text:
IX - RootsMature
Back to the story.
So, As I got out of the church-loft-sanctum, with Edward just vanishing into thin air before me, I saw that a duffle bag had be left there, the tools had been stashed inside. "Thanks Eddy." I murmured to the wind.
"Hey Bookguy? You hear me?" I asked loudly.
"Indeed I do." Said the voice... From right next to me. Startled I turned around and saw him.
Bizarro me wore the same clothes as me but looked like f'in shite, his eyes all baggy and tired looking, his hair and clothes dirty and ragged. "Man, you look horrible."
"The grimoire is a powerful shock onto an unawakened mind. It can hurt the psyche." He replied neutrally.
"What does that mean? And how are you no longer a voice?"
"You were unawakened. Your mind had yet the ability to understand the truth. As for the second question. I am still in you. This whole sanctum is yours, it has became part of you."
"Alright... What's the truth?"
"That there is nothing to understand. Everything is fluid and it is only beliefs that shape your world not the contrary."
"I'm pretty sure that's not the case. There is a real world you know, with consistant rules"
"There is because you think it is. Not just you, the whole human race believes and confirm that reality. Take for examples Dragons do they exist?"
"Yet they did. They appeared in many legends all over the globe for a reason. Dinosaurs as you call them, are their remains that mutated to fit the new edited version of reality."
"But I believe you have had enough of the theory. What is it you wanted?"
"I need help finding someone. Now."
"I am at your disposal. Take the tool with you. There is a vantage point about 10 minutes away that overlook the sea. I'll be waiting there." Just like the servant he vanished, no transition or anything. He just wasn't there anymore.
Somehow, I just knew where the thing was despite the size of the sanctum grounds. It was part of me he said, that was probably why. I shrugged.
Honestly by that point my head had started spinning. Yeah, ghosts and psychic vision are something but learning that everything you know is a frabication made my head hurt. It's even worse when it's your doppleganger that informs you.
There was a small white stone gazebo waiting there for me on a part where the cliff formed a V over the ocean. A large silver ring ran in the ground, a lectern next to it facing the ocean.
"Welcome" the bookman said, making a theatrical sweep of the arm to show the surroundings. He had changed from the exact same clothes to a semi-buttoned shirt and jeans. He had also made his or my hair into a ponytail. While I let mine hangs strait down touching the shoulder.
"Alright. I'm here. What now?"
"You will learn to cast your first spell." He snapped his fingers and a marble covered table appeared on the edge of the circle. "Empty your bag on the table. Done? Now, take them in your hand one by one."
"Okay" I took the closest and biggest, the staff. Infront of my eyes it changed into a new form. Gone was the quartz tip and in came another, purely transparent one. The feathers turned into an eagle's and symbolds ran down the lenght of wood.
"Ah Cherokee eh? Well, you do have it in your blood. Quite an interesting choice."
"What happened?" I asked him in confusion.
"The tools are adapting to you. Just go on." He said dismisingly like nothing had happened.
The samething happened to all items, in the end, I held an obscidian dagger, a cedar carved bowl, the staff and the pentacle had turned into a circle of wood holding a piece of colored cloth on it's edge. The seven sacred direction I remembered from my grandma's tales.
"Alright now that's done what happens?"
The book-me stood imobile for a second, his image glitching, for a lack of a better term.
"We will require a few reagent. A cedar cone, a handful blackberries, some tobaco leaves and an Item that belonged to your sister."
I searched around in my head for the last one... until I remembered the ring that clad my pinky. She had bought it for me when she was seven on my birthday, using a good part of her allowance... But where did it go? The greys had stripped me down...
"Looking for something, Sir?" Came the empty voice of the Phastasmic servant who had popped behind me, two inches away from my ear..
"Gah!" I screamed in surprise "Don't do that Ed"
"My most dearest apologies." He responded not exactly sounding sincere, but I doubted he could. He was a spirit, not a human afterall. Still, I was begining to like the guy.
"Wow... Thanks" I said as he handed me the tiny ring that had been polished.
He went away and I was left with myselves. "Where do I get the herbs?"
"We are in the woods. I think you'll figure it out." He said, turning his back on me, reading from a book. I left without a word, I had work to do.


