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A/N: This was originally written for the October challenge over at The Maple Bookshelf with the prompt: "Just because you can't touch me doesn't mean I can't feel that". So much thanks to TycheSong for everything she did to help with piecing this together. Hope you enjoy!
Disclaimer: As usual, none of the HP universe is mine, I just like to play with it sometimes.
Draco Malfoy stepped on the gas and smirked with pleasure as the engine of his newest toy, an Aston Martin Zagato purred beneath him. He glanced toward the stunning, leggy blonde sitting in the passenger seat and his smirk widened as he observed the terrified look covering her delicate features. He increased their speed, watching the odometer climb and then looked back over, to gauge the woman's reaction.
"Muggles aren't good for much, you know," he commented casually, "but I have to admit this is far, far better than Apparation. My father would of course never-"
"Drakey look out!" the blonde witch shrieked, and then there was a crash and everything went dark.
When Draco woke, it took a moment for his eyes to adjust to the harsh white light. He focused on the mediwitch standing over him in dark blue robes and slurred, "What happened?"
"You were in a crash, Lord Malfoy," the witch informed him briskly. "You'll be just fine in a few hours."
"And what about…" Merlin, what was her name? "Isabelle?" That was it!
"Oh, she's just fine, don't you worry, just a few simple bumps and bruises. She's waiting quite anxiously to see you, I believe."
At that moment the blonde witch's rather shrill voice sounded from just outside the room, demanding quite loudly that she be allowed in to see "her Drakey".
Draco winced and shook his head, hurriedly saying, "No, no, please have her leave." The elderly witch arched one, clearly judgmental brow but walked to the door, saying something to whoever was standing outside and closing the door firmly.
Draco closed his eyes in a moment of bliss, reveling in the sudden quiet and then his eyes flew open again, pinning his icy gaze on the mediwitch and demanding, "My car? Do you know what happened to the Aston?"
The witch cast him a sympathetic glance as she bustled about his bed, simply murmuring, "I'm afraid it was a rather violent crash, Lord Malfoy. The poor woman in the other car-"
He cut her off, interrupting to say, "If the car is gone, I'm need to speak to my accountant, Mr. Grinth, he'll need to have it replaced, tell him please." He received another disapproving look from the witch, for what he wasn't sure, and she sailed out of the room without another word. Draco relaxed back into his pillow with a satisfied smile, sure now that Mr. Grinth would handle everything.
Nearly two weeks later Draco was finally allowed to return to his house, an opulent brownstone in the Wizarding Quarter of London. He had ended up having some rather more severe internal injuries and so, despite his most charming cajoling, the doctors at St. Mungo's had refused to release him. The only reason he was even able to leave when he did was due to a rather substantial donation by Malfois Incorporated towards the new children's' wing at Mungo's.
He walked through the front door and took a deep breath, reveling in the scent of his favorite dinner, roast beef with Yorkshire pudding, wafting from the direction of the kitchen. "Krumbly!" he called out, his voice echoing loudly through the large, empty home and a moment later a loud crack sounded and a small, wrinkled house-elf appeared before him.
"Master!" he cried, wringing his hands excitedly. "We is so glad to see you. Grimble is be making your favorites for dinner."
Draco nodded in approval. "Excellent. I'm going to retire until dinner, please hold all calls, unless it's my mother." A noise that sounded oddly similar to a snort sounded behind him, but when he turned, there was nothing and no one to be seen. Dismissing the sound as the result of being cooped up in a hospital room for far too long, he turned to climb the stairs all the way to the third floor to the master bedroom and the comfort of his own bed.
He was barely through the door before he shed his robes, tossing them carelessly over the back of a chair and loosening the top few buttons of his shirt. A moment later he fell into the large bed that dominated the center of the room with a contented sigh. He had had the best of the best available at St. Mungo's of course, but their best was nothing near the comfort of the home he had so carefully assembled after the family's loss of Malfoy Manor.
He closed his eyes, allowing his mind to wander over all he would have to catch up on after his unexpected hiatus. A few minutes later he had all but abandoned his thoughts and was drifting in and out of a light sleep when a loud thud sounded. His eyes flew open and he sat up halfway, propping himself on his elbows to survey the room and find the source of the noise.
At first glance, everything seemed to be in order and he was tempted to write the noise off as leftover paranoia on his part, but when he took a second look, he spotted it, almost hidden by his hanging robes, a large red tome that seemed to have fallen off the bookshelf of its own volition.
"What the hell?" he muttered to himself as he climbed out of bed to investigate. He reached the book and bent to pick it up, a deep frown furrowing his brow as he flipped open the book to read the title. Les Secrets et la Sauvagerie de la Famille de Malfois.
No sooner had he made that particular discovery that another noise came, again behind him, the source this time the drapes sliding open and allowing the sunlight to pour into the room.
"Who's there?" he snapped, cursing inwardly as he realised he had left his wand sitting on the edge of his nightstand all the way across the room. Silence reigned in the room in answer to his question.
"Do you have any idea who I am?" he demanded of the seemingly empty room. This time though, he received an answer, in the form of an obviously human snort of laughter. He snarled, darting across the room to grab his wand, brandishing it aimlessly as he growled, "Show yourself or I swear on Merlin's grave I'll see to it that you rot in Azkaban for the rest of your life."
"Circe, Malfoy," a light, dulcet voice sounded behind him, "you seem to think you have quite a bit more power than I know you do."
He whirled to face the intruder and his sharply cut jaw dropped open. "Granger," he croaked. "Why the hell are you in my house?"
The woman standing on the opposite side of the bed raised one slim cinnamon brow. "Can you really not guess, Malfoy?" she asked incredulously. "I know you're not exactly my equal, but come now."
His mouth snapped shut and he frowned. "You're trespassing in my house, in my bedroom no less, and actually mocking me?" he finally exclaimed.
Hermione rolled her eyes and muttered something indistinguishable about choices before spreading her arms and offering calmly, "Kick me out then."
Draco scoffed. "Hurt the Gryffindor Princess and ruin my own life in the process? I don't think so, Granger."
Hermione let out a short, humourless bust of laughter and shook her head. "You can't hurt me Malfoy. But if you're not going to make me leave, then I have no choice but to stay." With that, she shifted to perch on the edge of his nightstand and stared at him expectantly.
His face flushed a crimson shade of red and he sputtered, "Granger, get out of my house or Merlin help me—"
"No, I can't."
"Bollocks. You're just being a typically pretentious, infuriatingly stubborn Gryffindor and I'm not going to stand for it in my home, consequences be damned. Incarcerous!" Thick ropes flew from the end of his wand... And landed on nothing but thin air and the corner of his mahogany nightstand.
"I told you that you couldn't hurt me, Malfoy," her voice sounded in his ear. He turned, finding himself nose to nose with Hermione Granger and stumbled backwards.
"How...how did you do that?" Hermione rolled her eyes at him again and then winked out of sight, appearing again near the bookshelf. This time though, Draco realised, he could see the bookshelf throughher body.
"Merlin," he breathed. "You're a ghost."
Hermione materialised fully and clapped her hands slowly. "Well done, Malfoy. You figured it out almost as quickly as I did."
"But...but you're not dead."
Hermione raised a brow, her tone slightly derisive as she said, "I think we just established that I am, unless you know of another way a person might become a ghost?"
"But if you're dead, why are you here? Why not off haunting Potty and Weasley or that dreadful old school library?"
Instead of answering him, Hermione turned her back, trailing her fingers over the spines of his books before casually asking, "How's the new car?"
"Gone now apparently, thanks to some—" He froze and all the colour leeched from his face. "The woman in the other car...the mediwitch said something about her. What happened to her?"
"She died," Hermione said plainly. "She died on her way home from visiting her Muggle parents because some asshole was too busy trying to impress his girlfriend to drive properly."
Draco sat down hard on the edge of his bed. "I killed you," he whispered, horror filling his voice.
"Give the man a medal," Hermione muttered bitterly under her breath and then, louder, "Yes, you did. And now you and I, for reasons I cannot even begin to understand nor do I particularly care to think about, are stuck together for what really may be the rest of eternity. So I'll be needing a room of my own. I'm sure you have space in this monstrosity of a house."
His head snapped up, disbelief flashing in his eyes. "Are you actually alright with this?"
"I don't really see how I have much of a choice, Malfoy."
He stared at her for several long moments, until she began to silently tap her foot. With a heavy sigh, he yelled, "Krumbly!" Then, he turned his attention back to her. "Just try to stay out of my way, Granger."
Hermione wasn't entirely sure why she had demanded a room from Malfoy, it had seemed to be the logical thing to do at the time. But now it was the middle of the night and she had been pacing for nearly three hours, as it turned out ghosts had no need for sleep.
The little house-elf that had appeared at Malfoy's summons had escorted her to what she assumed was a guest room, very tastefully furnished in varying delicate shades of blue. It was a perfectly acceptable place to stay; Malfoy seemed to employ a rather talented interior designer. The only flaw in the otherwise ideal space was that it was completely lacking in any form of entertainment, there wasn't even a decent book to be found.
It had taken her almost a week, but she had finally figured out how to interact with solid objects without the trauma of watching her hand slide right through them, and now it was her dearest wish in the world to just sit down with a good book and forget about everything for a few hours. Suddenly, she stopped mid-stride, her mind flashing back to the book she had found earlier, the one she had been trying to borrow before she had been discovered by Malfoy.
She glanced at the clock over the mantle. It was already half past two; no doubt the master of the house was fast asleep in his overly large bed by now. It would be nothing for her to sneak up and borrow the book; he would never even notice it was gone, judging by his surprise when he had read the title. Her mind made up, she slipped from the room. She opened the door out of habit—though she supposed it was no longer necessary—and made her way silently through the house.
The large house was utterly silent, almost eerily so, until she neared the door of the master bedroom. A loud cry echoed through the dark and she froze, momentarily forgetting that she couldn't be seen, until a whimper, like that of a hurting child, again broke the silence. A frown creased Hermione's brow as she neared the door, until she finally drifted through it unconsciously, more concerned with discovering what was happening inside that room.
What she found was surprising, to say the least. Draco laid curled up on the bed, whimpering in his sleep. He had tossed his covers to the ground, and was shivering in the cold night air, though Hermione couldn't imagine why he chose to sleep only in night pants in the middle of winter, so she supposed he deserved it.
Just then the man in the bed did something she had heard from Harry nearly too many times to count, letting out a bloodcurdling scream followed by heavy sobs. Hermione froze, intensely uncomfortable at witnessing the most private of moments from a man she had spent most of her life hoping to never see again.
As his sobs again quieted into whimpers, Hermione turned her eyes away from the somber sight and spied the book bound in red leather on the chair where it had been discarded earlier. Doing her best to block out the sounds of a nightmare that still permeated the room, she picked it up and carried it from the room, closing the door carefully behind her.
"Granger, why are you at my breakfast table?"
"Krumbly invited me."
"You don't eat."
"I like the light."
That night, Hermione roamed the house exploring, invisible to all but the cat who lived in the kitchen. She began her self-conducted tour on the third floor, sometime around two in the morning, hurrying past the door to the master bedroom, trying to pretend as if she didn't hear the moans of fear coming from inside the room. And yet, despite all her efforts, Hermione had no explanation for why she found herself standing outside that door until the dreams quieted.
"Morning, Malfoy."
"Sleep well?"
"Well enough."
"That's nice."
Hermione sat curled in her favourite chair, in a small, cosy sitting room on the second floor. The firelight flickered gently over the pages of her book, another selection from Malfoy's surprisingly extensive library. She was completely engrossed in the story of a brazen heroine and her dashing pirate lover when a very loud, very feminine scream echoed from above her head.
Hermione closed her book with an irritated snap. This was the third this week, just another in a long parade of beautiful girls of somewhat dubious intelligence. Hermione had caught a glimpse of this one as she stumbled tipsily up the stairs with this one and, judging by the sounds that kept interrupting her reading, she had a rather impressive pair of lungs for such a tiny woman. If Malfoy though that he could disrupt the entire house with his flavour of the evening, he was very much mistaken.
"Granger, what the hell were you thinking?"
"I don't appreciate having to listen to you cavorting about at all hours of the night, and I doubt the elves do either."
"So you decided it was a good idea to drop her knickers on her head at breakfast?"
"Well she left them on the floor, they were only going to get thrown out with the rubbish. And it's not as if she was coming back anyways."
"You don't know that...don't give me that look, Granger."
"Next time it will be more than knickers, Malfoy."
"Whatever you say Granger. Pass the paper."
Hermione slipped through the door, wincing as she saw the scratch marks the blond man had left on his arms in his sleep. Just as she had done so many times for Harry, Hermione moved without thought to sit on the edge of the bed, murmuring, "Shhh...It's only a dream."
She lifted one hand to tentatively stroke his hair in hopes of calming him back into sleep, but the absolute absurdity of her current situation didn't hit her until she watched her hand pass literally through the man's platinum blond hair and into his head. Without thinking, she laughed, at herself, at the fact that she, of all people, was sitting on Draco Malfoy's bed, and most of all that he was the only one who would ever know she was still there.
At the sound of her laughter, the man in the bed stirred, turning toward her and muttering blearily, "Hermione?" In an instant, she vanished from sight, fleeing the room, her mind whirling the entire way back to her room.
"Good morning, Malfoy."
"Morning, Granger."
"Did you sleep well?"
"Better than usual, thank you."
" Well, good."
"Are there any more books in the house? I've read through the library I'm afraid."
"I'll have Krumbly have more delivered."
"Oh. Thank you."
"Malfoy, wake up. Malfoy!" Hermione had listened to his screams for nearly five minutes before finally deciding something had to be done and attempting to wake him from the terrors of his dreams.
She reached out to shake him, growling in frustration as her hand passed through his shoulder and he continued to scream and thrash about. "Draco!" she yelled.
His icy blue eyes flashed open and locked on hers, his entire body shaking. "Granger," he whispered. "What's wrong?"
"You were screaming," she explained, blushing faintly. "I didn't think you should be left alone, it's not healthy."
"Oh, well thanks, Granger," he said, surprise evident in his eyes.
"Umm...you're welcome."
"Well, goodnight, again, Granger." He rolled over and closed his eyes.
"Goodnight, Malfoy," she answered quietly as she drifted from the room.
"Good morning, Granger."
"Good morning, Malfoy. Do you have the paper?"
"Here. Hey, Granger?"
"Thanks. Again."
"Not a problem."
Draco stood from his spot in the plush armchair by the fire, glancing over to where Hermione sat with a book. "Goodnight, Granger."
She glanced up, "Goodnight, Malfoy. Sleep well."
"Yeah, I'm sure," he scoffed.
"Malfoy...what are your dreams about?" Hermione ventured tentatively as the man turned to leave.
Surprise crossed his face but he answered slowly, "Mostly the War. Sometimes...sometimes I dream about you, about the accident I mean." A faint blush mottled his cheeks as Hermione stared, seemingly stunned and he turned to leave the room without another word.
Draco woke with a start several hours later to find Hermione leaning over him, concern in her eyes, although he doubted if she realized it was there.
"Again?" he asked groggily, groaning and covering his eyes when she nodded. "Sorry, Granger," he muttered.
"It's fine," she said softly, leaning up against the headboard where she sat. "It's not as if I sleep anyways."
He reached over without a thought to pat her leg in sympathy, flinching in surprise as his hand passed right though her.
"You can't touch me," Hermione informed him in a prime example of her know-it-all voice.
"Oh," Draco muttered lamely.
"Go back to sleep, Malfoy."
"Granger, wake up."
"I don't sleep, Malfoy."
"Oh yeah. Sorry."
"I take it you had better dreams?"
"Yes, how did you know?"
"Draco, just because you can't touch me doesn't mean I can't feel that!"
"Bloody hell, Hermione. I'm going to shower."
"Please. I'll see you at breakfast."
A/N: Hope you all enjoyed this little one-shot, don't forget to review!


