing tools can greatly facilitate relevant brain investigations and are being increasingly applied. In this review, we briefly summarize these parallel workflow tools and discuss relevant issues. PMID:26029043

Barriers and Facilitators Affecting Patient Portal Implementation from an Organizational Perspective: Qualitative Study.

PubMed

Kooij, Laura; Groen, Wim G; van Harten, Wim H

2018-05-11

(eg, strict regulations). Both similarities and differences were found between stakeholder groups and hospital types. For example, managers and information technology employees mainly considered guaranteeing privacy and security as a predominant barrier. Financial difficulties were particularly mentioned by medical professionals and managers. Patient portal implementation is a complex process and is not only a technical process but also affects the organization and its staff. Barriers and facilitators occurred at various levels and differed among hospital types (eg, lack of accessibility) and stakeholder groups (eg, sufficient resources) in terms of several factors. Our findings underscore the importance of involving multiple stakeholders in portal implementations. We identified a set of barriers and facilitators that are likely to be useful in making strategic and efficient implementation plans. ©Laura Kooij, Wim G Groen, Wim H van Harten. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 11.05.2018.

Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

PubMed Central

Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian

2015-01-01

ABSTRACT The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCE Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural

Toward Facilitative Mentoring and Catalytic Interventions

ERIC Educational Resources Information Center

Smith, Melissa K.; Lewis, Marilyn

2015-01-01

In TESOL teacher mentoring, giving advice can be conceptualized as a continuum, ranging from directive to facilitative feedback. The goal, over time, is to lead toward the facilitative end of the continuum and specifically to catalytic interventions that encourage self-reflection and autonomous learning. This study begins by examining research on…

Amyloplast movement and gravityperception in Arabidopsis endoderm

NASA Astrophysics Data System (ADS)

Tasaka, M.; Saito, T.; Morita, M. T.

Gravitropism of higher plant is a growth response regulating the orientation of organs elongation, which includes four sequential steps, the perception of gravistimulus, transduction of the physical stimulus to chemical signal, transmission of the signal, and differential cell elongation depending on the signal. To elucidate the molecular mechanism of these steps, we have isolated a number of Arabidopsis mutants with abnormal shoot gravitropic response. zig (zigzag)/sgr4(shoot gravitropism 4) shows little gravitropism in their shoots. Besides, their inflorescence stems elongate in a zigzag-fashion to bend at each node. ZIG encodes a SNARE, AtVTI11. sgr3 with reduced gravitropic response in inflorescence stems had a missense mutation in other SNARE, AtVAM3. These two SNAREs make a complex in the shoot endoderm cells that are gravity-sensing cells, suggesting that the vesicle transport from trans-Golgi network (TGN) to prevacuolar compartment (PVC) and/or vacuole is involved in gravitropism. Abnormal vesicular/vacuolar structures were observed in several tissues of both mutants. Moreover, SGR2 encodes phospholipase A1-like protein that resides in the vacuolar membrane. Endodermis-specific expression of these genes could complement gravitropism in each mutant. In addition, amyloplasts thought to be statoliths localized abnormally in their endoderm cells. These results strongly suggest that formation and function of vacuole in the endoderm cells are important for amyloplasts sedimentation, which is involved in the early process of shoot gravitropism. To reveal this, we constructed vertical stage microscope system to visualize the behavior of amyloplasts and vacuolar membrane in living endodermal cells. We hope to discuss the mechanism of gravity perception after showing their movements.

Facilitators for practice change in Spanish community pharmacy.

