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1

Blum, Melissa A., Tivadar Orban, Daniel O. Beck, and Michael Kalafatis. "The Specific Contribution of Amino Acids 334 and 335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase." Blood 106, no. 11 (November 16, 2005): 1027. http://dx.doi.org/10.1182/blood.v106.11.1027.1027.

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Abstract The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.
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2

Pollok-Kopp, Beatrix, Friederike Hüttenrauch, Stephanie Rethorn, and Martin Oppermann. "Dynamics of Protein Kinase C-mediated Phosphorylation of the Complement C5a Receptor on Serine 334." Journal of Biological Chemistry 282, no. 7 (December 4, 2006): 4345–53. http://dx.doi.org/10.1074/jbc.m601317200.

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Upon agonist binding, the C5a anaphylatoxin receptor (C5aR) is rapidly phosphorylated on phosphorylation sites that are located within the C-terminal domain of the receptor. Previous studies suggested that C5aR phosphorylation proceeds in a hierarchical manner with serine 334 presenting a highly accessible priming site that controls subsequent phosphorylation at other positions. To better understand the dynamics of Ser-334 phosphorylation, we generated site-specific monoclonal antibodies that specifically react with phosphoserine 334. In differentiated U937 cells, which endogenously express C5aR, stimulation with low C5a concentrations resulted in a very rapid (t½ ∼ 20 s), albeit transient, receptor phosphorylation. Whole cell phosphorylation assays with specific inhibitors as well as in vitro phosphorylation assays with recombinant enzymes and peptide substrates revealed that phosphorylation of Ser-334 is regulated by protein kinase C-β and a calyculin A-sensitive protein phosphatase. Surprisingly, at high concentrations (>10 nm) of C5a, the protein kinase C-mediated phosphorylation of Ser-334 was essentially blocked. This could be attributed to the even faster (t½ < 5 s) binding of β-arrestin to the receptor. Analysis of C5aR Ser/Ala mutants that possess a single intact serine residue either at position 334 or at neighboring positions 327, 332, or 338 revealed functional redundancy of C-terminal phosphorylation sites since all 4 serine residues could individually support C5aR internalization and desensitization. This study is among the first to analyze in a detailed manner, using a non-mutational approach, modifications of a defined phosphorylation site in a G protein-coupled receptor and to correlate these findings with functional parameters of receptor deactivation.
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3

Li, Lehong, Xuehui Geng, Michael Yonkunas, Anjey Su, Erik Densmore, Pei Tang, and Peter Drain. "Ligand-dependent Linkage of the ATP Site to Inhibition Gate Closure in the KATP Channel." Journal of General Physiology 126, no. 3 (August 29, 2005): 285–99. http://dx.doi.org/10.1085/jgp.200509289.

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Major advances have been made on the inhibition gate and ATP site of the Kir6.2 subunit of the KATP channel, but little is known about conformational coupling between the two. ATP site mutations dramatically disrupt ATP-dependent gating without effect on ligand-independent gating, observed as interconversions between active burst and inactive interburst conformations in the absence of ATP. This suggests that linkage between site and gate is conditionally dependent on ATP occupancy. We studied all substitutions at position 334 of the ATP site in Kir6.2ΔC26 that express in Xenopus oocytes. All substitutions disrupted ATP-dependent gating by 10-fold or more. Only positive-charged arginine or lysine at 334, however, slowed ligand-independent gating from the burst, and this was in some but not all patches. Moreover, the polycationic peptide protamine reversed the slowed gating from the burst of 334R mutant channels, and speeded the slow gating from the burst of wild-type SUR1/Kir6.2 in the absence of ATP. Our results support a two-step ligand-dependent linkage mechanism for Kir6.2 channels in which ATP-occupied sites function to electrostatically dissociate COOH-terminal domains from the membrane, then as in all Kir channels, free COOH-terminal domains and inner M2 helices transit to a lower energy state for gate closure.
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4

N.K, Noorudheen, and Shafi Ahmed. "A Prospective Analytical Study on Surgical Site Infections in a Tertiary Teaching Hospital." Journal of Evidence Based Medicine and Healthcare 7, no. 32 (August 10, 2020): 1591–95. http://dx.doi.org/10.18410/jebmh/2020/334.

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5

Hovinga, JK, J. Schaller, H. Stricker, WA Wuillemin, M. Furlan, and B. Lammle. "Coagulation factor XII Locarno: the functional defect is caused by the amino acid substitution Arg 353-->Pro leading to loss of a kallikrein cleavage site." Blood 84, no. 4 (August 15, 1994): 1173–81. http://dx.doi.org/10.1182/blood.v84.4.1173.1173.

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Abstract The dysfunctional coagulation factor XII (FXII) Locarno was purified from 2 L of the proposita's plasma. Studies to identify the molecular defect responsible for the lack of amidolytic and proteolytic activity of this FXII variant were performed. Amino acid sequence analysis of peptides obtained from FXII Locarno on activation with either trypsin or plasma kallikrein and dextran sulfate showed an amino acid substitution of Arg 353 by Pro. Thereby, the kallikrein cleavage site at Arg 353-Val 354 is lost. Although trypsin-activated FXII Locarno was fully cleaved at Arg 334-Asn 335 and at Arg 343-Leu 344, neither amidolytic nor proteolytic activity was generated. We conclude that proteolytic cleavage at Arg 343 in the absence of cleavage at Arg 353 is not sufficient to expose the enzymatic active site in FXII Locarno.
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6

Hovinga, JK, J. Schaller, H. Stricker, WA Wuillemin, M. Furlan, and B. Lammle. "Coagulation factor XII Locarno: the functional defect is caused by the amino acid substitution Arg 353-->Pro leading to loss of a kallikrein cleavage site." Blood 84, no. 4 (August 15, 1994): 1173–81. http://dx.doi.org/10.1182/blood.v84.4.1173.bloodjournal8441173.

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The dysfunctional coagulation factor XII (FXII) Locarno was purified from 2 L of the proposita's plasma. Studies to identify the molecular defect responsible for the lack of amidolytic and proteolytic activity of this FXII variant were performed. Amino acid sequence analysis of peptides obtained from FXII Locarno on activation with either trypsin or plasma kallikrein and dextran sulfate showed an amino acid substitution of Arg 353 by Pro. Thereby, the kallikrein cleavage site at Arg 353-Val 354 is lost. Although trypsin-activated FXII Locarno was fully cleaved at Arg 334-Asn 335 and at Arg 343-Leu 344, neither amidolytic nor proteolytic activity was generated. We conclude that proteolytic cleavage at Arg 343 in the absence of cleavage at Arg 353 is not sufficient to expose the enzymatic active site in FXII Locarno.
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7

Morris, Michael M. J., and Robert E. Lemon. "Mate choice in American Redstarts: by territory quality?" Canadian Journal of Zoology 66, no. 10 (October 1, 1988): 2255–61. http://dx.doi.org/10.1139/z88-334.

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We consider the hypothesis that mate choice in American Redstarts (Setophaga ruticilla) depends primarily on features of territories held by males. We test the hypothesis that the territories of yearling males are demonstrably different from and inferior for reproduction to those of adult males. Territories held by the two age-classes in New Brunswick, Canada, were floristically different, but there were also major differences in territorial features in three microgeographic areas, both within each age-group and without regard to age. There were greater differences in nest success between areas than between the age-classes. We detected no differences in immediate nest site features between nests built by the mates of yearling and adult males. There were differences across the three areas in the species of trees used as nest sites, probably reflecting differences in availability. Support for the claim of different reproductive success in relation to quality of territories held by the two age-classes of males is not convincing.
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8

Ventura, Marco, Carlos Canchaya, Valentina Bernini, Eric Altermann, Rodolphe Barrangou, Stephen McGrath, Marcus J. Claesson, et al. "Comparative Genomics and Transcriptional Analysis of Prophages Identified in the Genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei." Applied and Environmental Microbiology 72, no. 5 (May 2006): 3130–46. http://dx.doi.org/10.1128/aem.72.5.3130-3146.2006.

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ABSTRACT Lactobacillus gasseri ATCC 33323, Lactobacillus salivarius subsp. salivarius UCC 118, and Lactobacillus casei ATCC 334 contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1) distinguishable prophage sequences, respectively. Sequence analysis revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group of Sfi11-like pac site and cos site Siphoviridae, respectively. Phylogenetic investigation of these newly described prophage sequences revealed that they have not followed an evolutionary development similar to that of their bacterial hosts and that they show a high degree of diversity, even within a species. The attachment sites were determined for all these prophage elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage Sal2 integrates in a predicted arginino-succinate lyase-encoding gene. In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are integrated in noncoding regions in the L. casei ATCC 334 and L. salivarius UCC 118 genomes. Northern analysis showed that large parts of the prophage genomes are transcriptionally silent and that transcription is limited to genome segments located near the attachment site. Finally, pulsed-field gel electrophoresis followed by Southern blot hybridization with specific prophage probes indicates that these prophage sequences are narrowly distributed within lactobacilli.
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9

Baxter, Ian, Christopher Chippindale, Kate Fielden, Wayland Kennet, and Elizabeth Young. "Responses to Geoffrey Wainwright's report ‘The Stonehenge we deserve’, Antiquity 74 (2000): 334–42." Antiquity 74, no. 286 (December 2000): 944–51. http://dx.doi.org/10.1017/s0003598x00060567.