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Post-'Lucha', Argentina's Leonas target elusive gold medal
By Miguel Lobianco
BUENOS AIRES (Reuters) - Argentina have the ability to win an elusive Olympic gold medal in women's hockey at the Rio Games despite losing talismanic, eight-times world player of the year Luciana Aymar, coach Gabriel Minadeo said.
Aymar, famed and feared for her weaving runs and dribbling skills, retired at the end of 2014 after a medal-studded career - though without ever having won a gold.
"Argentina must learn to play without her, she wasn't going to be eternal ... I think Argentina has potential and the players to be able to replace 'Lucha' with another gameplan,' Minadeo told Reuters in an interview.
"We're going to be able to show Argentina are a power without having 'Lucha'," said Minadeo, whose team will take part in the Champions Trophy in England next month.
"What she had was that she resolved things for you when they got complicated, with an individual move, pulling something out of the hat," he said after a practice at the Cenard national athletics training center in the capital.
However, for all her achievements, Aymar never managed to add the gold medal to two silver and two bronzes she won at the previous four Games starting with silver at Sydney 2000 when the team earned their nickname Las Leonas (lionesses).
Forward Carla Rebecchi, who took over from Aymar as team captain, said they had set themselves the target of the gold medal after winning a second silver at the London Games in 2012.
"We don't want to put pressure on ourselves but we know it's the medal we're missing and it's also a dream, we know it's difficult but we're doing everything we can ... to compete at the highest level," she told Reuters. Continued...

subset: commoncrawl


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Festive's Red Wired Star Garland is a simple but effective decoration for any room in the house; accessorize picture frames, doorways or even stair banisters with this sparkly treat! Festive's Star Garland is wired to ensure maximum staying power!
Important Features:
Length: 7.5m
Colour: Red
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cigar socket with battery tester 12V
- Description
This compact cigarette lighter power supply socket is equipped with a battery tester.
In addition to the power supply for the satnav, mobile phone, etc. this battery interface can naturally also be employed for plugging in battery chargers.
The integrated battery tester does not require constant current (meaning there are no unnecessary demands placed on the battery) and only displays one of five different states of charge for the battery when the button is pushed.
Constant green = fully charged
Flashing green = still okay
Red/green = charge or drive
Red/green = flat – needs to be charged
Constant red = must be charged immediately (voltage is falling below 12.3 V; this means the battery is becoming sulphated and will start incurring damage)
The "insides" of the German-made socket are comprised of high-quality components that are also used by the European automotive industry.
This offers an optimal snug fit in the casing.
The electronics are also "made in Germany" and have been over-moulded with a waterproof cover.
Thanks to its integrated mounting bracket, this combination product can be easily attached to the frame
or handlebars using cable ties.
Naturally it can also be installed in the interior trim etc.
The long, 1.2 m connection cables are long enough to reach the handlebars (a red/black cable is all that has to be run to the battery).
The accessory fuse can be mounted as desired in the best possible location on the vehicle.
– extremely compact design in an aluminium casing
– high-quality plug contacts
– long connection cable: 1,2 m/1 mm²
– integrated fixing bracket for the handlebars or frame
- Shipping & delivery
We always give our best to handle your order as fast as possible. Orders which are placed before 2 pm on a work day are usually handed over to our logistics partner (DHL) on the same day if in stock.
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4.95 Euro within Germany, Poland
5.95 Euro in Austria, Belgium, Denmark, Luxembourg, Netherlands
9.95 Euro Spain, Finland, United Kingdom, Italy, Sweden
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In our online shop, you can choose from various payment options. You can pay by invoice, direct debit, cash on delivery, credit card or finance.
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Bordesteckdose funktioniert, Batterietester nicht,
Der Batterietester testet gar nichts. die grüne Leuchte leuchtet nicht auch bei frisch geladener Batterie. Die rote leuchtet nur bei eingeschalteter Zündung .
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Here you can ask public questions which can be answered not only by us, but also by other customers. If you have a private question, please use our contact form.