PubMed

Gastelurrutia, Miguel A; Benrimoj, S I Charlie; Castrillon, Carla C; de Amezua, María J Casado; Fernandez-Llimos, Fernando; Faus, Maria J

2009-02-01

To identify and prioritise facilitators for practice change in Spanish community pharmacy. Spanish community pharmacies. Qualitative study. Thirty-three semi-structured interviews were conducted with community pharmacists (n = 15) and pharmacy strategists (n = 18), and the results were examined using the content analysis method. In addition, two nominal groups (seven community pharmacists and seven strategists) were formed to identify and prioritise facilitators. Results of both techniques were then triangulated. Facilitators for practice change. Twelve facilitators were identified and grouped into four domains (D1: Pharmacist; D2: Pharmacy as an organisation; D3: Pharmaceutical profession; D4: Miscellaneous). Facilitators identified in D1 include: the need for more clinical education at both pre- and post-graduate levels; the need for clearer and unequivocal messages from professional leaders about the future of the professional practice; and the need for a change in pharmacists' attitudes. Facilitators in D2 are: the need to change the reimbursement system to accommodate cognitive service delivery as well as dispensing; and the need to change the front office of pharmacies. Facilitators identified in D3 are: the need for the Spanish National Professional Association to take a leadership role in the implementation of cognitive services; the need to reduce administrative workload; and the need for universities to reduce the gap between education and research. Other facilitators identified in this study include: the need to increase patients' demand for cognitive services at pharmacies; the need to improve pharmacist-physician relationships; the need for support from health care authorities; and the need for improved marketing of cognitive services and their benefits to society, including physicians and health care authorities. Twelve facilitators were identified. Strategists considered clinical education and pharmacists' attitude as the most important, and

Producing Gestures Facilitates Route Learning

PubMed Central

So, Wing Chee; Ching, Terence Han-Wei; Lim, Phoebe Elizabeth; Cheng, Xiaoqin; Ip, Kit Yee

2014-01-01

The present study investigates whether producing gestures would facilitate route learning in a navigation task and whether its facilitation effect is comparable to that of hand movements that leave physical visible traces. In two experiments, we focused on gestures produced without accompanying speech, i.e., co-thought gestures (e.g., an index finger traces the spatial sequence of a route in the air). Adult participants were asked to study routes shown in four diagrams, one at a time. Participants reproduced the routes (verbally in Experiment 1 and non-verbally in Experiment 2) without rehearsal or after rehearsal by mentally simulating the route, by drawing it, or by gesturing (either in the air or on paper). Participants who moved their hands (either in the form of gestures or drawing) recalled better than those who mentally simulated the routes and those who did not rehearse, suggesting that hand movements produced during rehearsal facilitate route learning. Interestingly, participants who gestured the routes in the air or on paper recalled better than those who drew them on paper in both experiments, suggesting that the facilitation effect of co-thought gesture holds for both verbal and nonverbal recall modalities. It is possibly because, co-thought gesture, as a kind of representational action, consolidates spatial sequence better than drawing and thus exerting more powerful influence on spatial representation. PMID:25426624

Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

PubMed

Mizuno, Tadahaya; Hayashi, Hisamitsu; Kusuhara, Hiroyuki

2015-06-01

By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRβ inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRβ interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRβ and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRβ from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system. © 2015 American Heart Association, Inc.

Facilitating classroom based interprofessional learning: a grounded theory study of university educators' perceptions of their role adequacy as facilitators.

PubMed

Derbyshire, Julie A; Machin, Alison I; Crozier, Suzanne

2015-01-01

The provision of inter professional learning (IPL) within undergraduate programmes is now well established within many Higher Education Institutions (HEIs). IPL aims to better equip nurses and other health professionals with effective collaborative working skills and knowledge to improve the quality of patient care. Although there is still ambiguity in relation to the optimum timing and method for delivering IPL, effective facilitation is seen as essential. This paper reports on a grounded theory study of university educators' perceptions of the knowledge and skills needed for their role adequacy as IPL facilitators. Data was collected using semi structured interviews with nine participants who were theoretically sampled from a range of professional backgrounds, with varied experiences of education and involvement in facilitating IPL. Constant comparative analysis was used to generate four data categories: creating and sustaining an IPL group culture through transformational IPL leadership (core category), readiness for IPL facilitation, drawing on past interprofessional learning and working experiences and role modelling an interprofessional approach. The grounded theory generated from this study, although propositional, suggests that role adequacy for IPL facilitation is dependent on facilitator engagement in a process of 'transformational interprofessional learning leadership' to create and sustain a group culture. Copyright © 2014 Elsevier Ltd. All rights reserved.

Facilitating Cognitive Development.