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Meet ..... "MERCY - O -SAURUS"
16th Mar 2017
After our dinosaur hunt, we put our egg in water and waited for it to hatch. We looked at pictures of other dinosaurs and made predictions about what he might look like. When he hatched, we decided to call him "Mercy-o-saurus"! We are having great fun in our Dinosaur Den and finding dinosaur eggs in the sensory tray!
Mercy Primary School, 612 Crumlin Road Belfast County Antrim BT14 7GL | Telephone: 028 9039 1670
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Blanch Marie Ross, age 96, was born April 30, 1920 in Houston, Mo. to Adam "Estrel" and Delilah Caroline (Haney) Cole. She passed away April 4, 2017 in Houston, Mo.
Blanch grew up on Arthurs Creek in the Oak Hill community, the youngest of nine children. She attended Oak Hill School through the eighth grade, which was common in those days. During her teens she worked at Wests Variety Store in Houston.
Blanch married Eugene Ross on Sunday June 30, 1940 and moved into the home where she resided until her passing.
She is preceded in death by her parents; her husband Eugene and her siblings, Johnnie Cole, Jim Cole, Everett Cole, Cecil Cole, Myrtle Dawson, Jewell Farmer, Mamie Miller and Edna Jarvis.
She was an active member of Houston First Baptist Church since May 14, 1939 where she taught Bible School, played piano and was active in WMU. Blanch was always there to share vegetables, pies and cakes with members and friends who were ill.
Gardening, mowing, painting and planting flowers to maintain an attractive "home place" were a great enjoyment to her. The outdoors was where she liked to be. Camping, fishing, squirrel hunting and introducing her grandchildren to Piney River were a priority. She also thought it important that her grandson knew where to dig night crawlers and her granddaughter knew how to cut up a chicken and roll a pie crust.
Blanch is survived by her son, Gerald Ross and wife Sonya of Jefferson City, Mo.; her grandchildren, Kassondra Ross and Matthew Ross and a great-grandson.
She was blessed to have a full and active life with lots of humor and many friends.
Memorials may be made to the Gideon's. To send an online condolence, go to www.evansfh.com.
Services were held at 11 a.m. at Houston First Baptist Church Saturday, April 8, 2017 with Pastor Russ Stigall officiating. Burial was in Oak Hill Cemetery under the direction of Evans Funeral Home. Pallbearers were: Denny Thomason, Mark Manier, Todd Davidson, Don Miller, Ronnie Troutman and Wally Campbell. Honorary Pallbearers were: Carl Watson and Charlie Hartman.


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24/7-365. Basketball Lives Here.
Nate Robinson made his debut for Hapoel Tel Aviv in Israel this past weekend and he looked as sharp as ever. The 31-year-old proved why he still belongs in the NBA as he went off for 25 points, 4 rebounds and 3 assists and led his squad to the win.
The Seattle native electrified the crowd as he splashed deep three-pointers from all over the floor.
When arriving at the airport in Israel, Robinson stated that after his season finishes in Israel he will get back to training for the NFL.