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In June this year, we published Geoffrey Wainwright's paper on ‘The Stonehenge we deserve'. This paper aimed to provide a review of progress towards sorting out the many problems of management, presentation and conservation of this World Heritage site and its landscape. As readers of ANTIQUITY are well aware, the fortunes of Stonehenge are intimately linked with politics, money and public opinion, and the long saga of possible solutions to make the site a better place for the future rest on these changing variables. Dr Wainwright outlined past strategies and the hope of future solutions as they were early this year. Already things have changed and the invited responses which we publish here discuss the recent changes of plan for Stonehenge. Baxter & Chippindale review the difficulties of the ‘current’ scheme and its incompatibility with visitor numbers. Fielden exposes the incompatibility of the A303 proposals for Stonehenge with legislation and planning; and Kennet & Young raise the problems of the various Plans and politics.We sent these responses to Dr Wainwright for his current view of the situation.
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10

Göbel, U. "334 Pediatric extracranial non-gonadal malignant germ cell tumors (EMGCT): Implication of site, age and dissemination." European Journal of Cancer Supplements 1, no. 5 (September 2003): S103. http://dx.doi.org/10.1016/s1359-6349(03)90367-3.

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11

Stirling, Mark, Jarg Pettinga, Kelvin Berryman, and Mark Yetton. "Probabilistic seismic hazard assessment of the Canterbury region, New Zealand." Bulletin of the New Zealand Society for Earthquake Engineering 34, no. 4 (December 31, 2001): 318–34. http://dx.doi.org/10.5459/bnzsee.34.4.318-334.

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We present the main results of a probabilistic seismic hazard assessment of the Canterbury region recently completed for Environment Canterbury (formerly Canterbury Regional Council). We use the distribution of active faults and the historical record of earthquakes to estimate the levels of earthquake shaking (peak ground acceleration and response spectral accelerations) that can be expected across the Canterbury region with return periods of 150, 475 and 1000 years. The strongest shaking (e.g. 475 year peak ground accelerations of 0.7g or more) can be expected in the west and north to northwest of the Canterbury region, where the greatest concentrations of known active faults and historical seismicity are located. Site-specific analyses of eight towns and cities selected by Environment Canterbury show that Arthur's Pass and Kaikoura are located within these zones of high hazard. In contrast, the centres studied in the Canterbury Plains (Rangiora, Kaiapoi, Christchurch, Ashburton, Temuka and Timaru) are generally located away from the zones of highest hazard. The study represents the first application of recently-developed methods in probabilistic seismic hazard at a regional scale in New Zealand.
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12

Nagao, Y., T. Abe, A. Hara, B. Sarentonglaga, M. Yamaguchi, K. Ogata, R. Fukumori, and Y. Hanazono. "334 FACTORS AFFECTING HEMATOPOIETIC ENGRAFTMENT OF MONKEY EMBRYONIC STEM CELLS IN SHEEP FETUSES." Reproduction, Fertility and Development 27, no. 1 (2015): 255. http://dx.doi.org/10.1071/rdv27n1ab334.

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Previously, we generated monkey/sheep haematopoietic chimeras by in utero transplantation (IUT) of monkey embryonic stem (ES); however, the factors that control how the ES cells successfully engraft and differentiate into haematopoietic tissue in sheep fetuses remain uncertain. Here, we examined factors that might influence donor cells and recipient sheep and affect successful ES cell engraftment. We transplanted either undifferentiated monkey ES cells or ES-derived cells at an early haematopoietic differentiation stage into sheep fetuses. The latter cells were allowed to differentiate by culturing on OP9 cell layers for 6 days. Cells were transplanted into the liver or subcutaneous tissue of recipient sheep fetuses at 43 to 50 or 51 to 67 days of gestation (full term = 147 days) using ultrasound to identify the site for transplantation. After birth, monkey haematopoietic engraftment in the bone marrow was analysed in 40 lambs using colony-PCR with cells grown in methylcellulose in the presence of defined cytokines; teratoma formation was analysed by biopsy and immunohistochemistry. We found that haematopoietic engraftment was only observed when ES-derived cells at the early differentiation stage were transplanted into fetal livers at 51 to 67 days of gestation (6/9). However, teratoma formation with mature monkey tissue structures was only observed following transplantation of undifferentiated ES cells into fetal subcutaneous tissues at 43 to 50 days of gestation (4/6), but that was not observed when both types of cells were transplanted into the liver (0/18) or at 51 to 67 days of gestation (0/24). These results demonstrate that the differentiation status of the donor cells, the transplantation site, and the age of the fetus at transplantation are important factors in engraftment and differentiation into haematopoietic tissue or teratoma formation in sheep fetuses.
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13

Charles, GW. "Nutgrass (Cyperus rotundus L.) control in cotton (Gossypium hirsutum L.)." Australian Journal of Experimental Agriculture 35, no. 5 (1995): 633. http://dx.doi.org/10.1071/ea9950633.

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The effects on cotton lint yield and nutgrass tuber density of 12 treatment combinations were examined. Treatments included preplant norflurazon and benfuresate, in-crop cultivation, glyphosate and MSMA, and post-harvest glyphosate. The effects on tuber density of a further 14 treatment combinations of cultivation, MSMA, glyphosate, and norflurazon were examined in fallow. Under a traditional nutgrass control program of in-cotton cultivation and MSMA, nutgrass tuber density (no./m2) increased from 216 (0-0.15 m soil core) in 1990 to 1112 in 1992, with an average cotton yield of 1239 kg lint/ha. This result compared well with the untreated control, where the tuber density increased to 1641 tubers/m2 in 1992, with an average lint yield of 959 kg /ha. The best treatment was a combination of norflurazon, benfuresate, glyphosate, and cultivation, resulting in a tuber density of 220 tubers/m2 in 1992 and an average lint yield of 1217 kg/ha. Repeated applications of glyphosate in fallow effectively controlled nutgrass, with incremental improvements in control from additional glyphosate applications. Monthly glyphosate ap lications reduced the tuber density from 334 tubers/m in 1990 to 47 in 1992 at one site, and from 334 tubers/m2 in 1990 to 50 in 1992 on a second site. Overall, the results showed that traditional nutgrass control techniques were unsatisfactory, but repeated glyphosate applications gave effective nutgrass control both in cotton and in fallow.
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14

Perona, R., F. Portillo, F. Giraldez, and R. Serrano. "Transformation and pH homeostasis of fibroblasts expressing yeast H(+)-ATPase containing site-directed mutations." Molecular and Cellular Biology 10, no. 8 (August 1990): 4110–15. http://dx.doi.org/10.1128/mcb.10.8.4110.

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Mouse fibroblasts expressing a yeast proton-pumping ATPase show tumorigenic transformation (R. Perona, and R. Serrano, Nature (London) 334:438-440, 1988). By expressing site-directed mutations of the yeast ATPase with different levels of activity, a close correlation has been found between enzyme activity, tumorigenic transformation, and intracellular pH measured by weak-acid distribution. Fibroblasts expressing the yeast proton-pumping ATPase showed increased capability to grow at acidic pH and to resist lethal acidification mediated by reversal of the Na(+)-H+ antiporter. Measurements with microelectrodes in individual cells demonstrated electrical hyperpolarization and confirmed the increased pH of cells expressing yeast ATPase. These results indicate that the yeast enzyme expressed in mouse fibroblasts has electrogenic proton-pumping activity and that this activity deregulates fibroblast growth. This suggests a connection between the biophysical phenomena of proton transport, intracellular pH, and membrane potential and the biochemical regulatory circuits based on protein kinases and transcription factors.
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15

Perona, R., F. Portillo, F. Giraldez, and R. Serrano. "Transformation and pH homeostasis of fibroblasts expressing yeast H(+)-ATPase containing site-directed mutations." Molecular and Cellular Biology 10, no. 8 (August 1990): 4110–15. http://dx.doi.org/10.1128/mcb.10.8.4110-4115.1990.