Fragen zum Produkt


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Elegant International
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Glycolite skin lightening & rejuvenating Face Wash is a gentle cleanser. Its contents like Glycolic Acid and Lactic Acid are generally used as natural skin exfoliates and moisturizers. This Alfa-Hydroxy face wash helps remove sebum build up and dead skin cells that can cause acne lesions or accentuate fine lines. This content reacts with the upper layer of the epidermis weakening the binding properties of the lipids that hold the dead skin cells togetther. The other ingredients carry the individual flakes away and a water rinse neutralizes the remaining acid. The result is a much smoother skin surface, a more youthful appearance & texture. It may reduce wrinkles, acne scarring, hyperpigmentation and improve many other skin conditions. This face wash is valued for its high quality & effective usage. This face wash is laboratory-tested by our quality experts to ensure its quality. The given face wash is made accessible in numerous packaging options to suit various demands of our customers.


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July 14 2016
One of the biggest dark-horse house influencers The Magician has built a formidable reputation over the past decade as a DJ, Producer and renowned label head. The Belgian hit maker was previously apart of the popular DJ duo Aeroplane before announcing his solo work in 2010 under The Magician. From there, his track record continued to flourish with a bunch of successful records from labels like Kitsune and Parlophone before taking on his biggest venture to date with his own label 'Potion'. 2014 was his year after making waves with a worldwide dance hit in 'Sunlight' featuring Years and Years. From there on in, it has been countless international tours and European residencies along with nurturing talent from his own label like Aussie pals The Aston Shuffle. His latest release 'Shy' features the vocals of Brayton Bowman and has received support from the world's biggest DJs like Oliver Heldens, Martin Solveig and Lost Frequencies.
We caught up with the man himself to grab his top 10 tracks right now, some of which are unreleased gems classed as 'the next big thing' - so make sure to keep an ear out.
Tune Of The Week: White N3rd - Freaky With You
5 Reasons To Listen To Music While You Work Out


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Carson Valley Arts Council hosts exhibit of watercolors
December 21, 2012
The Carson Valley Arts Council will feature an exhibition of the watercolor paintings of Marge Buttles and Helen Martin, members of the Carson Valley Art Association. The exhibit will be up through Feb. 28 at the Copeland Cultural Arts Building at 1572 Highway 395 in Minden. CVAC will host an artists reception from 5 p.m. to 7 p.m. Friday, with wine (for a donation) and non-alcoholic beverages and snacks. The gallery is open from 10 a.m. to 5 p.m. Monday through Saturday, except for holidays.Buttles, born in Berkeley, Calif., grew up and was educated in the San Joaquin Valley and the San Francisco Bay area. She and her husband, Harold, moved to Carson Valley in 1992. She became a member of the Carson Valley Art Association as well as the Nevada Art Association and the East Fork Gallery. She mainly enjoys painting landscapes and old barns that pique her interest.Martin is originally from Santa Cruz County, Calif., and has been drawing and painting since she was a child. She and her husband, Dave, have lived in many places, including Western Australia, and have traveled extensively through Mexico, the Caribbean and the Pacific Islands. They now live out in Fish Springs on an old homestead that they've been renovating for the past seven years. Martin is a member of the Gardnerville Woman's Literary Club as well as the Carson Valley Art Association, and is the editor of their monthly newsletter. Her art can viewed online at http://artbyhelen.googlepages.com.The Carson Valley Arts Council is currently in a capital campaign to retire the note on its home in the historic Copeland Lumber Warehouse. For a contribution of $25 or more, donors will receive an original hand-painted pin or pendant by artist Pam Brekas, and have their picture taken. For more information, visit http://www.cvartscouncil.com or call 775-782-8207.
Trending In: Local
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June 20 2016
This morning the lineup for Australia's much loved boutique festival, Listen Out, dropped!! - and it's a huge one. With a gang of talent coming from all around the globe and representing everything from house to UK Grime, we know that Listen Out 2016 is going to be bigger and better than ever.
Off of the back of their MASSIVE world tour, Rufus will be returning to home soil for their last Aussie shows of the year, alongside fellow Aussies L D R U, Cosmos Midnight, JOY and Ngaiire among many. In typical Listen Out fashion, they have invited some of the biggest international names in electronic music to grace our decks, and this year the honours go to superstars Jauz, Baauer and Tchami as well as Claptone & Gorgon City. No Listen Out festival would be complete without a bit of Rap and UK Grime, which is why Listen Out has invited ASAP Ferg & Stormzy to the stage along with one of the most in-demand artists of the year in Anderson .Paak & The Free Nationals.