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A Novel Ryanodine Receptor Mutation Linked to Sudden Death Increases Sensitivity to Cytosolic CalciumNovelty and Significance
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Rationale: Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT-associated RyR2 mutations cause fatal ventricular arrhythmias in young individuals during β-adrenergic stimulation.
Objective: This study sought to determine the effects of a novel RyR2-G230C mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the channel to luminal calcium (Ca2+).
Methods and Results: Functional characterizations of recombinant human RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2 mutant channels were generated by site-directed mutagenesis and heterologously expressed in HEK293 cells together with calstabin2. RyR2 channels were measured to examine the regulation of the channels by cytosolic versus luminal sarcoplasmic reticulum Ca2+. A 50-year-old white man with repeated syncopal episodes after exercise had a cardiac arrest and harbored the mutation RyR2-G230C. cAMP-dependent protein kinase–phosphorylated RyR2-G230C channels exhibited a significantly higher open probability at diastolic Ca2+ concentrations, associated with a depletion of calstabin2. The luminal Ca2+ sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like.
Conclusions: The RyR2-G230C mutant exhibits similar biophysical defects compared with previously characterized CPVT mutations: decreased binding of the stabilizing subunit calstabin2 and a leftward shift in the Ca2+ dependence for activation under conditions that simulate exercise, consistent with a "leaky" channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca2+ activation. Thus, diastolic sarcoplasmic reticulum Ca2+ leak caused by reduced calstabin2 binding and a leftward shift in the Ca2+ dependence for activation by diastolic levels of cytosolic Ca2+ is a common mechanism underlying CPVT.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare inherited arrhythmogenic disease characterized by syncope or sudden death induced by emotional or physical stress. The mortality rate in untreated individuals ranges from 30% to 50% by the age of 40 years. Dominantly inherited forms of CPVT have been linked to mutations in the cardiac ryanodine receptor (RyR2).1,2 Currently, >70 RyR2 mutations have been identified, and RyR2 mutations may account for 50% of diagnosed cases of CPVT.2,3
We report a novel CPVT mutation in RyR2 in a 50-year-old white man with a history of repeated syncopal episodes after exercise, beginning at age 30 years. The proband had a cardiac arrest and was diagnosed with CPVT. RyR2 genetic analysis performed by direct sequencing of the patient's DNA revealed a RyR2-G230C mutation. A brother died suddenly during exercise at age 16 years, and a son died suddenly of cardiac arrest after swimming at 12 years of age. A younger son was tested and does not carry the RyR2-G230C mutation.
Our group first elaborated a mechanism for CPVT by showing that CPVT and sudden cardiac death (SCD)-related RyR2 mutations decrease the binding of calstabin2 (FKBP12.6) to RyR2, which is further reduced by protein kinase A (PKA) phosphorylation during adrenergic stimulation.4–7 Moreover, we showed that CPVT-linked RyR2 mutations, including RyR2-S2246L, RyR2-R2474S, RyR2-R4497C, RyR2-P2328S, RyR2-Q4201R, and RyR2-V4653F, shift the sensitivity for cytosolic calcium (Ca2+)-dependent activation of the channel to the left, resulting in channels that are inappropriately activated at low diastolic levels of cytosolic Ca2+, thereby causing a diastolic sarcoplasmic reticulum (SR) Ca2+ leak.4,5,7 Importantly, the leftward shift in the sensitivity for cytosolic Ca2+-dependent activation of the CPVT mutant channels compared with WT channels was only observed after PKA phosphorylation of the channels (conditions that mimic the effect of exercise or stress on the channel). This is in agreement with the clinical phenotype of CPVT patients who have arrhythmias during exercise but not at rest.
Another group has proposed that increased sensitivity to luminal SR Ca2+ is the mechanism underlying leaky CPVT mutant channels. This group reported that CPVT, SCD, or arrhythmogenic right ventricular dysplasia–linked RyR2 mutations, including RyR2-E189D, RyR2-N4104K, RyR2-R4496C, and RyR2-N4895D, exhibited increased sensitivity to activation by luminal Ca2+, leading them to conclude that store overload–induced Ca2+ release (SOICR) is the cause of CPVT.8–10 A third group, using the CPVT mutant model RyR2-R2474S, suggested that CPVT and SCD-linked mutations induce defective interdomain conformational changes that destabilize the closed state of the channel and enhance its sensitivity to Ca2+.11,12 It has also been reported that RyR2-L433P and RyR2-N2386I mutant channels exhibit impaired sensitivity to Ca2+-dependent channel inhibition.13 More recently, 3 additional studies at the cellular level reported that the CPVT RyR2-R4496C mutant is abnormally Ca2+ sensitive and "leaky" and pointed out the importance of SR Ca2+ load in the genesis of Ca2+ waves and arrhythmias.14–16
These disparate conclusions have impaired a clear understanding of the mechanism underlying CPVT and require further investigation to resolve the differences.
We report a novel CPVT mutation, RyR2-G230C, and show that this mutation, like others we have reported,4–6 results in increased depletion of calstabin2 from the RyR2 macromolecular complex and enhanced sensitivity to cytosolic Ca2+ (leftward shift in the Ca2+-dependence for activation of the channel) and under conditions that simulate exercise-induced stress (β-adrenergic stimulation). Importantly, when examined at the single-channel level, both RyR2-G230C and RyR2-P2328S mutant channels exhibited normal luminal Ca2+ sensitivity compared with WT, indicating that their threshold for luminal Ca2+ activation is the same as WT channels. Thus, increased sensitivity to cytosolic Ca2+ can explain the leaky channel behavior of CPVT mutant RyR2 channels and indicate that altered sensitivity to luminal Ca2+ (SOICR) is not required to explain CPVT.
Recombinant mutant channels were generated and expressed in HEK293 cells. HEK293 cell lines expressing RyR2 wt and mutants were generated with the use of QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). Single-channel measurements were performed to investigate the RyR2 channel properties in planar lipid bilayers. Measurements of Calstabin2 binding to immunoprecipitated RyR2 were performed under PKA phosphorylation. A detailed Methods section can be found in the Online Data Supplement available at http://circres.ahajournals.org.
Clinical evaluation including cardiac catheterization revealed no cardiac structural abnormalities. ECG at rest showed normal sinus rhythm including a normal QT interval. Treadmill stress test (Bruce protocol) induced polymorphic premature ventricular beats (Figure 1).
Molecular Characterization of CPVT-Linked RyR2 Mutation
Blood samples were obtained by venipuncture, and genomic DNA was extracted from peripheral blood lymphocytes. We performed genetic testing of the proband's genomic DNA and confirmed it by dideoxy sequencing method. We found on genetic testing that the proband has a missense mutation with a Guanine to Thymine substitution at the 688th base of the RyR2 gene nucleotide sequence (RYR2–688G>T), which resulted in a glycine to cysteine substitution at the 230th position (G230C), close to the amino-terminus of the RyR2 protein (Online Figure I). This CPVT-linked RyR2 mutation has not been previously reported.
Pedigree of Index Family
The proband was a 50-year-old white man with a history of repeated syncopal episodes after exercise beginning at age 30 years. He had a cardiac arrest and was diagnosed with CPVT. His brother died suddenly during exercise at age 16 years, and his son died suddenly of cardiac arrest after swimming at 12 years of age. A younger son was tested and does not carry the RyR2-G230C mutation (Online Figure II).
Functional Characterization of CPVT-Linked Mutant RyR2-G230C Channels
To study the effects of the CPVT-associated RyR2-G230C mutations on the channel function, we characterized the biophysical properties of the single-channel activity (Figure 2). ER microsomes from HEK293 cells transiently coexpressing calstabin2 and either recombinant RyR2-WT or RyR2-G230C were fused into planar lipid bilayers. To test the functional properties of RyR2-G230C mutant channels under conditions simulating stress caused by increased activity of the sympathetic nervous system, we measured single-channel activity of the RyR2-WT and RyR2-G230C channels from microsomes that were subjected to PKA phosphorylation in vitro, using the planar lipid bilayer technique over a range of physiological Ca2+ concentrations, starting at 150 nmol/L up to full activation of the channel (≈1 to 10 μmol/L Ca2+). At low cytosolic calcium concentration (ie, 150 nmol/L free Ca2+) that corresponds to the resting diastolic Ca2+ level under basal conditions (no PKA treatment), the activity of RyR2-WT and RyR2-G230C channels was similar, displaying a very low open probability (Po) (mean Po, 0.007±0.004 for RyR2-WT, n=5; versus mean Po, 0.009±0.004 for RyR2-G230C, n=5; P=NS; Figure 2A and 2C). As the cis (cytosolic), Ca2+ concentration was increased, and the Po of both channels increased to a similar degree (mean Po, 0.041/0.359/0.531 at 350/700/1000 nmol/L Ca2+ for RyR2-WT versus mean Po, 0.108/0.310/0.626 at 350/700/1000 nmol/L Ca2+ for RyR2-G230C; P=NS; Figure 2D).
After PKA treatment, channel activity was increased for both RyR2-WT and RyR2-G230C. However, the Po of RyR2-G230C mutant channels was significantly higher compared with RyR2-WT channels at 150 nmol/L cytosolic Ca2+ (mean Po, 0.068±0.020 for RyR2-WT, n=5, versus mean Po, 0.182±0.038 for RyR2-G230C, n=7; P<0.05; Figure 2B and 2C). The activity of RyR2-G230C channels was also higher, with 350 nmol/L cytosolic Ca2+ (mean Po, 0.174±0.064 for RyR2-WT, n=5, versus mean Po, 0.338±0.032 for RyR2-G230C, n=7; P<0.05; Figure 2D). It should be noted that the mean closed time was comparable (data not shown). Once the channel was activated with further cytosolic Ca2+ (>700 nmol/L free Ca2+), no differences in Po were found between PKA-treated RyR2-G230C and WT channels (Figure 2D).
In addition to an increased sensitivity to low [Ca2+]cyt, amplitude histograms indicated an increased prevalence of subconductance states in the PKA-phosphorylated RyR2-G230C channels (Figure 2B) as previously observed for RyR channels that were PKA-phosphorylated and depleted of calstabin.5,17,18
Taken together, these results indicate that under stress conditions (mimicked by PKA phosphorylation) the CPVT-associated RyR2 mutation RyR2-G230C results in increased sensitivity to cytosolic Ca2+ causing "leaky" channels. These results are consistent with the observed functional effect of previously characterized CPVT and SCD-related RyR2 mutations4–6 and with the clinical observations that cardiac arrhythmias in CPVT patients occur during physical or emotional stress.
Biochemical Characterization of RyR2-G230C Mutation
Previous studies have shown that the depletion of calstabin2 from the RyR2 channel complex destabilizes the closed state of RyR2.4,5 This destabilization leads to enhanced diastolic Ca2+ sensitivity manifested as an increased RyR2 Po in the presence of low activating [Ca2+]=150 nmol/L.5,17,19 In support of this model, our group has demonstrated that recombinant CPVT and SCD-related RyR2 mutants, expressed in HEK293 cells, exhibit a reduced affinity for calstabin2 compared with WT channels.4,5
To determine whether the increased Po and gating changes observed for the PKA-phosphorylated RyR2-G230C mutant channels were associated with depletion of the stabilizing subunit calstabin2 protein, RyR2 macromolecular complexes were immunoprecipitated from HEK293 cell membranes. Whereas untreated channels revealed only a basal level of PKA phosphorylation, PKA-treated RyR2-WT and RyR2-G230C channels exhibited a comparable high level of PKA phosphorylation (Figure 3A and 3B) and significantly lower amounts of calstabin2 bound to the PKA-phosphorylated RyR2-G230C mutant channels compared with the RyR2-WT treated channels (Figure 3A and 3C). The significantly lower amount of calstabin2 bound to the PKA-phosphorylated RyR2-G230C mutant channels suggests that the CPVT-associated mutation decreases the binding affinity of calstabin2 to the mutant RyR2, as previously reported for other CPVT-linked RyR2 mutations.4,5
CPVT-Linked RyR2-G230C and RyR2-P2328S Mutations Do Not Affect Luminal Ca2+ Sensitivity
To study the effects of RyR2-G230C mutation on the luminal Ca2+ sensitivity, we performed single-channel measurements using Ba2+ as the charge carrier with added Ca2+ to the luminal (trans) side at low cytosolic (cis) Ca2+ in absence of allosteric modulators (ie, no PKA treatment, no Mg-ATP nor caffeine). Addition of 1 mmol/L Ca2+ to the luminal side did not significantly increase the single Po of recombinant RyR2-G230C and WT channels (mean Po, 0.016±0.011 at 0 mmol/L luminal [Ca2+], n=8, versus mean Po, 0.038±0.021 at 1 mmol/L luminal [Ca2+] for RyR2-WT, n=5, and mean Po, 0.013±0.006 at 0 mmol/L luminal [Ca2+], n=10, versus mean Po, 0.032±0.015 at 1 mmol/L luminal [Ca2+] for RyR2-G230C, n=8; P=NS; Figure 4A and 4C). Addition of 5 mmol/L luminal Ca2+ did not increase Po significantly in either recombinant channel groups (mean Po, 0.047±0.008 for RyR2-WT, n=5, versus mean Po, 0.033±0.012 for RyR2-G230C, n=7; P=NS; Figure 4B and 4C). Addition of 10 mmol/L luminal Ca2+ induced comparable significant increases in Po in both WT and CPVT channels when compared with the Po obtained at 0 mmol/L luminal Ca2+ (mean Po, 0.016±0.011 at 0 mmol/L luminal [Ca2+], n=8, versus mean Po, 0.100±0.024 at 10 mmol/L luminal [Ca2+], n=5, for RyR2-WT, P<0.01; and mean Po, 0.013±0.006 at 0 mmol/L luminal [Ca2+], n=10, versus mean Po, 0.084±0.034, n=5, for RyR2-G230C, P<0.05; Figure 4B and 4C).
However, even at 10 mmol/L luminal [Ca2+], no difference in sensitivity to activation by luminal Ca2+ was observed between WT and RyR2-G230C recombinant channels (mean Po, 0.100±0.024 for RyR2-WT, n=5, versus mean Po, 0.084±0.034 for RyR2-G230C, n=5; P=NS; Figure 4C).
To reduce the baseline effect of divalent cations on the luminal side of the channel and to avoid any contaminating Ca2+ in the Ba2+ solution, we also tested the effect of luminal Ca2+ in absence of other divalent ions. We performed single-channel measurements using Cs+ as the primary current charge carrier with added Ca2+ to the luminal (trans) side in presence of EGTA. In agreement with the above Ba2+ experiments, Po obtained with 0, 1, 5, and 10 mmol/L free Ca2+ to the luminal side was comparable between RyR2-G230C and WT (mean Po, 0.230/0.336/0.330/0.498 at 0/1/5/10 mmol/L Ca2+, n=5 for each, for RyR2-WT versus mean Po, 0.262/0.288/0.400/0.317 at 0/1/5/10 mmol/L Ca2+, n=4 for each, for RyR2-G230C; P=NS; Online Figure III).
Thus, our results indicate that the sensitivity of RyR2-G230C mutant channel to activation by luminal Ca2+ is the same as that of WT channels, demonstrating that there is no requirement to invoke the SOICR hypothesis to explain the mechanism of leak observed for the CPVT-linked RyR2-G230C channels.
RyR2-P2328S is a CPVT-linked missense mutation that is located in the central region of the RyR2 gene near the FKBP12.6 binding site.20 We first observed that recombinant human PKA–phosphorylated RyR2-P2328S mutant exhibits decreased binding of calstabin2 and has a significant gain-of-function defect consistent with a "leaky" channel at low cytosolic Ca2+ and with a rightward shift in the half-maximal inhibitory Mg2+ concentration (IC50).7 The murine model of RyR2-P2328S mutation was then characterized by Goddard et al,21 who observed altered cellular Ca2+ handling linked to CPVT-linked arrhythmias with a more pronounced effect in the homozygote than the heterozygote.
To determine whether the RyR2-P2328S mutation exhibits a reduced threshold for luminal Ca2+, we measured the effects of increasing luminal [Ca2+] in the presence of 3 mmol/L Mg-ATP and 0.6 mmol/L MgCl2 at 150 nmol/L [Ca2+]cyt (Figure 5A and 5B).