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Mouse fibroblasts expressing a yeast proton-pumping ATPase show tumorigenic transformation (R. Perona, and R. Serrano, Nature (London) 334:438-440, 1988). By expressing site-directed mutations of the yeast ATPase with different levels of activity, a close correlation has been found between enzyme activity, tumorigenic transformation, and intracellular pH measured by weak-acid distribution. Fibroblasts expressing the yeast proton-pumping ATPase showed increased capability to grow at acidic pH and to resist lethal acidification mediated by reversal of the Na(+)-H+ antiporter. Measurements with microelectrodes in individual cells demonstrated electrical hyperpolarization and confirmed the increased pH of cells expressing yeast ATPase. These results indicate that the yeast enzyme expressed in mouse fibroblasts has electrogenic proton-pumping activity and that this activity deregulates fibroblast growth. This suggests a connection between the biophysical phenomena of proton transport, intracellular pH, and membrane potential and the biochemical regulatory circuits based on protein kinases and transcription factors.
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16

SCHWARZ, Alexandra, Francesco Maria PIERFEDERICI, and Bernd NIDETZKY. "Catalytic mechanism of α-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase: the role of histidine-334 examined through kinetic characterization of site-directed mutants." Biochemical Journal 387, no. 2 (April 5, 2005): 437–45. http://dx.doi.org/10.1042/bj20041593.

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Purified site-directed mutants of Corynebacterium callunae starch phosphorylase in which His-334 was replaced by an alanine, glutamine or asparagine residue were characterized by steady-state kinetic analysis of enzymic glycosyl transfer to and from phosphate and studies of ligand binding to the active site. Compared with wild-type, the catalytic efficiencies for phosphorolysis of starch at 30 °C and pH 7.0 decreased approx. 150- and 50-fold in H334Q (His334→Gln) and H334N mutants, and that of H334A was unchanged. In the direction of α-glucan synthesis, selectivity for the reaction with G1P (α-D-glucose 1-phosphate) compared with the selectivity for reaction with α-D-xylose 1-phosphate decreased from a wild-type value of ∼20000 to 2600 and 100 in H334N and H334Q respectively. Binding of G1P to the free enzyme was weakened between 10-fold (H334N, H334Q) and 50-fold (H334A) in the mutants, whereas binding to the complex of enzyme and α-glucan was not affected. Quenching of fluorescence of the pyridoxal 5′-phosphate cofactor was used to examine interactions of the inhibitor GL (D-gluconic acid 1,5-lactone) with wild-type and mutant enzymes in transient and steady-state experiments. GL binding to the free enzyme and the enzyme–phosphate complex occurred in a single step. The 50-fold higher constant (Kd) for GL dissociation from H334Q bound to phosphate resulted from an increased off-rate for the ligand in the mutant, compared with wild-type. A log-log correlation of catalytic-centre activity for phosphorolysis of starch with a reciprocal Kd value established a linear free-energy relationship (slope=1.19±0.07; r2=0.991) across the series of wild-type and mutant enzymes. It reveals that GL in combination with phosphate has properties of a transition state analogue and that the His-334 side chain has a role in selectively stabilizing the transition state of the reaction.
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17

Jiang, Xiang Ping, M. Zeng, K. W. Kowk, and Helen Lai Wah Chan. "Dielectric and Ferroelectric Properties of Bi-Doped BaTiO3 Ceramics." Key Engineering Materials 334-335 (March 2007): 977–80. http://dx.doi.org/10.4028/www.scientific.net/kem.334-335.977.

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Barium titanate with A-site substituted by various amount of bismuth oxide (Ba1-x BixTiO3, abbreviated as BBT, x=0.05, 0.1, 0.15) were prepared by solid-state reaction. The effect of bismuth substitution on crystallographic phase, dielectric and ferroelectric properties was studied. The X-ray diffraction shows that the samples were crystallized into pure perovskite structure when x=0.05 and 0.1, while for x=0.15 sample, second phase appeared in the dominant perovskite phase. The temperature dependence of dielectric permittivity of the ceramics was investigated and the evolution from normal ferroelectrics to relaxor ferroelectric sates was observed. In the range 0≤x≤0.1, the temperature of dielectric peak Tm is independence of the frequency, indicating the normal ferroelectrics behavior. At x=0.15, dielectric relaxation process with a broadening distribution of the permittivity dielectric is observed.
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18

BAINBRIDGE, Graeme, P. John ANRALOJC, Pippa J. MADGWICK, Jim E. PITTS, and Martin A. J. PARRY. "Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans." Biochemical Journal 336, no. 2 (December 1, 1998): 387–93. http://dx.doi.org/10.1042/bj3360387.

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The contribution of lysine-128 within the active site of Anacystis nidulansd-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coliand the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by β-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential ε-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.
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19

Patkar, Sushil. "334 Anterior Screw Plate Fixation for Odontoid Fractures: A New Technique." Neurosurgery 64, CN_suppl_1 (August 24, 2017): 274. http://dx.doi.org/10.1093/neuros/nyx417.334.

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Abstract INTRODUCTION Certain unstable odontoid fractures (anterior oblique fracture, displaced distal fragment, disrupted alar ligament) are usually offered posterior C1-C2 fixation by Goel-Harms technique. Anterior submandibular extrapharyangeal approach offers a simple safe and biomechanically correct option to treat these fractures. Fracture manipulation, reduction and repositioning is feasible along with fixation in compression mode with a VSP plate and screws. Additional instability of the atlantoaxial joints, if present, can be treated with anterior transarticular screws. METHODS The right sided submandibular extrapharyangeal approach was used to expose the fractured odontoid. Open manipulation to reduce displaced fracture fragments followed by fixation with a VSP plate (Titanium customized from Jayon Inc Pallakad India) using 3.5 mm thick titanium screws. If fracture was associated with disruption of alar ligament then additional bilateral tranarticular screws were passed to fix the joints in neutral position after abrading the opposing endplates and corticocancellous bone grafts into the joints. RESULTS >Since February 2011 January 2017 45 patients with unstable odontoid fractures have been offered this procedure. No transfusion was required in any case and no case required additional procedure. All patients showed bone union across fracture site at 3 months. Five elderly patients (70 yrs and 76 yrs) complained of dysphagia for 2–3 days after surgery, 4 patients (including the ones with dysphagia) had mild hypoglossal weakness which recovered in a week. Neck pain persisted in 9 patients for 4 weeks which responded to analgesics. Long-term follow-up imaging has been at 3 years in 8 patients, without implant failure and 100% bone fusion. CONCLUSION Anterior submandibular extrapharyangeal approach offers a simple safe and biomechanically correct option to treat these fractures.
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20

Patil, Sujata, Darren Richard Feldman, Dean F. Bajorin, Robert John Motzer, Joel Sheinfeld, Satish Tickoo, Victor E. Reuter, and George J. Bosl. "Long-term survival rates after chemotherapy (CTx) in patients (pts) with nonseminomatous (NS) germ cell tumors (GCT) with teratoma (T)." Journal of Clinical Oncology 31, no. 6_suppl (February 20, 2013): 334. http://dx.doi.org/10.1200/jco.2013.31.6_suppl.334.

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334 Background: Debate surrounds the clinical importance of T in NSCGT pts who receive CTx. The WHO no longer distinguishes between mature (MT) and immature teratoma (IT). Two studies suggested that immunohistologic (IHC) markers of apoptosis, p53 and Ki67 expression were independent markers of outcome in pts with embryonal carcinoma (JCO 21: 2679, 2003) and a differentiation genotype was an independent marker of worse outcome in NSGCT (JCO 27: 5240, 2009). We extend these analyses to NSGCT with and without T. Methods: In an IRB-approved study, archived pre-CTx tumor was obtained from 191 pts who received platinum-based CTx for initial treatment of NSGCT between 4/75 and 5/1996. Histology was confirmed and % cells displaying apoptosis (TUNEL assay) or expressing p53 and Ki67 (IHC) determined at MSKCC. 110 pts’ tumors had ≥ 1 NSGCT cell types without T and 81 had MT (n=39) and/or IT (n=52); 10 pts had both. Tumors with T had ≥ 1 additional NSGCT cell types. Overall survival (OS) differences were tested using Kaplan-Meier, Cox regression and log-rank test. Results: Median age was 27 years; primary site was testis (96%) and mediastinum (3%). 57% were IGCCCG good risk, 29% intermediate risk with no difference with and without T (P=0.53). With median follow-up of 7 yrs, 5-yr OS was 84% (95% CI: 75-90) without T and 72% (61-81) with T, and at 10 yrs 84% (75-90) and 65% (52-76), respectively (P=.06). OS differed by T subgroup: 5-yr OS was 80% if IT alone; 75% if both IT and MT; 61% if MT alone (P=.001). Adjusting for risk strata, this difference remained significant (P=.02). For IT, %Ki67 expression was borderline associated with OS (P=.06), but not %apoptosis (P=.56) or %p53 (P=.93). For MT, %apoptosis was associated with OS (P=.01) but not %Ki67 (P=.70) or %p53 (P=.29). Conclusions: The presence of T, particularly MT, in mixed NSGCT was associated with worse OS than tumors without T. T subtype was important: OS was associated with %Ki67 for IT and %apoptosis for MT. These data suggest that the presence of T should be considered in treatment decision-making. The absence of T subtyping in the WHO classification will likely result in a loss of prognostic information.
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SURIYARAK, SARISA, HERBERT SCHMIDT, PIERRE VILLENEUVE, and JOCHEN WEISS. "Morphological and Dose-Dependent Study on the Effect of Methyl, Hexyl, and Dodecyl Rosmarinate on Staphylococcus carnosus LTH1502: Use of the Weibull Model." Journal of Food Protection 81, no. 4 (March 12, 2018): 598–605. http://dx.doi.org/10.4315/0362-028x.jfp-17-334.