ERIC Educational Resources Information Center

Schwebel, Milton

1985-01-01

Human cognition research is shifting away from the importance of IQ and is emphasizing the stimulation and acceleration of a child's mental development. The emerging field of instructional psychology is trying to facilitate cognitive development. Current experimental programs--a university-school project in Belgium and a family project in…

Physically facilitating drug-delivery systems

PubMed Central

Rodriguez-Devora, Jorge I; Ambure, Sunny; Shi, Zhi-Dong; Yuan, Yuyu; Sun, Wei; Xu, Tao

2012-01-01

Facilitated/modulated drug-delivery systems have emerged as a possible solution for delivery of drugs of interest to pre-allocated sites at predetermined doses for predefined periods of time. Over the past decade, the use of different physical methods and mechanisms to mediate drug release and delivery has grown significantly. This emerging area of research has important implications for development of new therapeutic drugs for efficient treatments. This review aims to introduce and describe different modalities of physically facilitating drug-delivery systems that are currently in use for cancer and other diseases therapy. In particular, delivery methods based on ultrasound, electrical, magnetic and photo modulations are highlighted. Current uses and areas of improvement for these different physically facilitating drug-delivery systems are discussed. Furthermore, the main advantages and drawbacks of these technologies reviewed are compared. The review ends with a speculative viewpoint of how research is expected to evolve in the upcoming years. PMID:22485192

Kinetic Model Facilitates Analysis of Fibrin Generation and Its Modulation by Clotting Factors: Implications for Hemostasis-Enhancing Therapies

DTIC Science & Technology

2014-01-01

facilitates analysis of fibrin generation and its modulation by clotting factors : implications for hemostasis-enhancing therapies† Alexander Y...investigate the ability of fibrinogen and a recently proposed prothrombin complex concentrate composition, PCC-AT (a combination of the clotting factors II...kinetics. Moreover, the model qualitatively predicted the impact of tissue factor and tPA/tenecteplase level variations on the fibrin output. In the

SNAP-25 IN NEUROPSYCHIATRIC DISORDERS

PubMed Central

Corradini, Irene; Verderio, Claudia; Sala, Mariaelvina; Wilson, Michael C.; Matteoli, Michela

2009-01-01

SNAP-25 is plasma membrane protein which, together with syntaxin and the synaptic vesicle protein VAMP/synaptobrevin, forms the SNARE docking complex for regulated exocytosis. SNAP-25 also modulates different voltage-gated calcium channels, representing therefore a multifunctional protein that plays essential roles in neurotransmitter release at different steps. Recent genetic studies of human populations and of some mouse models implicate that alterations in SNAP-25 gene structure, expression and/or function may contribute directly to these distinct neuropsychiatric and neurological disorders. PMID:19161380

Making sense of complexity in context and implementation: the Context and Implementation of Complex Interventions (CICI) framework.

PubMed

Pfadenhauer, Lisa M; Gerhardus, Ansgar; Mozygemba, Kati; Lysdahl, Kristin Bakke; Booth, Andrew; Hofmann, Bjørn; Wahlster, Philip; Polus, Stephanie; Burns, Jacob; Brereton, Louise; Rehfuess, Eva

2017-02-15

The effectiveness of complex interventions, as well as their success in reaching relevant populations, is critically influenced by their implementation in a given context. Current conceptual frameworks often fail to address context and implementation in an integrated way and, where addressed, they tend to focus on organisational context and are mostly concerned with specific health fields. Our objective was to develop a framework to facilitate the structured and comprehensive conceptualisation and assessment of context and implementation of complex interventions. The Context and Implementation of Complex Interventions (CICI) framework was developed in an iterative manner and underwent extensive application. An initial framework based on a scoping review was tested in rapid assessments, revealing inconsistencies with respect to the underlying concepts. Thus, pragmatic utility concept analysis was undertaken to advance the concepts of context and implementation. Based on these findings, the framework was revised and applied in several systematic reviews, one health technology assessment (HTA) and one applicability assessment of very different complex interventions. Lessons learnt from these applications and from peer review were incorporated, resulting in the CICI framework. The CICI framework comprises three dimensions-context, implementation and setting-which interact with one another and with the intervention dimension. Context comprises seven domains (i.e., geographical, epidemiological, socio-cultural, socio-economic, ethical, legal, political); implementation consists of five domains (i.e., implementation theory, process, strategies, agents and outcomes); setting refers to the specific physical location, in which the intervention is put into practise. The intervention and the way it is implemented in a given setting and context can occur on a micro, meso and macro level. Tools to operationalise the framework comprise a checklist, data extraction tools for

Emotion processing facilitates working memory performance.