Check out the full lineup and ticket details below!
Anderson .Paak & The Free Nationals
A$AP Ferg
Claptone Immortal Live
Cosmo's Midnight
Gorgon City DJ Set
Rüfüs (Last Aus Shows For This Year)
Sui Zhen DJ Set
Tash Sultana
Willow Beats
Yung Lean
MELBOURNE | Sat 24th Sept @ Catani Gardens, St Kilda
PERTH | Sun 25th Sept (long weekend) @ Western Parklands – HBF Arena, Joondalup
SYDNEY | Sat 1st Oct (long weekend) @ Centennial Park
BRISBANE | Sun 2nd Oct (long weekend) @ The Sporting Fields, Victoria Park
Tickets go on sale midday Tuesday, June 23rd, for tickets or more info head over to the official Listen Out Site.
5 Reasons To Listen To Music While You Workout
Listen: Set Mo Bring The House Down In Their Mixmag Mixlab Set


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Challenge 2: hunting hunters
The first challenge was done after an hour and a half, almost one third of the total hunters and students were missing. We were now walking toward the site of our second challenge.
"The whip works perfectly," I offered Shizuma. She smiled and went back to her conversation with Koemi and Joanne. Albert and Kyre were trailing behind, talking. Albert had been in a separate platform from us, all I knew was that he overpowered his opponents in less than five minutes.
From behind me I heard bodies clashing and scraping the ground. I turned and saw Neil had tackled Axel to the ground, Neil was now holding Axel's shirt collar.
"How dare you break my father's arm and throw him off the platform," Neil was enraged, he punched Axel on the face. I walked towards them and yanked Neil off Axel.
"Chill," I said, "its just a game, he is going to be all right."
Neil shook my hands off his shoulder and shoved me back. "Don't mess with me," he warned us, then stormed away.
I shrugged and lend a hand to Axel, he pushed my hand away and stood on his own. "I didn't need your help," he said, "I could've taken Neil down this moment."
I rolled my eyes, "save it for the next challenge, if you get lucky he might be your opponent..."
He stared at me with cold eyes and he started walking, catching up with the rest of our friends. I walked silently beside him, in silence we agreed to leave our pending battle after the challenge.
We reached a closed canopy forest, trees were towering in front of us. The close canopy impede the growth of a jungle and sunlight was scarce.
"Welcome to your second challenge," Salazar announced, "unlike the first challenge, this one will be timed." He snapped his finger and his assistant brought forth a bag, "inside there are different colours of handkerchiefs, each of you are going to get one and once inside the forest you will tie it on your body. This challenge will eliminate half of the hunters and/or students. Only the remaining 30 will proceed to the third challenge."
His assistant walked around the hunters, handling each one of them a black bag with the piece of fabric inside. I felt strange having Cadravar competing, by far he was he oldest, that scroll must be very important. I was also wondering where were the eliminated competitors.
"The first was a dueling challenge, the second one is about hunting," Salazar continued, "each one will hunt down the one having their same handkerchief colour. You have thirty minutes to hunt your prey and bring back the handkerchiefs of the same colour back." The sun was now high on the sky, signaling noon time.
The second challenge started, all of us ran inside the forest, to different directions. I stayed with Shizuma.
"What you got?" I asked her.
"Nervous?" she taunted, taking out an orange kerchief and tying it on her shoulder.
I took out a green one and tied it around my forehead. Shizuma laughed at me.
"You will be such an easy target," she said, "see you out there in thirty minutes." She jumped up to a branch and swung her lithe body from branch to branch. I ran aimlessly around me, this was going to be tough. Anybody who didn't make it before the set time will be disqualified. I jumped toward a tree and with a kick I impulsed my body up, I used several branches for support as I jumped farther up. I was certain many of the competitors were up on trees searching for their prey.
I stood on the edge of the tallest branch, this was indeed a thick canopy, nothing could be devised from this height. After standing motionless for more than fifteen minutes my eyes swept the area once more before coming down, I saw droplets of blood escaping from an abrasion on my shoulder as a small arrow brazed against my flesh.