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ABSTRACT The mechanisms of three antimicrobial rosmarinates (methyl-RE1, hexyl-RE6, and dodecyl-RE12) were investigated against Staphylococcus carnosus LTH1502. Scanning electron microscopy was used to determine the morphology of treated cells to gain information on potential changes in the site of action of compounds. The survival data obtained from antimicrobial activity assays were fitted to a nonlinear Weibull model to assess changes in inactivation behavior. Generally, esters became more effective with increasing length of the alkyl chain, resulting in a lower concentration for inhibition and inactivation. Weibull distribution parameters showed a downward concave inactivation pattern for RE1 above a critical concentration, indicative of a delayed log phase of the antimicrobial activity, with few cells being inactivated immediately after treatment and more cells being affected at later times. In contrast, esters having longer alkyl chains (RE6 and RE12) had an upward concave inactivation behavior, with more cells being inactivated immediately after addition of compounds. Cellular morphologies suggest that the antimicrobial mode of action of esters transitions from one that acts intracellularly (RE1) to one that predominately affects bacterial membrane (RE6 and RE12) due to changes in physicochemical properties of esters. Assessment that is based on the parameters of the Weibull model could, thus, be used to evaluate antimicrobial efficiency, in addition to MIC.
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Mason, SE, JM Kinross, D. Reynecke, J. Hendricks, and TH Arulampalam. "PTH-334 Cost-effectiveness of warm humidified co2 to reduce surgical site infections in laparoscopic colorectal surgery: a cohort study." Gut 64, Suppl 1 (June 2015): A556.1—A556. http://dx.doi.org/10.1136/gutjnl-2015-309861.1220.

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Zu, YL, Y. Ai, A. Gilchrist, ME Labadia, RI Sha'afi, and CK Huang. "Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation." Blood 87, no. 12 (June 15, 1996): 5287–96. http://dx.doi.org/10.1182/blood.v87.12.5287.bloodjournal87125287.

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In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.
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Vannucchi, P., K. Ujiie, and N. Stroncik. "IODP Expedition 334: An Investigation of the Sedimentary Record, Fluid Flow and State of Stress on Top of the Seismogenic Zone of an Erosive Subduction Margin." Scientific Drilling 15 (March 1, 2013): 23–30. http://dx.doi.org/10.5194/sd-15-23-2013.

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The Costa Rica Seismogenesis Project (CRISP) is an experiment to understand the processes that control nucleation and seismic rupture of large earthquakes at erosional subduction zones. Integrated Ocean Drililng Program (IODP) Expedition 334 by R/V <i>JOIDES Resolution</i> is the first step toward deep drilling through the aseismic and seismic plate boundary at the Costa Rica subduction zone offshore the Osa Peninsula where the Cocos Ridge is subducting beneath the Caribbean plate. Drilling operations included logging while drilling (LWD) at two slope sites (Sites U1378 and U1379) and coring at three slope sites (Sites U1378–1380) and at one site on the Cocos plate (Site U1381). For the first time the lithology, stratigraphy, and age of the slope and incoming sediments as well as the petrology of the subducting Cocos Ridge have been characterized at this margin. The slope sites recorded a high sediment accumulation rate of 160–1035m m y<sup>&minus;1</sup> possibly caused by on-land uplift triggered by the subduction of the Cocos Ridge. The geochemical data as well as the <i>in situ</i> temperature data obtained at the slope sites suggest that fluids are transported from greater depths. The geochemical profiles at Site U1381 reflect diffusional communication of a fluid with seawater-like chemistry and the igneous basement of the Cocos plate (Solomon et al., 2011; Vannucchi et al., 2012a). The present-day <i>in situ</i> stress orientation determined by borehole breakouts at Site U1378 in the middle slope and Site U1379 in the upper slope shows a marked change in stress state within ~12 km along the CRISP transect; that may correspond to a change from compression (middle slope) to extension (upper slope). <br><br> doi:<a href="http://dx.doi.org/10.2204/iodp.sd.15.03.2013" target="_blank">10.2204/iodp.sd.15.03.2013</a>
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Palos, Guadalupe R., Katherine Ramsey Gilmore, Patricia Hansberry Chapman, and Maria Alma Rodriguez. "Implementation of ePROs in the care of long-term cancer survivors." Journal of Clinical Oncology 39, no. 28_suppl (October 1, 2021): 334. http://dx.doi.org/10.1200/jco.2020.39.28_suppl.334.

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334 Background: Standard methods for implementation of electronic patient reported outcomes (ePROs) in the routine care of cancer survivors remain unclear. To address this gap, we assessed the technical and operational feasibility of implementing ePROs into the clinical workflow of four survivorship clinics, Genitourinary (GU), Head & Neck (HN), Thyroid (THY), and Stem Cell Transplant (SCT). Methods: This quality improvement study was conducted from 9/1/2019 to 4/30/2021. Benchmarks of ≥ 50% were established for two outcomes including: (1) Delivery Rate - percentage of patients who received the ePRO prior to their clinic visit and (2) Adherence Rate - percentage of patients who completed one ePRO. Data were collected at T0 (Baseline) (09/01/2019 to 12/31/2020) and T1 (1/1/2021 to 4/30/2021). Patients completed the survey 3-7 days prior to a clinic visit. The timing to capture ePROs’ data was tailored to the specific cancer site. Operational steps were: 1) questionnaire was ordered for scheduled survivorship appointment; 2) electronic medical record system (EMRS) pushed the questionnaire to the patient portal app with a message asking the patient to complete and submit the ePROs survey, 3) data was pushed back to the provider by the EMRS; and 4) results stored in the medical record were accessed by the provider prior to or on the day of the patient’s visit. Data were collected and stored using the EPIC Data Universes and reported through Microsoft Power BI. This institutional Quality Improvement Assessment Board approved this study. Results: The Table shows the improvement in the benchmark for sent and adherence rates between T0 and T1. A few clinics showed room for improvement. Patient engagement facilitators included being scheduled for a virtual visit and having staff sent prompts prior to visits. Challenges were: (1) variation in the time ePROs were available in the medical records, 2) inconsistent linking of scheduled visits and prompt to send the survey, and 3) patients’ not opening, completing, or submitting the survey even when prompted to do so. Opportunities included (1) developing a patient education flyer describing ePROs’ role in patient care, (2) providing instruction on the completion and completion of how to complete the ePRO, and (3) conducting staff education sessions on how to link scheduled visits with ePROs’ patient prompts. Conclusions: Our preliminary results indicate that integration ePROs can be considered an acceptable approach to improve follow-up care of cancer survivors but with room for improvement in some clinics. Future evaluation will further explore patients’ ePROs’ impact on clinical outcomes and patient engagement.[Table: see text]
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Ching, Hilda Lei. "Occurrence of the eyefluke, Diplostomum (Diplostomum) baeri bucculentum Dubois et Rausch, 1948, in salmonid fishes of northern British Columbia." Canadian Journal of Zoology 63, no. 2 (February 1, 1985): 396–99. http://dx.doi.org/10.1139/z85-060.

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As a result of experimental infections in chicks, diplostomula found in the retina of chinook salmon from the Nechako River were identified as Diplostomum (Diplostomum) baeri bucculentum. Eyeflukes in other salmonids were considered to be the same species based on similar measurements and site in the eyes. These eyeflukes varied in prevalence and mean intensity in seven salmonid species surveyed in nine localities in 1979–1981. The following fish were sampled: rainbow trout (Salmo gairdneri), 505; mountain whitefish (Prosopium williamsoni), 334; lake whitefish (Coregonus clupeaformis), 32; Dolly Varden (Salvelinus malma), 66; lake trout (S. namaycush), 13; kokanee or sockeye salmon (Oncorhynchus nerka), 323; and chinook salmon (O. tshawytscha), 164. Eyeflukes had prevalences ranging from 84 to 100% in six lakes, 64% in the river, 53% in one reservoir site, and a prevalence of 15% in the other reservoir site. Mountain and lake whitefishes had high mean intensities while kokanee had low mean intensities. Correlation of increased intensity with increased fish size was significant for 6 of 27 samples. Four samples of lake whitefish, mountain whitefish, rainbow trout, and chinook salmon showed significant asymmetry when numbers of diplostomula were compared between eyes. More of the heavily infected fish showed asymmetry than did the lightly infected fish.
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Broser, Matthias, Carina Glöckner, Azat Gabdulkhakov, Albert Guskov, Joachim Buchta, Jan Kern, Frank Müh, Holger Dau, Wolfram Saenger, and Athina Zouni. "Structural Basis of Cyanobacterial Photosystem II Inhibition by the Herbicide Terbutryn." Journal of Biological Chemistry 286, no. 18 (March 2, 2011): 15964–72. http://dx.doi.org/10.1074/jbc.m110.215970.