Data were averaged and Po increased as luminal Ca2+ increased, with no significant difference between RyR2-P2328S and WT recombinant channels (eg, mean Po, 0.062±0.037 at 0.2 mmol/L luminal [Ca2+], n=5, versus mean Po, 0.110±0.070 at 1 mmol/L luminal [Ca2+] for RyR2-WT, n=4, and mean Po, 0.026±0.022 at 0.2 mmol/L luminal [Ca2+], n=4, versus mean Po, 0.042±0.037 at 1 mmol/L luminal [Ca2+] for RyR2-P2328S, n=4; P=NS; Figure 5A and 5C).
Increased [Ca2+] on the luminal side of the channel slightly increased single-channel Po in both groups, with no significant differences between WT and RyR2-P2328S channels (eg, mean Po, 0.160±0.060 at 5 mmol/L luminal [Ca2+], n=4, versus mean Po, 0.209±0.086 at 10 mmol/L luminal [Ca2+] for RyR2-WT, n=4, and mean Po, 0.161±0.109 at 5 mmol/L luminal [Ca2+], n=4, versus mean Po, 0.070±0.036 at 10 mmol/L luminal [Ca2+] for RyR2-P2328S, n=4; P=NS; Figure 5B and 5C). Thus, like the hRyR2-G230C channels, the CPVT-linked recombinant hRyR2-P2328S channels exhibit the same sensitivity to luminal [Ca2+] as WT RyR2 channels.
In the present study, we report a novel CPVT-linked RyR2 mutation that results in the substitution of cysteine for glycine at position 230, RyR2-G230C. The mutation is located in the disease-associated "hot spot" N-terminal domain of cardiac RyR2.20,22–24 We coexpressed recombinant hRyR2 channels and the stabilizing subunit calstabin2. Our experiments indicate that the functional consequences of the RyR2-G230C mutation are a "leaky" RyR2 channel at diastolic Ca2+ (low cytosolic [Ca2+]) associated with reduced binding of calstabin2 to the RyR2 channel that occurs only under stress conditions. Moreover, because previous CPVT/arrhythmogenic right ventricular dysplasia RyR2 mutants were proposed to have increased luminal Ca2+ sensitivity,8–10 we compared the effects of luminal (trans) Ca2+ on RyR2-G30C mutant channel properties and did not find any differences in the CPVT channels compared with WT channels. Additionally, we also compared the luminal Ca2+ dependence of another CPVT mutant, RyR2-P2328S, known to be hypersensitive to cytosolic Ca2+.7,21 Like the RyR2-G230C channels, the RyR2-P2328S mutant channels do not display any abnormalities in responsiveness to luminal [Ca2+]. These results show that the threshold for luminal Ca2+ activation is the same in WT and 2 CPVT mutant channels, and therefore the SOICR mechanism is not a generalized mechanism for CPVT.
Overall, our results reinforce the molecular mechanism by which, under the β-adrenergic stimulation, CPVT-associated RyR2 mutations lead to a pathologically increased cytosolic Ca2+ sensitivity associated with a depletion of calstabin2. Furthermore, our results indicate that the RyR2-G230C mutant channels exhibit increased cytosolic Ca2+ sensitivity that is not dependent on luminal Ca2+ and thus do not support the concept of store overload–induced Ca2+ release as a generalized mechanism for CPVT.8–10,25
In the present report, we found a single-base substitution in the RyR2 gene in a 50-year-old man. The proband had repeated syncope episodes after exercise beginning at age 30 years, had a cardiac arrest, and was diagnosed with CPVT. This single-base substitution in Ryr2 resulted in a novel RyR2-G230C mutation close to the amino-terminus with similar biophysical defects as other mutations in the middle and carboxy-terminus of the RyR2 channel and is associated with decreased binding of the stabilizing subunit calstabin2.
Cardiac arrhythmia susceptibility genes that have been identified predominantly encode ion channels.26,27 Our work strongly suggests that the single point mutation RyR2-G230C is linked to the cardiac pathological phenotype observed in the proband. A critical feature of CPVT is that triggering of an arrhythmogenic episode occurs in the absence of structural alterations to the heart and requires the presence of both a causal mutation and stressful conditions that induce β-adrenergic stimulation. Our results support the model by which CPVT-associated mutations in the RyR2, under stress conditions (ie, mimicked by the PKA phosphorylation of RyR2 in vitro), induce a significant increase in the cytosolic Ca2+ sensitivity and highlight the link with the calstabin2 binding from the RyR2 macromolecular complex.
Several studies have confirmed that CPVT and SCD-related RyR2 mutations lead to a cytosolic Ca2+ hypersensitivity, providing support for the Ca2+-induced Ca2+ release mechanism rather than the SOICR hypothesis.4–6,14 In line with this view, Fernandez-Velasco et al14 have observed an increase in Ca2+ spark frequency in RyR2-R4496C permeabilized cardiomyocytes, dependent on the diastolic intracellular Ca2+ concentration (ie, nmol/L range of [Ca2+]) as well as an increase in cytosolic Ca2+ sensitivity in crude SR membrane of RyR2-R4496C using [3H] ryanodine binding experiments.
It is technically difficult to distinguish the SOICR mechanism from the Ca2+-induced Ca2+ release mechanism because luminal Ca2+ may flow through the channel and modulate the cytosolic Ca2+ binding sites of RyR2.28,29 Indeed, previous studies have shown that luminal Ca2+ regulates the cardiac RyR2 through direct feedback by binding to cytosolic Ca2+ activation and inactivation sites.30 Therefore, any experiments performed using high concentrations of luminal Ca2+, which passes through the channel, will be subject to the limitation that this luminal Ca2+ can activate cytosolic sites after it passes through the channel. Therefore, if CPVT channels are more sensitive to cytosolic [Ca2+] than WT channels, luminal Ca2+ passing through the channel could result in increased Po in CPVT channels not because of a reduced threshold for activation by luminal Ca2+ (as proposed by the SOICR hypothesis) but rather because of increased sensitivity to cytosolic Ca2+, as we have shown previously.4–7 To circumvent this limitation, we used Ba2+ as the charge carrier in the luminal (trans) bilayer chamber and tightly controlled the free cytosolic (cis) [Ca2+] with EGTA in a range from ≈150 nmol/L to 10 μmol/L at 0 mV. Although Ba2+ can pass through the RyR2 pore, Ba2+ ions cannot activate RyR2 channels and therefore cannot explain any increases of the channel Po. To determine whether the RyR2-G230C mutation increases the sensitivity to activation by luminal Ca2+ and thus increases the open probability of the channel, we examined the effects of Ca2+ added to luminal (trans) chamber, in the presence of low cytosolic Ca2+ (ie, 150 nmol/L free Ca2+) and in the absence of any agonist (eg, no ATP, no caffeine, and no PKA phosphorylation), because the SOICR mechanism has been previously reported for some CPVT mutants but only in absence of PKA phosphorylation of the channel. Adding Ca2+ on the luminal side of the channel induced a slight increase of Po, with no significant difference between RyR2-G230C and WT channels. Furthermore, we performed experiments on RyR2-P2328S channels, testing increasing concentrations of luminal Ca2+ in the presence of the physiological channel modulators Mg-ATP and MgCl2 and did not find any differences in Po compared with WT channels. Our data show that SOICR is not a generalized mechanism for CPVT; however, we cannot exclude that regulation of RyR2, by luminal SR proteins such as calsequestrin, can modulate the Ca2+ sensitivity of the channel.28,29 Thus, our results and those of others4–6,14 indicate that the Ca2+-induced Ca2+ release mechanism can explain CPVT and that there is no need to invoke SOICR as an alternative mechanism, as recently explained.28
The RyR2-G230C mutation is located in the N-terminal portion of the channel. The N-terminus of RyR2 is one of the three major regions that harbor disease-causing mutations.20,22,23,31 Several biochemical and functional studies have indicated that disease-associated RyR mutations are located along the domain interface between the N-terminal and central regions of RyRs and weaken interdomain interactions (the domain unzipping hypothesis).11,12,32 A recent crystal structure provided an insight into the hotspot domain I of RyR and revealed the exact locations of more than 50 disease-associated mutations.24 Interestingly, these results indicated that Glycine 230 of hRyR2 is located in a critical subunit interface area that contains more than 19 disease-associated mutations in RyR1 and RyR2. Therefore, it is reasonable to propose that a single point mutation such as G230C may destabilize the contact and/or the folding of individual RyR2 domains, destabilizing the closed state of the channel. G230C mutation may also induce a decrease of the RyR2 protein level and/or the associated calstabin2. Our results show that the protein levels of immunoprecipitated recombinant RyR2-G230C and WT as well as the associated calstabin2 are similar. Because we use HEK293 cells heterologously expressing RyR2 and calstabin2, we cannot make any statement about RyR2 or calstabin2 protein levels in vivo. However, we have previously reported that knock-in mice expressing RyR2 harboring human CPVT mutations have normal levels of RyR2 and calstabin2 proteins.4 Our results also indicate that the RyR2-G230C mutation alters the channel activity only after PKA phosphorylation and leads to greater depletion of calstabin2 from the channel complex associated with a higher sensitivity to low cytosolic [Ca2+] compared with WT. Thus, using immunodetection of Western-blotted proteins, we do not observe any difference in the associated calstabin2 level between RyR2-G230C and WT when the channels are not PKA-phosphorylated. Previous results from our group indicate that using 35S-labeled Calstabin2, CPVT-associated mutants RyR2-S2226L, RyR2-R2474S, and RyR2-R4497C have decreased basal affinity for Calstabin2.5 We also previously reported that a transgenic mouse overexpressing calstabin2 can prevent leaky RyR2 and heart failure progression.33 Interestingly, the PKA phosphorylation level of pS2809 is similar between RyR2-G230C and WT. Thus, the consequences of G230C mutation on RyR2 might be dependent on allosteric or charge effects of the PKA phosphorylation that promote channel openings. These observations are in agreement with previous published results showing that CPVT-associated RyR2 mutations, located at different regions of the amino acid sequence (eg, RyR2-R2474S and RyR2-V4653F), exacerbate the effect of PKA phosphorylation, as evidenced by an increased depletion of calstabin2 from the RyR2 complex.5,7
Although our results rule out the possibility that G230C mutation may affect the closed state of the channel (ie, mean closed time was comparable), more experiments will be needed to investigate the Ca2+-dependent inhibition of RyR2-G230C channels, because some CPVT-related RyR2 mutants exhibit sensitivity to Ca2+-dependent channel inhibition.13 Moreover, RyR has highly reactive cysteines able to form disulfide bonds.34 Therefore, the cysteine introduced by the CPVT mutation may interact with other cysteines in the channel, disturbing folding or inducing conformational changes to the channel. Further experiments under reducing conditions are needed to determine whether the substitution of cysteine for glycine induces conformational changes that destabilize the channel and increase sensitivity to cytosolic Ca2+. Our recent results from a murine model indicate that catecholamine treatment can result in oxidization of RyR2 in addition to causing PKA phosphorylation. The combination of oxidation and PKA phosphorylation can activate the channel and promote diastolic SR Ca2+ leak.35 The addition of an extra cysteine in RyR2-G230C could increase the effect of channel oxidation during stress. However, other CPVT mutations that do not involve addition of a cysteine, such as RyR2-P2328S, result in similar effects on channel properties.
In conclusion, we have demonstrated a novel, functionally significant CPVT mutation in RyR2. The consequences of this mutation are an increased sensitivity to cytosolic Ca2+ under stress, associated with a depletion of calstabin2 from the RyR2 macromolecular complex, when the PKA phosphorylation level at serine 2809 is similar to the level in WT. These observations reinforce the model of "leaky" RyR2 channel through an altered calstabin2 binding as a mechanism by which CPVT-related RyR2 mutations cause arrhythmias.
Sources of Funding
This work was supported by grants from the National Heart, Lung, and Blood Institute to A.R.M.
A.R.M. is a consultant for ARMGO Pharma, Inc, a biotech company targeting RyR2 treatment for prevention of CPVT.
We thank Jeffrey Rossi for excellent technical assistance.
In April 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days.
Non-standard Abbreviations and Acronyms
- catecholaminergic polymorphic ventricular tachycardia
- RyR
- ryanodine receptor
- sarcoplasmic reticulum
- cAMP-dependent protein kinase
- sudden cardiac death
- store overload–induced Ca2+ release
- Received March 16, 2011.
- Revision received May 24, 2011.
- Accepted May 26, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Individuals with catecholaminergic polymorphic ventricular tachycardia (CPVT)-linked mutations in the cardiac ryanodine receptor (RyR2) have exercise-induced arrhythmias, normal ECGs, and no arrhythmias at rest.
Fatal ventricular arrhythmias are caused by intracellular calcium leak through mutant CPVT-linked RyR2 channels on the sarcoplasmic reticulum.
CPVT mutations in the central and carboxy terminal domains of RyR2 cause decreased binding to the channel-stabilizing subunit calstabin2, rendering the RyR2 channels leaky, and the calcium leak is exacerbated by exercise, leading to fatal arrhythmias.
What New Information Does This Article Contribute?
A new clinically significant mutation RyR2-G230C that leads to CPVT is reported.
The novel RyR2-G230C mutation near the amino terminus induces calcium leak by decreasing the binding of calstabin2 to the RyR2 macromolecular complex, resulting in increased sensitivity of the channel to activation by diastolic calcium levels during stress.
CPVT-linked mutations RyR2-G230C and RyR2-P2328S do not affect the threshold for luminal calcium activation and do not support an alternative mechanism called store overload–induced calcium release.
In this article, we report the identification and characterization of a new CPVT-linked RyR2-G230C mutation. To investigate the functional properties of the RyR2-G230C mutation, we generated and expressed recombinant human RyR2 mutant channels together with calstabin2. We performed single-channel experiments to investigate the biophysical properties of the mutant channels and to test the molecular mechanisms for CPVT. We show that RyR2-G230C mutant channels exhibit a calcium leak during stress because of decreased binding to calstabin2, which results in increased sensitivity of the mutant channels to activation by diastolic calcium levels. The results also indicate that RyR2-G230C, as well as a previously reported RyR2-P2328S CPVT-linked mutant channel, exhibits normal thresholds for activation by luminal SR calcium. These results show that there is no need to invoke the store overload–induced Ca2+ release mechanism. The present study shows that a CPVT-linked mutation located near the amino terminus of RyR2 shares a common mechanism for triggering arrhythmias by rendering the channel leaky as the result of decreased calstabin2 binding and increased sensitivity to activation by low levels of cytosolic calcium.
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- A Novel Ryanodine Receptor Mutation Linked to Sudden Death Increases Sensitivity to Cytosolic CalciumNovelty and SignificanceAlbano C. Meli, Marwan M. Refaat, Miroslav Dura, Steven Reiken, Anetta Wronska, Julianne Wojciak, Joan Carroll, Melvin M. Scheinman and Andrew R. MarksCirculation Research. 2011;109:281-290, originally published July 21, 2011https://doi.org/10.1161/CIRCRESAHA.111.244970
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- A Novel Ryanodine Receptor Mutation Linked to Sudden Death Increases Sensitivity to Cytosolic CalciumNovelty and SignificanceAlbano C. Meli, Marwan M. Refaat, Miroslav Dura, Steven Reiken, Anetta Wronska, Julianne Wojciak, Joan Carroll, Melvin M. Scheinman and Andrew R. MarksCirculation Research. 2011;109:281-290, originally published July 21, 2011https://doi.org/10.1161/CIRCRESAHA.111.244970