I lost balance and fell, breaking branches and twigs with my back. Finally I fell on the thickest branch, holding my bleeding arm. With a quick scan around me I saw my prey, she had a green bandana tied on her thigh, a crossbow strapped on her right forearm. I recognized her from the academy, Elena.
"You are such an unlucky boy," Elena said, aiming her crossbow at my forehead, "you had to be my prey. Give me the kerchief Wilcaust."
"Catch me first," I said and quickly jumped off the branch to fall into another one, her arrow missing my inches. I flipped out of view around the tree trunk.
I pressed my back against the trunk on the shadowy part of the tree, straining my ears to hear her approaching steps. If I knew anything about Elena, it was her stealthy and quiet prowl. I heard the scraping of tree bark above me and quickly jump away as Elena descended rapidly on me, shooting arrows. Violet spiritual energy engulfed her crossbow. Elena then disappeared around the trunk.
I had to be faster. I slid behind a trunk, took out one of my swords, and encrusted it deep on the tree trunk. I placed an ear close to the hilt, my hands cupping my ears to the hilt. I could hear the vibrations of the tree, giving me a clue as to where Elena was. I dislodged the sword and jumped higher. I had located Elena, she was now some branch levels below me. I sheathed my sword quietly and jumped down on her.
As I landed on the branch, she turned around, her crossbow ready to fire, but I was faster and took hold of her wrist, turning her crossbow away from me. She screamed in pain as I twisted her arm outward, "bastard.." she muttered. She was trying to wriggle free, kicking me and stomping on my foot. Finally I pressed her against the trunk, my hands reached for her thigh and tear the kerchief away. I took arrows from her crossbow and furnished them with my own spiritual energy, then I nailed her loose garment to the tree, burying the arrow deep in the tree.
"Sorry Elena," I said, "hope you can forgive me."
"Get me out of this Wilcaust!" she screeched as I ran away from her, victory in my hands.
I was running out of the forest, when something caught my eye. I hid behind a tree and saw Neil take the white bloody kerchief from a dead hunter. Neil had killed a hunter! Was that allowed? Then I saw Neil walk past another corpse nearby and out of view. I rush out of the forest to found some of the competitors who had finished. I went up to Salazar.
"Are we allow to kill?" I asked in a demanding voice.
Salazar gave me an ill look, "of course not," he said, "this is a healthy competition. I don't favour hunters killing hunters." There was feign honour in his voice.
"I saw some dead hunters!"
"Nonsense," he said, "I have a special force patrolling the forest to keep the game under control, if there were deaths they would've report back..."
"What if they are dead too?" there was impatience in my voice.
"I assure you, young man," Salazar eyes were furious, "there will be no deaths tolerated. Now, leave and enjoy your victory over your prey."
I opened my mouth in protest, but a hand pressed my shoulder, it was Candravar.
"Aldrest, trust in Mr. Dreix words," he said in a low tone. There was something in Candravar's voice and attitude that silenced me and follow him. "Headmaster I am glad you made it out." Once we were separated I told him about the dead hunters.
"Keep an eye open," he suggested and left my side. There were ten minutes left, where were the rest?


text:
Big Media Is Watching You
They controlled transmission:
Attkisson: I found out that we had to quit pursuing the story more or less due to lack of interest well before we found answers to a lot of questions. Including what about all the other cases besides the one you know as Fast and Furious that were also using similar strategies to transfer weapons down to Mexico. And how did this, if at all, play into a strategy the United States may be using to draw support or give support towards one of the cartels in Mexico against one of the others much like they had done in Columbia and other places.They didn't need the government to do it for them...
O'Reilly: Playing one off against the other. You said something interesting that you had to abandon the story for lack of interest. Can you clarify that?
Attkisson: It just came to be that, I don't think on the viewers' part, but on the people that decide what stories go into the broadcast and what there is room for, they felt fairly early on that this story was over when I felt as though we had barely begun to scratch the surface. They didn't ask me what was left to report. They decided on their own the story was done.