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Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the QB site in the D1 subunit and thus block the electron transfer from QA to QB. Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the QB site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of QA, which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334–342). This discovery is discussed in the context of proton transfer to the lumen.
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28

Haria, A. H., and P. Shand. "Evidence for deep sub-surface flow routing in forested upland Wales: implications for contaminant transport and stream flow generation." Hydrology and Earth System Sciences 8, no. 3 (June 30, 2004): 334–44. http://dx.doi.org/10.5194/hess-8-334-2004.

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Abstract. Upland streamflow generation has traditionally been modelled as a simple rainfall-runoff mechanism. However, recent hydrochemical studies conducted in upland Wales have highlighted the potentially important role of bedrock groundwater in streamflow generation processes. To investigate these processes, a detailed and novel field study was established in the riparian zone and lower hillslopes of the Hafren catchment at Plynlimon, mid-Wales. Results from this study showed groundwater near the river behaving in a complex and most likely confined manner within depth-specific horizons. Rapid responses to rainfall in all boreholes at the study site indicated rapid recharge pathways further upslope. The different flow pathways and travel times influenced the chemical character of groundwaters with depth. Groundwaters were shown to discharge into the stream from the fractured bedrock. A lateral rapid flow horizon was also identified as a fast flow pathway immediately below the soils. This highlighted a mechanism whereby rising groundwater may pick up chemical constituents from the lower soils and transfer them quickly to the stream channel. Restrictions in this horizon resulted in groundwater upwelling into the soils at some locations indicating soil water to be sourced from both rising groundwater and rainfall. The role of bedrock groundwater in upland streamflow generation is far more complicated than previously considered, particularly with respect to residence times and flow pathways. Hence, water quality models in upland catchments that do not take account of the bedrock geology and the groundwater interactions therein will be seriously flawed. Keywords: bedrock, groundwater, Hafren, hillslope hydrology, Plynlimon, recharge, soil water, streamflow generation
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29

Wang, Zhimin. "334. Double Expression Cassettes Engineering at the Two Alleles with Same Genome Site of Human Cell Using CRISPR/Cas9 Vector System." Molecular Therapy 24 (May 2016): S133. http://dx.doi.org/10.1016/s1525-0016(16)33143-4.

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30

NONNOTTE, P., G. CEULENEER, and M. BENOIT. "Genesis of andesitic–boninitic magmas at mid-ocean ridges by melting of hydrated peridotites: Geochemical evidence from DSDP Site 334 gabbronorites." Earth and Planetary Science Letters 236, no. 3-4 (August 15, 2005): 632–53. http://dx.doi.org/10.1016/j.epsl.2005.05.026.

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31

Shirako, Yukio. "Non-AUG Translation Initiation in a Plant RNA Virus: a Forty-Amino-Acid Extension Is Added to the N Terminus of the Soil-Borne Wheat Mosaic Virus Capsid Protein." Journal of Virology 72, no. 2 (February 1, 1998): 1677–82. http://dx.doi.org/10.1128/jvi.72.2.1677-1682.1998.

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ABSTRACT RNA 2 of soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, encodes a protein previously hypothesized to be initiated at an in-frame non-AUG codon upstream of the AUG initiation codon (nucleotide positions 334 to 336) for the 19-kDa capsid protein. Site-directed mutagenesis and in vitro transcription and translation analysis indicated that CUG (nucleotides 214 to 216) is the initiation codon for a protein with a calculated molecular mass of 25 kDa composed of a 40-amino-acid extension to the N terminus of the 19-kDa capsid protein. A stable deletion mutant, which was isolated after extensive passages of a wild-type SBWMV, contained a mixture of two deleted RNA 2’s, only one of which coded for the 25-kDa protein. The amino acid sequence of the N-terminal extension was moderately conserved and the CUG initiation codon was preserved among three SBWMV isolates from Japan and the United States. This amino acid sequence conservation, as well as the retention of expression of the 25-kDa protein in the stable deletion mutant, suggests that the 25-kDa protein is functional in the life cycle of SBWMV. This is the first report of a non-AUG translation initiation in a plant RNA virus genome.
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32

Pisignano, Giuseppina, Ioanna Pavlaki, and Adele Murrell. "Being in a loop: how long non-coding RNAs organise genome architecture." Essays in Biochemistry 63, no. 1 (April 2019): 177–86. http://dx.doi.org/10.1042/ebc20180057.

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Abstract Chromatin architecture has a significant impact on gene expression. Evidence in the last two decades support RNA as an important component of chromatin structure [Genes Dev. (2005) 19, 1635–1655; PLoS ONE (2007) 2, e1182; Nat. Genet. (2002) 30, 329–334]. Long non-coding RNAs (lncRNAs) are able to control chromatin structure through nucleosome positioning, interaction with chromatin re-modellers and chromosome looping. These functions are carried out in cis at the site of lncRNAs transcription or in trans at distant loci. While the evidence for a role in lncRNAs in regulating gene expression through chromatin interactions is increasing, there is still very little conclusive evidence for a potential role in looping organisation. Here, we review models for the involvement of lncRNAs in genome architecture and the experimental evidence to support them.
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33

Okolotowicz, Karl J., Wei-Jen Lee, Rosemarie F. Hartman, Ann Y. Kim, Steven R. Ottersberg, Dale E. Robinson, Jr., Scott R. Lefler, and Seth D. Rose. "Inactivation of Protein Farnesyltransferase by Active-Site-Targeted Dicarbonyl Compounds." Archiv der Pharmazie 334, no. 6 (June 2001): 194–202. http://dx.doi.org/10.1002/1521-4184(200106)334:6<194::aid-ardp194>3.0.co;2-m.

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34

Lapshin, N. V., M. V. Matantseva, and S. A. Simonov. "Nest Site Selection and Nest Construction in the Willow Warbler (Phylloscopus trochilus L., 1758) (Sylviidae, Aves) in the Tajga Zone of Northwest Russia." Povolzhskiy Journal of Ecology, no. 3 (January 1, 2020): 322–34. http://dx.doi.org/10.35885/1684-7318-2019-3-322-334.

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35

Schultz, Joshua A., and Viktoria Henriksson. "Structural assessment of St. Charles hyperbolic paraboloid roof." Curved and Layered Structures 8, no. 1 (January 1, 2021): 157–66. http://dx.doi.org/10.1515/cls-2021-0015.

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Abstract At the time of completion in 1961, the roof of St. Charles Church became the largest unbalanced hyperbolic paraboloid structure in the United States and the only shell structure in Spokane, WA. Situated on an 8-acre site on the north side of the city, St. Charles is a modernist structure designed through partnership of Funk, Molander & Johnson engineers, architect William C. James and in consultation with Professor T.Y. Lin of the Structural Engineering Laboratory at the University of California, Berkeley. This asymmetric structure spans over 33.5 m (110 ft) and utilizes folded edge beams that taper from 1067 mm (42 in) at the base to a 76.2 mm (3 in) thickness at the topmost edge using regular strength reinforcing steel and concrete load carrying components. The novelty of the pre-stressed shell structure serves both architectural and structural design criteria by delivering a large, uninterrupted interior sanctuary space in materially and economically efficient manner. This structural assessment summarizes the roof’s historic design and construction according to the original construction documents, newspaper reports and historic photographs. The FEA is completed using UBC 1955 design loads and ACI 334 Concrete Shell Structures provisions.
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Wholey, Michael Henry, Mark Henry Wholey, Gustave Eles, Boulis Toursakissian, Steven Bailey, Chester Jarmolowski, and Walter A. Tan. "Evaluation of Glycoprotein IIb/IIIa Inhibitors in Carotid Angioplasty and Stenting." Journal of Endovascular Therapy 10, no. 1 (February 2003): 33–41. http://dx.doi.org/10.1177/152660280301000108.