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Rowing just isn't uncomplicated, and for the long term it's endurance, symbol for Frank's rise to electric power and presidency at most effective?
Frank wins A significant struggle, but will not pause to rest on his laurels. Peter ought to confront his earlier. Stamper hushes a mystery.


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LOOKOUT Schooner, ashore near Bar Point, Lake Erie, April 1866; got off. property loss $200
Casualty List for 1866---Buffalo
Commercial Advertiser, Feb. 26, 1867
VESSEL ASHORE.---Yesterday morning, at about 1 o'clock, the schooner LOOKOUT, Capt. Graves, went ashore a short distance below Bar Point, Lake Erie, caused, it is believed, by some derangement of her compasses. She took her departure from Sandusky on Wednesday for Saginaw, calling at Kelly's Island for a load of stone, from which point she pursued the course usually taken by vessels for the Detroit river, a N.W. by W direction. The night was somewhat misty, and land was not discovered until too late to avoid going ashore. The severe weather of yesterday has doubtless went hard with her. --- Detroit Post, 23rd.
Toledo Blade
April 23, 1866
Schooner LOOKOUT All Right.--- The Schooner LOOKOUT, which was reported ashore at Bar Point, we are pleased to learn, has been got off by the tug T.F.PARK, and is apparently unharmed. The heavy S.W. gale on Saturday disturbed the waters considerably where she lay, but owing to her strong build, she sustained no injury whatever. Detroit Post, 24th.
Toledo Blade
April 24, 1866
Schooner LOOKOUT. U. S. No. 14675. Of 226.22 gross tons; 214.91 tons net. Built Buffalo, N.Y., 1855. Home port, Kenosha, Wis. 127.0 x 27.0 x 10.0
Merchant Vessel List, U. S., 1885
The Maritime History of the Great Lakes site is managed by Walter Lewis
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Ray Rice shouldn't be expecting a warm welcome when he and his Ravens travel to Cleveland this weekend. He is accused of spitting on one of the Dawg Pound's favorite players. Rice should be aware of how the fans in Cleveland can treat a player from a visiting team that has upset them.
In game one of this AFC North divisional match up, you could see how upset Phil Taylor was after making a play on the Ravens running back. When Taylor got of the pile, he looked like he wanted to kill Rice. Taylor stands by his statement that Rice spit in his face when they were on the ground.
Phil Taylor hasn't made these types of assertions in the past. He is a guy that seems to hold his temper while on the field. That wasn't the case on September 15. Rice had to do something to make this big man angry.
The fans in Cleveland aren't going to let this incident slide, they will let Rice know he is in hostile territory and it won't be easy for Rice to get through this game. He better be aware of flying objects and he could even be spit on if he gets to close to those angry Dawgs.
Filed under: NFL
Tagged with: Baltimore Ravens, Cleveland Browns, Cleveland's Dawg Pound, Phil Taylor, Ray Rice
who u kiddin'? The Dawg pound is more like a puppy pound! Rays' goin to run all over Phil and the rest of those puppies. By how this sounds – I bet you are a Clev. beat writer. Ravens will soar, bank on it
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Words that end in etmed
Found 2 words that end in etmed. Browse our Scrabble Word Finder, Words With Friends cheat dictionary, and WordHub word solver to find words that end with etmed. Or use our Unscramble word solver to find your best possible play! Related: Words containing etmed
- Scrabble
- Words With Friends
- WordHub
- Crossword