text:
http://fightnetwork.com/ - Fight Network host Sarah Davis along with analysts John Ramdeen and Robin Black speak on the latest news in mixed martial arts, featuring the headliner of UFC 158 on March watch video >>
http://www.MMAWeekly.com/ -- Nick Diaz is a different kind of character.As a fighter, he's one of the most polarizing figures in the sport. People love to love him, or love to hate him, there's not a watch video >>
GSP vs Nick Diaz Full Fight Prediction by Paulie G -This is one of the craziest tightest ever in UFC history. GSP vs Nick Diaz will be an Epic battle. The pothead vs the Athlete. watch video >>
In a welterweight-heavy card, heated rivals Georges St-Pierre and Nick Diaz square off for the UFC welterweight title. Plus Johny Hendricks meets Carlos Condit, and Jake Ellenberger faces Nate Marqua watch video >>
Does Nick Diaz deserve to get beat down by GSP? watch video >>
http://fightnetwork.com/ - UFC 158: Georges St-Pierre vs. Nick Diaz heats up in this exchange during the media conference call. For additional coverage of MMA, boxing and pro-wrestling check out htt watch video >>
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http://fightnetwork.com - Fight Network analysts John Ramdeen and Robin Black bring us the headlines in mixed martial arts featuring Nick Diaz skipping the UFC 158 open workouts and the possibilities watch video >>
http://fightnetwork.com - Fight Network analysts John Ramdeen and Robin Black bring us the headlines in mixed martial arts featuring Georges St-Pierre's tolerance of the media wearing thin and Nick D watch video >>
Listen to the complete audio from the UFC 158 pre-fight press call with Nick Diaz and Georges St. Pierre, Carlos Condit and Johnny Hendricks. watch video >>


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There was little doubt, as the first poll numbers started trickling in Tuesday, that Americans are angry at the cadre of House Republicans who shut down the government this week.
Here in Connecticut, there is only fringe support for the ransom-demanding Republicans. Sensible people see the political terrorism for what it is.
Imagine if things were reversed and a small group of Democrats were holding the country hostage by demanding, for instance, tough new gun laws that they don't have the votes to get through Congress.
Of course, sensible people here in the land of steady habits know you can't give in to ransom demands. The crazed Republicans now demand repeal of a law passed by Congress, signed by the president and approved by the Supreme Court.
What other things that they don't like could be next: Medicare? Social Security?
Even some moderate Connecticut Republicans might have felt a little tinge of pride, as I did, when Connecticut Rep. John Larson had a meltdown on the House floor Monday night, a short tirade that was captured in a video that then bounced all over national news websites.
"Do you stand by your country or do you want to take it down?" a red-faced Larson screamed into a microphone. He went on to suggest how simple it might be to just put it to a full vote in the House.
"Americans deserve to know where their members stand," Larson screamed.
Connecticut voters have already excised Connecticut Republicans out of their delegation to Washington. The few moderate Republicans, who were able to cling to the center and get elected, are now gone.
The only political frontier that has remained viable for state Republicans is state government. In fact, given the woeful pace of the recovery here and the arrogant stumbling of the administration of Gov. Dannel P. Malloy, Republicans have reasonably been able to set their sights on reclaiming the governor's mansion.
My guess, though, is that this week's shutdown is going to have repercussions for Republicans here in Connecticut for a while, quite possibly through the next gubernatorial election.
If I were Republican Tom Foley, who has apparently set his sights on a rematch with Malloy, I would have climbed up on some rooftop Tuesday to condemn what the Republican crazies in Washington were up to.
The worrisome part of the whole shameful thing is that the Republicans most directly responsible for the shutdown come from gerrymandered districts with hard right constituencies that will welcome their representatives' tough stand on Affordable Health Care.
They are insulated from the worst of the backlash.
You don't have to go far in Connecticut these days to see Republicans retreating from the party brand, even before this week's shutdown.
In New London, Republican candidates for City Council seem to have largely dropped their party affiliation from lawn signs. The only Republican-endorsed council candidate I've seen use party affiliation on a lawn sign is Marie Friess-McSparran. And her sign says Democrat, her original party, not Republican, the party now endorsing her.
I think New London Republicans might even be better off, by way of association, taking their chances with the ransom-demanding Republicans in Washington than endorsing the rejected Democrat at home.
This is the opinion of David Collins.