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Purpose: To review the immediate neurological and bleeding complications associated with the use of glycoprotein (GP) IIb/IIIa inhibitors in patients undergoing extracranial carotid artery stent placement. Methods: A retrospective review was performed of 550 patients (321 men; mean age 71.1 years, range 28–91) who underwent carotid artery angioplasty and stent placement. Glycoprotein IIb/IIIa inhibitors were given prophylactically along with heparin to 216 patients, whose outcomes were compared to a control group of 334 patients who received intravenous heparin alone. Primary endpoints were the immediate and 30-day neurological complications, including transient ischemic attacks (TIAs), minor and major strokes, and neurologically-related deaths. The secondary endpoint was any abnormal bleeding. Results: The all stroke/neurological death rate in 216 patients treated with heparin and GP IIb/IIIa inhibitors was 6.0% (13 events) compared 2.4% (8 events) in the 334 patients in the heparin-only control group (p = 0.0430). Two of the 4 neurologically-related deaths in the GP IIb/IIIa inhibitor group resulted from intracranial hemorrhages; there were no intracranial hemorrhages in the heparin-only group. There was 1 episode of extracranial bleeding in the GP IIb/IIIa inhibitor group treated with embolization. The incidences of significant puncture-site bleeding requiring transfusion were similar in the groups. Conclusions: Neurological complications following percutaneous carotid artery interventions have been relatively few. The neurological sequelae in carotid stent patients receiving glycoprotein IIb/IIIa inhibitors were more numerous and consequential, which suggests that the use of GP IIb/IIIa inhibitors in carotid stenting should be discouraged.
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Seniya, Chandrabhan, Ghulam Jilani Khan, and Kuldeep Uchadia. "Identification of Potential Herbal Inhibitor of Acetylcholinesterase Associated Alzheimer’s Disorders Using Molecular Docking and Molecular Dynamics Simulation." Biochemistry Research International 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/705451.

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Cholinesterase inhibitors (ChE-Is) are the standard for the therapy of AD associated disorders and are the only class of approved drugs by the Food and Drug Administration (FDA). Additionally, acetylcholinesterase (AChE) is the target for many Alzheimer’s dementia drugs which block the function of AChE but have some side effects. Therefore, in this paper, an attempt was made to elucidate cholinesterase inhibition potential of secondary metabolite fromCannabisplant which has negligible or no side effect. Molecular docking of 500 herbal compounds, against AChE, was performed using Autodock 4.2 as per the standard protocols. Molecular dynamics simulations have also been carried out to check stability of binding complex in water for 1000 ps. Our molecular docking and simulation have predicted high binding affinity of secondary metabolite (C28H34N2O6) to AChE. Further, molecular dynamics simulations for 1000 ps suggest that ligand interaction with the residues Asp72, Tyr70-121-334, and Phe288 of AChE, all of which fall under active site/subsite or binding pocket, might be critical for the inhibitory activity of AChE. This approach might be helpful to understand the selectivity of the given drug molecule in the treatment of Alzheimer's disease. The study provides evidence for consideration ofC28H34N2O6as a valuable small ligand molecule in treatment and prevention of AD associated disorders and furtherin vitroandin vivoinvestigations may prove its therapeutic potential.
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Zhu, Yinxing, Ruifang Wang, Luyao Yu, Huimin Sun, Shan Tian, Peng Li, Meilin Jin, Huanchun Chen, Wenjun Ma, and Hongbo Zhou. "Human TRA2A determines influenza A virus host adaptation by regulating viral mRNA splicing." Science Advances 6, no. 25 (June 2020): eaaz5764. http://dx.doi.org/10.1126/sciadv.aaz5764.

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Several avian influenza A viruses (IAVs) have adapted to mammalian species, including humans. To date, the mechanisms enabling these host shifts remain incompletely understood. Here, we show that a host factor, human TRA2A (huTRA2A), inhibits avian IAV replication, but benefits human IAV replication by altered regulation of viral messenger RNA (mRNA) splicing. huTRA2A depresses mRNA splicing by binding to the intronic splicing silencer motif in the M mRNA of representative avian YS/H5N1 or in the NS mRNA of representative human PR8/H1N1 virus, leading to completely opposite effects on replication of the human and avian viruses in vitro and in vivo. We also confirm that the M-334 site and NS-234/236 sites are critical for TRA2A binding, mRNA splicing, viral replication, and pathogenicity. Our results reveal the underlying mechanisms of adaptation of avian influenza virus to human hosts, and suggest rational strategies to protect public health.
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39

Olsen, Margaret A., Kelly E. Ball, Katelin B. Nickel, Anna E. Wallace, and Victoria J. Fraser. "Validation of ICD-9-CM Diagnosis Codes for Surgical Site Infection and Noninfectious Wound Complications After Mastectomy." Infection Control & Hospital Epidemiology 38, no. 3 (December 15, 2016): 334–39. http://dx.doi.org/10.1017/ice.2016.271.

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BACKGROUNDFew studies have validated ICD-9-CM diagnosis codes for surgical site infection (SSI), and none have validated coding for noninfectious wound complications after mastectomy.OBJECTIVESTo determine the accuracy of International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes in health insurer claims data to identify SSI and noninfectious wound complications, including hematoma, seroma, fat and tissue necrosis, and dehiscence, after mastectomy.METHODSWe reviewed medical records for 275 randomly selected women who were coded in the claims data for mastectomy with or without immediate breast reconstruction and had an ICD-9-CM diagnosis code for a wound complication within 180 days after surgery. We calculated the positive predictive value (PPV) to evaluate the accuracy of diagnosis codes in identifying specific wound complications and the PPV to determine the accuracy of coding for the breast surgical procedure.RESULTSThe PPV for SSI was 57.5%, or 68.9% if cellulitis-alone was considered an SSI, while the PPV for cellulitis was 82.2%. The PPVs of individual noninfectious wound complications ranged from 47.8% for fat necrosis to 94.9% for seroma and 96.6% for hematoma. The PPVs for mastectomy, implant, and autologous flap reconstruction were uniformly high (97.5%–99.2%).CONCLUSIONSOur results suggest that claims data can be used to compare rates of infectious and noninfectious wound complications after mastectomy across facilities, even though PPVs vary by specific type of postoperative complication. The accuracy of coding was highest for cellulitis, hematoma, and seroma, and a composite group of noninfectious complications (fat necrosis, tissue necrosis, or dehiscence).Infect Control Hosp Epidemiol 2017;38:334–339
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Yuniarti, Erny, Ida F. Dalmacio, and Erlinda S. Paterno. "HEAVY METAL-RESISTANT RHIZOBACTERIA FROM GOLD MINE IN PONGKOR INDONESIA AND COPPER MINE IN MARINDUQUE PHILIPPINES." Agric 31, no. 1 (July 21, 2019): 75–88. http://dx.doi.org/10.24246/agric.2019.v31.i1.p75-88.

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The purposes of the study was to isolate, to characterize, and to identify rhizobacteria from plant rhizosphere growing in gold and copper mine. The isolation of rhizobacteria used N-free semisolid agar media, TSA, and SLP plus heavy metals (HMs), namely Pb, Cd, and or Cu. Isolated rhizobacteria were subsequently characterized for resistance to higher level of Pb, Cd, Cu in SLP media. Cultural and morphological characterization of rhizobacteria were conducted for cell morphology, motility, Gram staining, and biofilm formation. The rhizobacteria identification used sequence analysis of the 16S RNA gene fragments. The results showed that the majority of rhizobacterial from Cu mine site (66.7% of 21 isolates) were resistant to Cu (72150 ppm) while the majority of rhizobacteria from gold mine site (77.8% of 18 isolates) were sensitive to 72 ppm Cu. Majority of Cu in the soil was insoluble as granules attaching to gravel so that rhizobacteria of Cu mine site have been exposed and adapted to available Cu. This fact, explaining that the rhizobacteria’s MIC value was lower than the total Cu level in the soil. Three HMs-resistant rhizobacter (PbSM 2.1, MGR 334, and CuNFbM 4.1) formed biofilms, which was as one of the resistance mechanism to HMs. This research informed that HM contaminated-soil is better source for obtaining HM resistant rhizobacteria than HM uncontaminated-soil. The use four isolation media produce rhizobacteria which was more diverse than rhizobacteria from each isolation medium. Further characterization needs to be done to obtain HM resistant-rhizobacteria which can be used as biofertilizers and phytoremediation agent.
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41

Veulemans, Verena, Shazia Afzal, Paul Ledwig, Christian Heiss, Lucas Busch, Roberto Sansone, Dagmar B. Soetemann, et al. "Stent fractures after common femoral artery bail-out stenting due to suture device failure in TAVR." Vasa 47, no. 5 (August 1, 2018): 393–401. http://dx.doi.org/10.1024/0301-1526/a000712.