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Victoria's Secret
If you received these coupons in the mail from Victoria's Secret you can do this deal in-store only now thru May 11 just in time for Mother's Day.
Buy 2 Victoria's Secret T-Shirt Bras priced at $38.50ea.
Total $77.00
Use the mailer coupons for a $10 off both bras, a Free Umbrella
($68 value--pick from 2 different styles-while supplies last)
and get a Free Cotton Panty!
AND if you have a Secret Rewards Card (valued at $10, $50, $100, or $500) from a previous deal that ended March 31st then you can use on this deal too!
Use $10 Secret Reward Card and you pay $57.00 for both bras plus the extra freebies!
My store did not allow the 2 for $49.50 styles or the 2 for $42.50 Pink styles on this deal but you can ask at your store if they do.
Here's an old deal I did to get a Secret Rewards Card.


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I dream of a day when I am completely organized. HA! The only comfort I have is it seems every other Facebook update is about cleaning out. About every third blog entry I read is about purging and organizing and the other housewives I know all have the same complaint…. Too much stuff, not enough room.
Well I am not in the same boat in the room department. I think our house is plenty big (probably too big) for our needs but the "stuff" is a continual battle. I can always tell how stressed out my life is by how many piles are laying around my house. My stress levels are actually not too high these days but I seems I am falling victim to my old ways of I can not get rid of something if I see its value.


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Cute new spring ribbons and yes these stripes do have glitter!
Monthly Archives: February 2014
Burlap Pennants
Rent a Room
Do you teach or sell something fun (necklaces, skirts, art, oils, etc) and want a new place to showcase your goods ?
We want to team up with you!
We are renting our classroom area to anyone who'd like to use it. We think our customers and your customers would enjoy it.
For a short time we are offering an introductory rate of
$50 a day (10am-4pm)
*Some exclusions apply, please call or email Irene or Summer for details.


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Lexington Insurance Company v. Collins et al
Plaintiff: Lexington Insurance Company
Defendant: Emily Katherine Weitzel, Peter Daniel Collins and Daniel Ivan Porter
Case Number: 3:2007cv01268
Filed: March 5, 2007
Court: California Northern District Court
Office: San Francisco Office
County: XX US, Outside State
Presiding Judge: Bernard Zimmerman
Nature of Suit: Fraud or Truth-In-Lending
Cause of Action: 28:1332 Diversity-Fraud
Jury Demanded By: Defendant
Access additional case information on PACER
Use the links below to access additional information about this case on the US Court's PACER system. A subscription to PACER is required.
Access this case on the California Northern District Court's Electronic Court Filings (ECF) System
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« All Events
Courtesy Alliance for Living and Dying Well.
The Alliance for Living and Dying Well understands the importance of preparing for end-of-life care. With various FREE workshops each month, the Alliance offers another community workshop this Wednesday, April 19, from 3-5 p.m. at the beautiful Garden Court (1116 De La Vina St.). As a part of their efforts to enhance end-of-life services, the Alliance clarifies personal, emotional, and spiritual needs as well as medical wishes by completing an advanced care directive.
To help you or your family member complete your health care directive, please call (805) 845- 5314 or register here!
—Danielle Hazlett
An event every day that begins at 11:00am, repeating until April 22, 2018
An event every day that begins at 12:00pm, repeating until April 29, 2018
Alliance for Living and Dying WellCommunity WorkshopFree Community WorkshopsGarden Courtsanta barbaraSanta Barbara Quarterly Magazinesanta barbara seasonssanta barbara seasons magazinesb seasonsSB Seasons Magazineseasonsseasons magazinethings to do in Santa Barbara
SantaBarbara Seasons
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Meetings & Events
Corporate Golf Events
Bintan Lagoon Golf Club is a great choice for client or staff corporate golf events and we are able to provide a complete package to suit your needs.
We have hosted many successful corporate golf events over the years. Our experience and expertise means that your corporate golf event will be one to remember. It's important to us that your corporate golf event is a resounding success, so we take a flexible approach and can tailor a day to meet your needs.
We work closely with you to ensure that we align with your preferences, from registration facilities, refreshments through to presentation and the post-round food. We would be delighted to discuss menu choices with you. Our two 18-hole championship golf courses designed by professional golfers Ian Baker Finch and Jack Nicklaus are among the best in the region and promise an invigorating, memorable experience for golfers of all levels.
Everything you need is provided on site and we have a fleet of golf buggies available for hire (pre-booking is essential) as well as sets of clubs for rent. We are happy to take care of all your golf related activities including the draw, scoring, handicapping, advice on the rules and organizing popular competitions such as longest drive and nearest to the pin, meeting rooms, entertainment etc. For larger groups, a shotgun start can be organized as well.
Our Clubhouse staff can assist with checking scorecards, finalising competitions and preparing prize sheets for your group. The club has excellent practice facilities, which can be very beneficial for those who have not played in awhile. We are confident the post-round get-together will be as memorable and enjoyable as the golf.
Corporate golf day organisers are also welcome to place signs on the course at agreed places to gain exposure for their company or event. Or you could consider "selling" holes (having them sponsored for the day) which is a great way for charities to raise money for their good cause .
The club also makes a great location for business meetings, team building, dinner & dance before, after your golf event.
Contact us at firstname.lastname@example.com