Stonington beats East Lyme in eight to win first ECC baseball tournament title
Police: Gales Ferry driver collides with 2 cars, hit by 2 cruisers during chase
Saint Bernard grads told 'people of faith can make a difference'
Dellacono wins second straight ECC girls' tennis title in matchup of friends
Foxwoods to detail zip line, 'other new developments' next week
Fitch makes it look easy in winning third straight ECC softball championship
R.J. Evans goes to Louisville and Roger Bidwell goes to the Hall of Fame


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|64 m²
|Tipo de imóvel
|Estadia Mínima
|2 noites
Você tem o seu próprio apartamento para max. 5 pessoas. Dois quartos de luxo, sala de estar separada com sua própria cozinha, casa de banho de luxo moderno / e WC separado. O apartamento está convenientemente localizado em frente ao mar, perto do centro de Amesterdão. O pequeno-almoço é possível por acordo.
Great apartment in a great location. It was just enough room for our family of five (our little one slept on a fold-out mattress on the floor). It was clean and the beds were comfortable with plenty of linens, though the kitchen items were a bit sparse if you wanted to cook a meal. I highly recommend it.
Appartement agréable dans un quartier calme, 2 lits confortables.
Martijn, est très serviable, accueillant, disponible.
Bon séjour
Very quiet, secure and lovely apartment 3 flights of spiral staircase to top loft. Great location
Owners live on second floor, so it's well supported.
We enjoyed our stay here. It was as advertised. We had no surprises except that it was very clean. The owner was very helpful in giving us a lesson in how to use the appliances. It was very quit to be in the city. Beautiful place to stay. Yes, we would go back there.
Thank you Martijn for allowing us to stay in your home. It was the perfect location for our 5 days in Amsterdam. We actually booked this property after we had found out our other property allowed smoking, and changed as we didn't want to stay in apartment that smelt like smoke. And we are so glad we did.
The apartment was very clean with nice sized bedrooms. It was super comfortable for our family of 3. We had a nice view of the canal as well. The stairs were a bit of a hike, but we got used to it by the end. The only thing was that the shower had no door so the floor got quite wet when we showers. However that was only a minor detail.
We loved that the apartment was only a few minutes walk to buses and trams, which was then only 5 minutes from central station. Our son loved the park nearby. It has fantastic climbing equipment for children over 4. We would stop there every morning and afternoon.
Martijn was great. He was quick to respond. Met us when we arrived and allowed us to leave our luggage there for the day before we checked in. He was even nice enough to print off our boarding passes as we had forgotten.
All in all we would highly recommend this property. We would definitely stay there again.
Good location, clean facility and a roof that opens up. We had a great time in Amsterdam and this apartment added to that experience. The owner was attentive and met us upon arrival. The only down side to the apartment was what the owner referred to as Amsterdam Stairs. Very steep and narrow. Overall a great experience for our family. Grocery stores and sites well within walking distance.
A partir da A10:
Pegue a saída Haarlem e vire para a Haarlemmerweg / S103 S104 direção / Floresta e Lommer- Vire à direita para Van Hall Street-Vire à esquerda na Primeira Kostverlorenkade-Pegue a segunda à direita na Fagelstraat. Você atingiu seu destino final.
Do aeroporto Schiphol:
Amsterdam Central trem a partir da estação central, apanhe o autocarro 18 ou 21. Saia na Frederik Hendrikplansoen. Por isso, é 2 min. Caminhe para o Fagelstraat
Possibilidade de viagem de barco. Completamente Amsterdam !!
|Validade da Tarifa
|por noite
|Noite (fim de semana)
|por semana
|por mês *
|Pacote ou Evento
Tarifa Básica
Estadia mínima: 2 noite(s)
A partir deR$ 452
|Observações: As tarifas levam em consideração a estadia de 2 hóspedes, adicione R$ 70 por noite, por pessoa adicional
|Imposto de hospedagem
|5.0% por noite, por pessoa. Esta quantia terá de ser paga ao proprietário na chegada. Esta taxa não se aplica a crianças com menos de 12 anos.
voorschot bij boeking. waarborgsom gevraagd. sleutels af te halen bij eigenaar. degressief tarief afhankijk van de verblijfsduur. tarief afhankelijk van het aantal personen
* Tarifa mensal aproximada. O valor real dependerá dos dias em que você de fato se hospedar no imóvel.


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A stylish haircut makes a good impression because it shows people you can be trusted around scissors. Inspire confidence with this Groupon.