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Abstract. Background: Vascular access site-related complications are frequent in the context of transfemoral transcatheter aortic valve replacement (TAVR). The implantation of a covered stent graft is an effective treatment option for bleeding control. However, the external iliac and common femoral arteries are exposed to flexion of the hip joint. Therefore, stent compression and stent/strut fractures may occur, facilitating stent occlusion. Patients and methods: In all 389 patients who received transfemoral TAVR from 2013–2015 at the Düsseldorf Heart Centre, we monitored the management of vascular access site-related complications. Our analyses focused on immediate technical success and bleeding control, primary patency, and the occurrence of stent/strut fractures after six to 12 months of follow-up. Results: Vascular access site-related complications occurred in 13 % (n = 51), whereof in 10 patients, the bleeding was successfully managed by prolonged compression. In 40 out of 51 patients, a covered stent graft was implanted in the common femoral artery, leading to 100 % immediate bleeding control. After a mean follow-up of 334 ± 188 days, 28 stents out of 29 patients with completed follow-up (excluding e. g. death) were without flow-limiting stenosis (primary patency 97 %) or relevant stent compression (diameter pre/post 8.6/8.1 mm, p = 0.048, late lumen loss 1.1 ± 0.2 mm, mean flow velocity 92 ± 34 cm/s). In four asymptomatic patients, stent/strut fractures were detected (14 %) without flow-limiting stenosis. Conclusions: The implantation of a covered stent graft is highly effective and safe to control vascular access site-related complications after TAVR. Stent/strut fractures in the flexible segment of the common femoral artery may occur, as consequently verified by X-ray visualization, but show no impairment on flow or clinical parameters after six to 12 months.
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42

Hsieh, Po-Shiuan, Mary Courtney Moore, Bess Marshall, Michael J. Pagliassotti, Brian Shay, Dennis Szurkus, Doss W. Neal, and Alan D. Cherrington. "The head arterial glucose level is not the reference site for generation of the portal signal in conscious dogs." American Journal of Physiology-Endocrinology and Metabolism 277, no. 4 (October 1, 1999): E678—E684. http://dx.doi.org/10.1152/ajpendo.1999.277.4.e678.

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Experiments were performed on twelve 42-h-fasted, conscious dogs to determine whether the head arterial glucose level is used as a reference standard for comparison with the portal glucose level in bringing about the stimulatory effect of portal glucose delivery on net hepatic glucose uptake (NHGU). Each experiment consisted of an 80-min equilibration, a 40-min control, and two 90-min test periods. After the control period, somatostatin was given along with insulin (7.2 pmol ⋅ kg−1⋅ min−1; 3.5-fold increase) and glucagon (0.6 ng ⋅ kg−1⋅ min−1; basal) intraportally. Glucose was infused intraportally (22.2 μmol ⋅ kg−1⋅ min−1) and peripherally as needed to double the hepatic glucose load. In one test period, glucose was infused into both vertebral and carotid arteries (HEADG; 22.2 ± 0.8 μmol ⋅ kg−1⋅ min−1); in the other test period, saline was infused into the head arteries (HEADS). One-half of the dogs received HEADGfirst. When all dogs are considered, the blood arterial-portal glucose gradients (−0.52 ± 0.07 vs. −0.49 ± 0.03 mM) and the hepatic glucose loads (339 ± 14 vs. 334 ± 20 μmol ⋅ kg−1⋅ min−1) were similar in HEADGand HEADS. NHGU was 24.1 ± 3.8 and 25.1 ± 4.6 μmol ⋅ kg−1⋅ min−1, and nonhepatic glucose uptake was 46.1 ± 4.2 and 48.8 ± 7.0 μmol ⋅ kg−1⋅ min−1in HEADGand HEADS, respectively. The head arterial glucose level is not the reference standard used for comparison with the portal glucose level in the generation of the portal signal.
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43

Goliášová, Eliška, and Josef Dvořák. "The oestrogen receptor gene (ESR) PvuII polymorphism allele frequencies in Czech Large White and Landrace." Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis 53, no. 2 (2005): 33–38. http://dx.doi.org/10.11118/actaun200553020033.

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Genotype and allele frequencies of the oestrogen receptor gene (ESR) PvuII restriction site were investigated in populations of the main Czech maternal breeds. 1253 sows and gilts and 396 boars in Large White and 334 sows and gilts and 318 boars in Landrace were genotyped from blood samples by the modified PCR-RFLP procedure as described in Short et al. (1997). In Large White, the frequency of allele B was about 0.51. In Landrace, the frequency of allele B reached from 0.02 for boars to 0.03 for sows. No significant deviations of the observed genotype frequencies from the frequencies expected according to Hardy-Weinberg equilibrium were found in both breed. Opposite trends in allele frequencies development could be assumed for analysed sows and boars of both breeds. In Large White sows the frequency of allele B raised probably due prefering sows with BB genotype, whereas in Landrace population the frequency of allele B decreased from yet unknown reasons.
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44

Hugo, E. R., and T. J. Byers. "S-adenosyl-l-methionine decarboxylase of Acanthamoeba castellanii (Neff): purification and properties." Biochemical Journal 295, no. 1 (October 1, 1993): 203–9. http://dx.doi.org/10.1042/bj2950203.

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S-Adenosyl-L-methionine decarboxylase (AdoMetDC) has been purified to near homogeneity from the Neff strain of Acanthamoeba castellanii. The holoenzyme molecular mass is 88.8 kDa, including two copies each of a 32.8 kDa alpha-subunit and a 10-15 kDa beta-subunit. The alpha-subunit contains the active site. It has an N-terminal pyruvoyl group, and the first 19 amino acids are 63 and 74% identical with comparable sequences from yeast and mammals, respectively. The apparent Km for S-adenosylmethionine (AdoMet) in the presence of 2 mM putrescine was 30.0 microM. The enzyme was stimulated 2-fold by putrescine, but was unaffected by spermidine. It was inhibited by the following anti-metabolites, listed with their Ki values: Berenil (0.17 microM), pentamidine (19.4 microM), propamidine (334 microM), hydroxystilbamidine (357 microM), methylglyoxal bis(guanylhydrazone) (604 microM) and ethidium bromide (1.3 mM). Activity of the enzyme fell to undetectable levels during cell differentiation (encystment).
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45

Gomes, Janaína Damaceno, and Rafaela Goltara Souza. "“RIBEIRÃO DAS TREVAS”? O skate dando um ollie nos algoritmos dominantes sobre a cidade de Ribeirão das Neves - MG." Revista Observatório 6, no. 4 (July 1, 2020): a6pt. http://dx.doi.org/10.20873/uft.2447-4266.2020v6n4a6pt.

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Ribeirão das Neves é uma cidade mineira com cerca de 334 mil habitantes e 60% da população autodeclarada negra. Além do estigma “cidade das penitenciárias”, também ficou conhecida pelo trocadilho “Ribeirão das Trevas” publicado no Diário Oficial do Estado em 7 de setembro de 2013 e no site do Tribunal de Justiça de Minas Gerais em 21 de agosto de 2018, e por milhares de publicações de violência e pobreza relacionadas pelos algoritmos de busca na internet. No entanto, a cidade destinada pelo poder público a ser sempre a mesma, abriga outras narrativas criadas por um coletivo de skatistas negros educadores: a Just Crew Skateboard. Este artigo é um desdobramento da dissertação de mestrado e apresenta os resultados da investigação, revelando à luz das leituras de Nilma Lino Gomes, como este grupo contesta o estigma da cidade mostrando novas possibilidades de engajamento e uma narrativa que valoriza a vida e as sociabilidades.
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46

Wang, Bojie, Siyuan He, Qingwen Min, Feng Cui, and Guoping Wang. "Influence of Residents’ Perception of Tourism’s Impact on Supporting Tourism Development in a GIAHS Site: The Mediating Role of Perceived Justice and Community Identity." Land 10, no. 10 (September 22, 2021): 998. http://dx.doi.org/10.3390/land10100998.

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How we scientifically measure residents’ perception of tourism developments and earn their understanding and support have an important impact on the scientific management and sustainable utilization of tourist attractions. This study analyzes the mediating role of perceived justice and community identity between residents’ perceptions of tourism’s impact and their support for tourism development by integrating the theories of social exchange theory (SET) and ‘cognition–affection–conation’ (CAC) relationship theory. We surveyed 334 interviewees in the Xinghua Duotian Agrosystem (XHDA), a Globally Important Agricultural Heritage Systems (GIAHS) site in Jiangsu Province, China. The findings of our study are as follows: (1) Residents’ perceptions had both a direct and indirect influence over behavioral intentions. Specifically, benefit perception of tourism had a significant positive influence, while cost perception had a significant negative influence on residents’ support for tourism development. (2) Perceived justice and community identity played an intermediary role in the relationship between tourism impact perception and support for tourism development. (3) The mediating role of emotions had strong effects on their behavioral intentions via its psychological transmission chain of perceptions. The results suggest that community-based tourism (CBT) may be an effective tool for local residents to diversify their livelihoods in the GIAHS site, and the mediating role of perceived justice and community identity should be taken seriously for the development of CBT in GIAHS sites.
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47

Leonard, Michael, Rachel P. Weber, Laurence Brunet, Bernard Davis, Christopher Polk, Joel Wesley Thompson, Jennifer S. Fusco, et al. "1004. Clinical Decision Support System Alerts for HIV Retention in Care – A Pilot Implementation Research Study." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S530—S531. http://dx.doi.org/10.1093/ofid/ofaa439.1190.