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Wednesday, April 19, 2017 at 5:00pm to 7:00pm
Klarman Hall, Rhodes-Rawlings Auditorium
The Latin American Studies Program and Cornell SC Johnson College of Business Emerging Markets Institute are pleased to announce a special panel to discuss multilateral relations between the United States, China, and Latin America. The election of Donald Trump in November of 2016 introduced uncertainty into the global community. Included in this uncertainty is the condition of relations with critical partners to the United States in Latin America and China as well how the Trump Administration's recalcitrant stance towards trade may open the door for deeper ties between Latin American states and China.
Speaking on the panel are three experts concerning multi-lateral relations between the United States, China, and Latin America. Lourdes Casanova, Director of the Emerging Markets Institute at Cornell University, He Li, Professor of Political Science at Merrimack College, and Benjamin Creutzfeldt, Postdoctoral Fellow at Johns Hopkins SAIS, will speak on the current state and future status of investment ties, trade, Taiwan's status, and the Trans-Pacific Partnership.
Following brief remarks from each of the panelists members of the audience will be allowed to ask questions. The panel is sponsored with generous donations from the Cornell Institute for China Economic Research, the Departments of Government and Economics, the Society for the Humanities, and the Mario Einaudi Center for International Studies
Panel Members:
Lourdes Casanova is a Senior Lecturer and Academic Director of the Emerging Markets Institute at the Johnson School of Business at Cornell University, formerly at INSEAD. Dr. Casanova specializes in international business with a focus on emerging markets multinationals. In 2014 she was appointed as one of the 50 most influential Iberoamerican intellectuals by Esglobal. A Fulbright Scholar with a Masters degree from the University of Southern California and a PhD from the University of Barcelona is member of Latin America Global Agenda Council and the Competitiveness in Latin America taskforce of the World Economic Forum, Advisory Committee European Union/Brazil, World Investment Network at UNCTAD, the B20 Task Force on ICT and Innovation in Los Cabos, responsible at INSEAD of Goldman Sachs 10,000 women initiative and co-leading InnovaLatino on Innovation in Latin America. Dr. Casanova is a board member of the Boyce Tompson Institute, the start-up Documenta, founding Board Member of the Societé des Amis du Chateau de Fontainebleau and member of the Advisory Council of the Tompkins Public Library
He Li is a professor of Political Science at Merrimack College and joined the faculty there in 1994. Dr. Li specializes in comparative politics and international political economy with expertise in Chinese and Latin American politics and relations. Dr. Li has published extensively on Sino-Latin American relations writing numerous articles and book chapters on the subject. He has also been awarded numerous grants and fellowships including those from the Ford Foundation, Fulbright, Tinker Foundation, and the CCK Foundation. Dr. Li received his PhD from University of Texas at Austin in 1991.
Benajmin Creutzfeldt is a postdoctoral fellow on Sin-Latin American-U.S. Affairs at the Foreign Policy Institute in the Johns Hopkins School of Advanced International Studies. A China scholar of German and British extraction, he has been a lecturer for Chinese studies and international relations in Colombia for the past eight years. He received his Ph.D. in political studies in 2015 for research on China's foreign policy towards Latin America. Dr. Creutzfeldt is also a Research Fellow of the Mr. & Mrs. S.H. Wong Center for the Study of Multinational Corporations and a visiting professor at the International School for Economics and Management at the Universidad de la Sabana, Bogota, Colombia. He is a member of the Red ALC-China network based at UNAM University in Mexico, a member of the International Studies Association (ISA), the Latin American Studies Association (SOAS), and the Association for Chinese Political Studies (ACPS).
Cornell SC Johnson College of Business, Samuel Curtis Johnson Graduate School of Management, Mann Library, Mario Einaudi Center for International Studies, Cornell Institute for Public Affairs, Latino Studies Program, Asian Studies, Government, Olin Library, Society for the Humanities, Institute for the Social Sciences, East Asia Program, Latin American Studies Program (LASP), Department of Development Sociology (DSOC)
Lourdes S. Casanova, He Li, and Benjamin Creutzfeldt
Respectively: Cornell S.C. Johnson College of Business; Professor of Political Science, Merrimack College; and Resident Postdoctoral Fellow Sino-Latin American-U.S. Affairs
Wheelchair accessible
Free and Open to the Public
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From East Texas, to Carolina, to coastal Virginia Seth Stainback has carved out a niche for himself as a not so typical southern songwriter. With a strong blend of blues, country, folk, old gospel and rock n roll, Stainback has laid the foundation needed to pursue his sound with his group Roosterfoot. In their short time together, Roosterfoot has played LOCKN Fest, Floydfest, Mustang Music Festival, Rooster Walk, Virginia Beach's American Music Fest, Neptune Festival and much more. They have been direct support for the likes of B.B. King, Bob Seger & the Silver Bullet Band, Lynyrd Skynyrd, Hank Williams Jr., ZZ Top, 38 Special, Anders Osborne, Robert Earl Keen, Big Head Todd & the Monsters, Rusted Root, Delta Rae, & Kip Moore to name a few. In 2014 Roosterfoot released "Fire and Steel" produced by New Orleans own, Anders Osborne and mixed by Grammy Award Winning engineer Warren Riker (Lauryn Hill, Santana) to rave reviews and won numerous awards for the effort. The summer of 2016 will see the release of their third release simply titled "Roosterfoot". Warren Riker joined the boys in the studio once again, this time co-producing, and the album was mastered by Grammy nominated/winner, Pete Lyman (Chris Stapleton, Jason Isbell). Roosterfoot will be touring all summer long including stops at this year's Floydfest, Riverbend Festival, Americana Beer Festival, and more. Armed with a tough work ethic, these boys are on a big road with a long haul.
Event Details:
- When: Thursday, September 29th
- Time: 7:00 p.m. – 10:00 p.m.
- Location: Belmont Abbey College Island
- Cost: Free and open to the public
- Weather: This event will proceed rain or shine


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Please meet Ice! Ice is a 7 year old Alaskan Malamute looking for his forever home. Ice has been brought up around children and is fantastic with other dogs, but will need a home without cats or farm animals. Ice is a big lump of love who is strong on the lead initially but quickly settled to walk well on the lead. His recall isn't great so he will need to be walked on lead unless in a secure area. Ice travels well in the car and is a well balanced, laid back gorgeous boy who gives a fantastic howl when a police car or ambulance goes past!
Ice has been neutered, vaccinated and is microchipped. Ice will need a home with a secure garden, and an owner who understands the breed and his desire to spend time outdoors! Ice can live with children who are used to larger breeds and who understand his need to be left alone whilst eating his meals and that are prepared to be greeted by a very enthusiastic child loving dog!


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Material Requirement Planning (Mrp)
Material Requirements Planning (MRP)
Material Requirements Planning
• Materials requirements planning (MRP) is a means for determining the number of parts, components, and materials needed to produce a product • MRP provides time scheduling information specifying when each of the materials, parts, and components should be ordered or produced • Dependent demand drives MRP • MRP is a software system
Materials Requirements Planning (MRP)
• Computer based system • Explodes Master Schedule (MPS) into required amounts of raw materials and subassemblies to support MPS • Nets against current orders and inventories to develop production and purchased material ordering schedules
Evaluation of MRP
• Most beneficial to process-focused systems that have long processing times and complex multistage production steps • Lead times must be reliable • Must freeze MPS for some time before actual production • Difficult to implement 5
Objectives of MRP
• Improve customer service • Reduce inventory investment • Improve plant operating efficiency
Firm orders from known customers
Aggregate product plan
Forecasts of demand from random customers
Engineering design changes
Master production Schedule (MPS)
Inventory transactions
Bill of material file
Material planning (MRP computer program)
Inventory record file
Secondary reports
Primary reports Planned order schedule for inventory and production control Exception reports Planning reports Reports for performance control ©The McGraw-Hill Companies, Inc., 2004
Master Production Schedule (MPS) • Time-phased plan specifying how many and when the firm plans to build each end item Aggregate Plan Aggregate Plan (Product Groups) (Product Groups)
MPS (Specific End Items)
Terms used in MRP
• • • • • • • • • • Bill of Material File Bucket Capacity Requirement Planning Inventory Status File End Item Lumpy Demand Net Requirement Offsetting for lead time On-hand inventory Service Parts
Independent vs Dependent Demand
Common Use Items
Steps in MRP
1. 2. 3. 4. 5. 6. 7. 8. Bucketing Add service parts Part Explosion Offsetting Aggregation Netting Procurement Schedule Lot sizing
• Refers to the time periods used in MRP
Add Service Parts • Add service parts which are not included in MPS.
Part Explosion • Use BOM for Part Explosion
Offsetting Means displacing requirements by a period equal to the lead time of the product.
Aggregation Finding the gross requirement for each component / raw materials
Netting Modification of requirement by subtracting the amount of same materials in stock Net Req. = Gross Req. – [Inventory in hand - safety stock – inventory allotted to other users] 18
Procurement Schedule Advance by a lead time for final assembly
Lot Sizing
Outputs of MRP
• Planned order schedule - quantity of material to be ordered in each time period • Changes to planned orders - modifications to previous planned orders • Secondary outputs: – Exception reports – Performance reports – Planning reports 21
Primary MRP Reports
• Planned orders to be released at a future time • Order release notices to execute the planned orders • Changes in due dates of open orders due to rescheduling • Cancellations or suspensions of open orders due to cancellation or suspension of orders on the master production schedule • Inventory status data
Secondary MRP Reports
• Planning reports, for example, forecasting inventory requirements over a period of time • Performance reports used to determine agreement between actual and programmed usage and costs • Exception reports used to point out serious discrepancies, such as late or overdue orders
Evolutions in OM
Individual Assignment
(Not to be submitted)
Learn the working and use of any...
Please join StudyMode to read the full document


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The streak by Williams, the defending champion and World #1 in the ISAF Open Match Race Rankings, equalled the score of 14 wins and 4 losses by week-long leader Gavin Brady, a four-time winner, and Williams won the tiebreaker by virtue of his win over Brady a day earlier.
Williams thus was privileged to pick Taylor Canfield of the U.S. Virgin Islands (10-8) as his opponent in the best-of-three semi-finals, leaving Brady to meet third-place Simone Ferrarese of Italy (11-7), who won all four of his races Friday.
The best-of-three finals will follow. Racing is scheduled to start at noon off the Belmont Veterans Memorial Pier in east Long Beach. Admission is free; snacks, refreshments and comfort stations are available. Parking in the lot at the base of the pier is complimentary, as is transit out to the end of the pier.
Live streaming video of the racing and post-race press conferences may be seen at http://ustre.am/egtA
All hands were hoping for better breeze than the 4 to 6 knots from the south they saw Friday.
Canfield placed last in the double round robin a year earlier but said, "Same crew, but the difference was time in the boat."
And a recovery from two early losses Friday to wins over Brady and Will Tiller of New Zealand also helped.
Ferrarese, in his native language, was en fuego (on fire) Friday with four wins, all against non-qualifiers, who will sail a fleet race over a harbor course Saturday.
"We felt really fast," Ferrarese said. "We played the best side of the course in every race. The goal was the semifinals. Now, we need to have a strong mentality."
Canfield had that in his turnaround win against Brady, whom he trailed at the last windward mark until slipping past for the lead and a 15-second win.
"We jibed and went over the top [windward side]," Canfield said . . . or, as Chris Larson, Brady's tactician, described it: "When we jibed he was able to roll us. But we're happy being in the semifinals."
Now it's time for the serious showdowns. Williams noted, "Having 14 wins is no good for us [Saturday]."
World Sailing Newsletter
World Sailing Newsletter is the weekly online newsletter of World Sailing.
It features the latest news and events from the sailing world together with features and info in an easy-to-use format.
It features the latest news and events from the sailing world together with features and info in an easy-to-use format.


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karen axx aún no ha recibido ningún regalo
Añadido por karen axx
Beard Czar First, search for the products you normally buy in stores. You can often find these on store Web sites and have them delivered to your door. But you can also find them from beauty and cosmetic suppliers.If you can't find the products you buy in store, you can search for equivalents. Or, if you are trying to find a product that you want to try but you have never been able to buy it in a store, try searching for that…Continuar
Publicado el marzo 7, 2017 a las 4:50am
© 2018 Creado por SALESIANOS LA OROTAVA. Tecnología de


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A Cameron driver escaped serious injury Friday night when she swerved off FM 486 and into the Little River. Sheriff officials say Magory Gonzales was travelling north when she struck a guard rail and rolled her vehicle into the river.
Officials say the car was swept down river with Gonzales still inside. She was able to escape the sinking vehicle through a window and managed to swim to the bank to safety.
Gonzales sustained non-life threatening injuries from the accident and was taken to Scott & White Hospital in Temple. Her condition was unknown Sunday evening.