PubMed

Lindström, Björn R; Bohlin, Gunilla

2011-11-01

The effect of emotional stimulus content on working memory performance has been investigated with conflicting results, as both emotion-dependent facilitation and impairments are reported in the literature. To clarify this issue, 52 adult participants performed a modified visual 2-back task with highly arousing positive stimuli (sexual scenes), highly arousing negative stimuli (violent death) and low-arousal neutral stimuli. Emotional stimulus processing was found to facilitate task performance relative to that of neutral stimuli, both in regards to response accuracy and reaction times. No emotion-dependent differences in false-alarm rates were found. These results indicate that emotional information can have a facilitating effect on working memory maintenance and processing of information.

Facilitating the Design of a Campus Leadership Team

ERIC Educational Resources Information Center

Meyers, Renee A.; Johnson, John R.

2008-01-01

This essay describes how we facilitated the design of a campus leadership team. What is particularly interesting about this consultative project is that both authors participated--one as facilitator and the other as participant. The facilitation included a needs assessment prior to the event, the use of structured controversy techniques,…

RISE-306; State Facilitator Program Evaluation, 1975-1976.

ERIC Educational Resources Information Center

Communication Technology Corp., Marlton, NJ.

As an arm of the National Diffusion Network, the Pennsylvania State Facilitator's responsibilities include informing the schools about Title III approved programs and aiding in the actual adoption of such programs by school districts. Two aspects of the facilitator's role were identified for evaluation: (1) whether the facilitator had implemented…

Broad attention to multiple individual objects may facilitate change detection with complex auditory scenes.

PubMed

Irsik, Vanessa C; Vanden Bosch der Nederlanden, Christina M; Snyder, Joel S

2016-11-01

Attention and other processing constraints limit the perception of objects in complex scenes, which has been studied extensively in the visual sense. We used a change deafness paradigm to examine how attention to particular objects helps and hurts the ability to notice changes within complex auditory scenes. In a counterbalanced design, we examined how cueing attention to particular objects affected performance in an auditory change-detection task through the use of valid or invalid cues and trials without cues (Experiment 1). We further examined how successful encoding predicted change-detection performance using an object-encoding task and we addressed whether performing the object-encoding task along with the change-detection task affected performance overall (Experiment 2). Participants had more error for invalid compared to valid and uncued trials, but this effect was reduced in Experiment 2 compared to Experiment 1. When the object-encoding task was present, listeners who completed the uncued condition first had less overall error than those who completed the cued condition first. All participants showed less change deafness when they successfully encoded change-relevant compared to irrelevant objects during valid and uncued trials. However, only participants who completed the uncued condition first also showed this effect during invalid cue trials, suggesting a broader scope of attention. These findings provide converging evidence that attention to change-relevant objects is crucial for successful detection of acoustic changes and that encouraging broad attention to multiple objects is the best way to reduce change deafness. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

COPD self-management supportive care: chaos and complexity theory.

PubMed

Cornforth, Amber

This paper uses the emergent theories of chaos and complexity to explore the self-management supportive care of chronic obstructive pulmonary disease (COPD) patients within the evolving primary care setting. It discusses the concept of self-management support, the complexity of the primary care context and consultations, smoking cessation, and the impact of acute exacerbations and action planning. The author hopes that this paper will enable the acquisition of new insight and better understanding in this clinical area, as well as support meaningful learning and facilitate more thoughtful, effective and high quality patient-centred care within the context of primary care.

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Electron Paramagnetic Resonance Spectroscopy of Vanadium (IV) Complexes and Related Species.