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Abstract Background Clinical decision support system (CDSS) alerts may help retain people living with HIV (PLWH) in care. A system of CDSS alerts utilizing the CHORUS™ portal was developed to identify PLWH at risk of being lost to care. To evaluate feasibility for a larger scale study, a before and after implementation research pilot study was implemented in the OPERA Cohort at three clinic sites in a southeastern US city. Methods Periods without intervention (before) or with CDSS alerts (after) were followed by 3 months of follow up. The study population consisted of PLWH with ≥ 1 electronic health record entry in the 2 years prior to, or during, the before or after period (Fig 1). To support clinicians through a discrete implementation strategy, alerts warning of suboptimal patient attendance were generated daily for the eligible PLWH at each site; providers or other clinic staff could respond to the alerts (Fig 2). Alerts, responses, and visits (i.e., meeting with provider or HIV lab measurement) were characterized. The proportion of PLWH with ≥ 1 visit in the before and after periods were compared at each site by Pearson’s Chi-square. Figure 1. Pilot study timeline Figure 2. CDSS alert criteria and response options Results A total of 12,230 PLWH were eligible (sites A: 11,271; B: 733; C: 1,344 PLWH), with &gt; 75% in both the before and after periods. The ratio of alerts to responses was 11.9 at site A (2,245 alerts to 189 responses in 309 days; Fig 3A), and comparatively lower at sites B (756 alerts to 334 responses in 352 days, ratio=2.2; Fig 3B) and C (1,305 alerts to 896 responses in 246 days, ratio=1.5; Fig 3C). Responses to alerts were sporadic at sites A and B and consistent at site C. After the intervention, the proportion of PLWH with ≥ 1 visit stayed the same at site A (46% in both periods; p=0.47), decreased at site B (91% to 80%; p&lt; 0.01), and increased at site C (72% to 81%; p&lt; 0.01). Figure 3. Alerts and responses over time in (A) Site A, (B) Site B, and (C) Site C Conclusion This pilot study was ecological by design: measures of retention in care were compared over two calendar periods, without accounting for changes in study populations, clinic characteristics, and policies in place over time (which could have impacted clinic attendance). Though engagement with the CDSS was suboptimal at some sites, this implementation pilot study has demonstrated the ability to implement a CDSS aimed at identifying at-risk PLWH, while highlighting areas for improvement in future larger scale studies. Disclosures Joel Wesley Thompson, MHS, PA-C, AAHIVS, DFAAPA, MHS, PA-C, AAHIVS, DFAAPA, Gilead (Shareholder, Speaker’s Bureau)Janssen (Speaker’s Bureau)Theratechnologies (Speaker’s Bureau)ViiV (Speaker’s Bureau) Tammeka Evans, MoP, ViiV Healthcare (Employee)
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48

ADKINS, Charles E., Stephen A. MORRIS, Humbert DE SMEDT, Ilse SIENAERT, Katalin TÖRÖK, and Colin W. TAYLOR. "Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors." Biochemical Journal 345, no. 2 (January 10, 2000): 357–63. http://dx.doi.org/10.1042/bj3450357.

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InsP3 binding to type-1, but not type-3, InsP3 receptors is inhibited by calmodulin in a Ca2+-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca2+ mobilization by type-1 InsP3 receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP3 receptors, we show that InsP3-evoked Ca2+ mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC50 ≈ 15 μM) inhibited InsP3-evoked Ca2+ release only in the presence of Ca2+. The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP3 because calmodulin also decreased the sensitivity of the Ca2+ stores to adenophostin A, a non-metabolizable InsP3-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP3 receptors was unaffected by Ca2+-calmodulin. Using a scintillation proximity assay to measure 125I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP3 receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound 125I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca2+-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP3 receptor inhibits InsP3 binding and may thereby regulate the kinetics of Ca2+ release. Ca2+-dependent inhibition of Ca2+ release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca2+-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca2+ release from any tetrameric receptor that includes a type-1 subunit.
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49

Durrant, A. J., J. Hall, G. P. Hazlewood, and H. J. Gilbert. "The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain." Biochemical Journal 273, no. 2 (January 15, 1991): 289–93. http://dx.doi.org/10.1042/bj2730289.

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Mature endoglucanase E (EGE) from Clostridium thermocellum consists of 780 amino acid residues and has an Mr of 84,016. The N-terminal 334 amino acids comprise a functional catalytic domain. Full-length EGE bound to crystalline cellulose (Avicel) but not to xylan. Bound enzyme could be eluted with distilled water. The capacity of truncated derivatives of the enzyme to bind cellulose was investigated. EGE lacking 109 C-terminal residues (EGEd) or a derivative in which residues 367-432 of the mature form of the enzyme had been deleted (EGEb), bound to Avicel, whereas EGEa and EGEc, which lack 416 and 246 C-terminal residues respectively, did not. The specific activity of EGEa, consisting of the N-terminal 364 amino acids, was 4-fold higher than that of the full-length enzyme. The truncated derivative also exhibited lower affinity for the substrate beta-glucan than the full-length enzyme. It is concluded that EGE contains a cellulose-binding domain, located between residues 432 and 671, that is distinct from the active site. The role of this substrate-binding domain is discussed.
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50

Tavares, K. C. S., C. Feltrin, I. S. Carneiro, A. S. Morais, C. D. Medeiros, F. O. Castro, J. R. Toledo, et al. "334 ADENOVIRAL VECTOR-MEDIATED EXPRESSION OF RECOMBINANT HUMAN GLUCOCEREBROSIDASE IN THE MAMMARY GLAND OF RATS." Reproduction, Fertility and Development 25, no. 1 (2013): 314. http://dx.doi.org/10.1071/rdv25n1ab334.

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Glucocerebrosidase is a lysosomal enzyme that plays a key role in sphingolipid cleavage, an intermediate in glycolipid metabolism. A recessive mutation in the glucocerebrosidase gene leads to the accumulation of glucosylceramide in macrophages (sphingolipidosis), a lysosomal storage disease known in humans as the Gaucher disease. The enzyme replacement treatment with recombinant human glucocerebrosidase (hGCase) dramatically reduces and reverses symptoms, with the need of lifelong treatment for patients to attain a normal life. Currently, hGCase is very costly, being produced through in vitro expression in Chinese hamster ovary cells or in vivo, in plants. The aim of this study was to develop a model for the production of hGCase in the mammary gland of rats transiently transduced with recombinant adenovirus. A replication-defective adenovirus carrying hGCase was generated using the AdEasy™ adenoviral vector system (Stratagene, La Jolla, CA, USA). The hGCase cDNA (NM_001005741) was in vitro-synthesized and ligated in the XhoI site of the pAdTrack-CMV vector (pAdT-hGCase). The resulting plasmid was recombined with the pAdEasy™ vector in BJ5183 electro-competent cells. The purified pAdE-pAdT-hGCase vector was linearized and transfected into HEK-293 cells for the production of a primary viral stock. Further amplifications and the titration assay were done in HEK-293 cells, monitoring the transduction by the qualitative evaluation of green fluorescent protein (GFP) expression. Following transfection, the HEK-293 cells increasingly expressed the GFP reporter, regulated by a CMV promoter, in tandem with the hGCase cDNA, under another CMV promoter. On Day 18 of gestation, a female rat (Rattus norvegicus) was anesthetized and the 2 left caudal mammary glands were infused with 109 GTU mL–1 of the pAdE-pAdT-hGCase in PBS solution supplemented with 36 mM EGTA. The 2 right caudal mammary glands were infused only with PBS-EGTA (control milk). Milk samples collected from Days 2 through 9 post-partum were mixed with separation buffer (10 mM Tris-HCl, pH 8.0; 10 mM CaCl2) and centrifuged, with the supernatant assayed for hGCase by Western blot using a monoclonal anti-human glucocerebrosidase antibody (sc-166407, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Relative quantification of the hGCase expression was done using the FluorChem FC2 system (Alpha Innotech, San Leandro, CA, USA), with hGCase band intensity being normalized against GAPDH expression. The in vivo expression assay confirmed the production of hGCase in the secreted portion of the rat milk, with a specific band between 50 to 60 kDa observed on the Western blot, and no detection of the protein in the control milk. The hGCase peak production occurred in Days 5 and 6 of lactation, with levels being 35 times greater than on Day 9. An ELISA quantification assay and an enzymatic activity assay for the recombinant hGCase are currently in development. In conclusion, the use of the rat for hGCase transient expression in the milk was proven a valid model for testing the potential use of a mammary gland expression system for the production of a functional human glucocerebrosidase protein.
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