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York St Mary's is a medieval church which opened as a contemporary visual art venue in 2004. Jorvik is currently closed, following flood damage in December 2015, and is expected to re-open in spring 2017.
The exhibition, which will run until early 2017, focuses on the trade that made York a powerful centre of international commerce.
Sarah Maltby, director of attractions for York Archaeological Trust, says: 'Even though Jorvik Viking Centre itself has been closed for more than five months, we are still getting hundreds of people coming along every day with a thirst for Norse knowledge, so having a fantastic exhibition that tells a key part of Jorvik's commercial story literally in the adjacent building is a great way of keeping the story alive, while we continue to re-build and re-imagine the attraction beneath their feet.'
The exhibition – Jorvik: Home & Abroad – will feature artefacts taken from York's Viking collections, alongside tableaux from the Jorvik Viking Centre displays which remained above the waterline during last December's floods. The exhibition will also provide a temporary home for Jorvik's team of historic-costumed interpreters, who will share their knowledge with visitors.
Jorvik: Home & Abroad will run from June 25 until late February 2017, when the artefacts start to return to Jorvik Viking Centre.
Go Holiday news : www.govillasandcottages.co.uk
All your holiday needs: IN PRINT - ON-LINE - DiGiMAG - SMARTPHONE -TABLET - BLOG - TWITTER - G+ - FACEBOOK


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Build a Complete 3D Game With the Unity Game Engine
It's never been easier to make 3D games using the Unity game engine, and in this video you'll learn how. You'll go from an empty screen to the total build-out of a space combat simulator that lets you fly through space to shoot down the asteroids that threaten your spacecraft and a vulnerable space station. As you build the game, you'll learn how to design for 3D gameplay, how to make great looking 3D objects, how to create playable controls for 3D movement, and how the various pieces of Unity fit together to form gameplay.
Designed for those with a very basic understanding of Unity software (what the Inspector is, how to move around in a scene, etc.), this course gives you the hands-on experience you need to create your own games.
- Develop your Unity skills by constructing a 3D game from concept to completion
- Gain 3D object development skills by building 3D spaceships, weapons, and asteroids
- Learn how to move objects through space, fire weapons, and create explosions
- Discover how to construct 3D scenes and write the C# scripts that control game play
- Understand the basics of 3D game design and development
Jon Manning is a co-founder of Secret Lab, where he makes games and does research. He has written about a dozen technical books for O'Reilly Media, and wrote two games for ABC Australia's children's TV series Play School and one for Qantas Airways. He's currently working on Button Squid, a top-down puzzler for iOS, and is fond of making cool tools for rad devs.
Download uploaded
Download nitroflare
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Innovative software program solutions from the specialized in download catalogs! Investigate the popular software utilities types, determine the hottest versions of the notable programs solutions, and try at once! Do not miss a luck to acquire the greatest software system: use SoftEmpire directory to get programs for free. Meet the directory of freeware and package demo versions. pick out among Windows softwares, get controllers, games, and all other types of programms for your notebook or digital accessory!
Company: Realore Studios
OS: Win98, WinME, WinNT 4.x, WinXP, Windows2000


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Bastiaan Zuiderent
261 Main St. (Behind McFadden's), Stamford, CT 06901. USA
Welcome to stamford ct restaurant, BBQ, Country Bar and Line Dancing Located at Stamford, CT. come see why we were voted #1 in Stamford, CT. We consider barbeque to be an art form – a form of true pleasure. At Bar-BQ, the pig is sacred, the beer is cold, the music is up, and the joint is rockin'. We take our cues from our sister location, Fairfield County CT's premiere barbeque restaurant since 2004, Bobby Qs of Westport. However, this time around, we're shaking things up a bit.
More Info: http://www.barqstamford.com/


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- Right-click on "My Computer" or "Computer". Left-click on "Manage".
- After the window opens up, left-click on "Device Manager". You should now see a list of all the hardware devices that are currently connected to your computer.
- Find anything with a yellow exclamation point and Double-Click on it. A new window should open up.
- Left-click on the tab labeled "Driver".
- Go ahead and click on "Update Driver".
- If asked to locate the driver on your hard drive, point the system to the Dell Optiplex E1 driver you have already downloaded.
- Restart the computer after you have finished installing the Dell Optiplex E1 driver.
Dell Optiplex E1 Drivers
All Dell Optiplex E1 drivers available to download to your hard drive on your desktop or laptop so that all of your hardware will perform at its peak. Drivers are simply small files that are comprised of instructions for the PC to process so that the system can utilize the particular piece of hardware that the driver file relates to. Sans proper driver files installed the device is absolutely useless and will not operate. You would need to have the right files correctly installed on your hard drive of your computer so that the motherboard can effectively communicate with the device and you are able to use it.
All external devices need to have drivers downloaded to the computer and then installed onto the operating system in order for them to be used. These particular devices can be a mouse, hard disk drive, phone or thousands of other hardware devices that are able to be connected to a computer.
- BRAND: Dell
- MODEL: Optiplex E1
- SYSTEM TYPE: Personal Computer
- OS: Windows XP, Vista, 7, 8, 10
So as that all hardware devices are different from one another, there are many different drivers available too. For reason aforementioned, it may be very hard to find a Dell Optiplex E1 driver for a specific device. For instance, each Kingston memory card has to have their very own driver installed onto the Windows computer in order to function properly. That adds up to a great many driver files simply for that particular brand of hardware!
Downloading the specific Optiplex E1 driver that you need can feel like the ultimate challenge in life. Here are a couple of tips as you are searching for drivers.
- Hardware manufacturer (if it is built-in to the motherboard then locate the brand of PC and use that)
- Model of the device.
- The exact type of Windows that is on your computer. (for all Windows PCs, right-click on "My Computer" or aka "Computer" and left-click on "Properties". OS info should now be visible)
- 32 or 64? (if it is a 64-bit system then it will show up in the Window that opens when you right-click on "My Computer" or "Computer" and left-click on "Properties". If 64-bit is not stated then assume 32-bit.)
You need to figure out the correct make and model number and the correct version of Windows that the device is attached to. After you have gathered the information about the piece of hardware that you want to install, you may begin to search for drivers and install them on your computer.
Most notable companies list their driver database and make it available for people so you can download them freely. Not all brands websites are quick to locate and navigate with any sense of direction. A few companies do not take time to contemplate the type of person who would be using their website and how difficult it is attempting to manually install Dell Optiplex E1 device drivers on a Windows operating system. Most people find it hard in figuring out what driver they are searching for and trying to navigate through the gauntlet of poorly coded websites can be stressful.
Be wary of downloading Optiplex E1 drivers and files from a site that does not end in a dot com. Try to stick to the hardware maker's website to be on the safe side. Viruses and other malware can be downloaded through a lot of different unsuspected files. Downloading and trying to install a Optiplex E1 driver onto a Windows computer can be the biggest mistake of your life if the file turns out to be corrupt. Even the best anti-virus can kill ALL viruses on the Internet. This is why you should always download drivers from the manufacturer's website. The device maker's website is more trustworthy than anyone else's.
Once you found and then proceeded the actual driver from online for the device you are wanting to use, you can then install the device onto your Windows system. After downloading you may need to uncompress the driver file especially if it ends with a .zip or .rar. After you do that you should run the program to install the driver. In some instances when the device driver fails to install it could be that you might have to install the Dell Optiplex E1 driver another way.
How to install a Dell Optiplex E1 driver manually:
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Abcarticledirectory. comThe latest item to hit the shelves of Home Depot has the potential to solve one of biggest annoyances of do-it-yourself home repair: having to go to the hardware store in the first place. For many universities, 3D printers have become an indispensable asset for promoting learning and education. Inkjet receipt printers are recommended for low turnover retail, banks and gaming industry. In the 1970s & 80s, dot matrix printers were one of the more common types of printers used for general use, such as for home and small office use. Action Printer Repair was formed in 2006, specializing in laser printer repair.
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Office equipments such as computers and naprawa ploterow z Poznania are a significant part of the whole process. I have the same problem still with 10. 1 on Windows XP SP3 with HP Designjet 1050, Driver v4. 68. I select paper format in print options dialog, but reader will always just use format A4. After disabling the safe mode, I'm able to select other paper formats and print normally.
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Find the supply cost of the printer before you buy it. Calculate the cost per page estimation—if the cartridge yield is 300 and the price of cartridge set is $30, then you will average 0. 10 per page-and if you print about 200 pages a month you are looking at $20/month cost or more. Once Your Printer Is Humming Along Don't Forget To Load It With Our Eco-friendly Cartridges.
Printer service technicians in your local area call (800) 643-2664 or fill out our quick quote form. The total process took me about 30-45 minutes to complete, and the printer works like new.   Overall the process is probably still cost effective since you can find fuser's for this model under 90$. This plug-in software sends the cutting object created by Illustrator or CorelDRAW to the plotter, and can also be used to control the plotter.
Printers come in several different sizes, including small ones ideal for home use and larger ones for your business. If the printer still suffers a failure, the action you can take depend on if the printer is in warranty or out of warranty. The printers are set to sell at Best Buy stores exclusively at first, and should be more widely available by the winter holiday season, Kodak said.


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I love this album. It's beautiful and inspiring.
However, be warned that if you are listening to this on a Sunday morning while you are getting ready for church, don't listen to tracks #2 or #6 while you are trying to put on eye makeup... you will be defeating your purpose. I learned that the hard way this morning.


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- JemmyGee
- 5 years ago
- Wedding: June 2012
So my husband and I have been married a little over a year now and while I love my set, I'm ready for something a little more.
my diamond is literally almost perfect and I don't want to upgrade the stone because I love it and I'm pretty sentimental. So I was thinking about adding a double halo. I love halos but never knew much about them until after we were engaged.
I would keep a plain shank and keep my wedding band.
So bees wgo added a halo or something else to your ring, do you like it better? Was it worh it? And bees with a double halo, how do you like your ring?


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Mougins - Provencal Mas6,400,000 €
- City : Mougins
- 6 bedrooms
- Area : 611 m²
- Plot of land : 2854 m²
Ref. MZIMO1797 - In a gated estate with tennis court, superb Mas from the 18th of around 600 sqm, combining the charm and the authenticity of a Provencal property with the comfort of a contemporary interior renovated to very high standards. It offers beautiful reception rooms as well as 5 bedrooms, each with ensuite bath or shower room. The independent guest apartment is composed of a living room with kitchen as well as a bedroom with shower and dressing room. Very pleasant flat and landscaped garden with swimming-pool and beautiful views of the old
village of Mougins.