Choose from Three Options
- $24.99 for a haircut (a $30 value) and conditioning treatment (a $25 value; a $55 total value)
- $49.99 for a haircut, partial highlights (a $75 value), and choice of lip or eyebrow wax (an $8 value; a $113 total value)
- $59.99 for a haircut, full highlight (a $105 value), and choice of lip or eyebrow wax (a $143 total value)
Lavish Design Studio at Little Flower Day Spa
Specializing in all things hair, Mindi Patterson relies on her Aveda Institute training and years of experience when snipping and coloring her clients' locks. At Little Flower Day Spa, Patterson attends to each visitor, beginning with a personal consultation to discuss style, color, and the contents of their fridge. Additionally, Patterson offers skincare and makeup-application lessons, using products from BeautiControl.
2816 Culver Road
Mountain Brook, AL 35223
+12057475388Get Directions


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Crazy Crysis screenshot: 25600 x 16000 pixels
400 megapixel screenshots of Crysis? That doesn't just sound crazy, it really is crazy. Nevertheless: Crysis in 25600 x 16000 is worth every single picture; or two perhaps.
User OC_Burner from the 3D Center Forum dealt with the Downsampling technique in order to create Crysis screenshots so big that they are almost obscene. While "normal" Downsampling was still playable, OC-Burner's method means: Taking screenshots on a wing and prayer. The high resolution in combination with the low framerate is definitely not playable for real. So the technique is only for taking screenshots. Two of the Ultra-High-Res pictures (each of the jpg files is almost 100 MByte big) can be found as downloads at the end of the page while the other pictures have been downsized to 1280 x 800 respectively 1280 x 720 pixels to fit into our gallery.
If you want to know exactly how OC-Burner took the screenshots, we recommend that you take a look at the posting in the 3D Center Forum (German language!). For more information about Downsampling take a look at the article Crysis at 6,400 x 4,000: Ultra High-Res Gaming with 25.6 megapixels.


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Ratings based on 10 Verified Reviews
4.4 Room cleanliness
4.2 Service & staff
4.0 Room comfort
4.1 Hotel condition
Expedia Verified Reviews
for Everyoneby A verified traveler
Perfect little hideawayPosted Dec 14, 2016
Lovely stay and fantastic people that really made my stay enjoyable!
by A verified traveler
Posted Sep 28, 2016 on Hotels
Really friendly owners and flexible too to customer requirements
for Everyoneby A verified traveler from london
Beautiful views in garden of riverPosted Jun 4, 2016
Lovely place. Shower room too small. Owners friendly.
for Everyoneby A verified traveler from UK
Fantastic views of the river SevernPosted May 22, 2016
Pros: Location - very peaceful and scenic surroundings. Friendly atmosphere and tasty breakfast.
Cons: Squeaky floorboards were a tad annoying, but nothing serious. The internet was slow, but hardly surprising given how far it is from the exchange.
Met with a very warm welcome and a fresh filter coffee! Very hospitable and helpful with advice and recommendations. The B&B is slightly off the beaten track, but is in reasonable proximity to Malvern, Worcester and others - plenty of local pubs and restaurants.
for Couplesby A verified traveler from stoke on trent
peaceful b & bPosted May 10, 2016
Pros: Quiet, peaceful, wonderful hosts
Location: There are various pubs also around a 15 minute drive.
Lovely b & b on the banks of the Severn. We used as a base for visiting Malvern. It's only a 15 minute drive from the town. We had the smallest room, but the bed was very comfortable with plentiful facilities of teas, coffee etc. Fresh milk was ro died in a thermos. Breakfast was stupendous and cooked to order
by A verified traveler
Highly recommended Severnside accommodationPosted Aug 24, 2015 on Hotels
We enjoyed a wonderful 4 nights' stay here. The setting by the River Severn is excellent. The room was spacious and very comfortable, and there is also a guest lounge. The owners were very friendly, and the breakfast excellent. The only couple of downsides were the very rough and steep access track for the last hundred yards or so, and, being a new property, noise travelled very easily. However neither of these should put you off staying here and enjoying Malvern and its wonderful countryside.
by James
Posted Jun 17, 2015 on Hotels
by LEE from Cardiff
5 STARSPosted Apr 17, 2015 on Hotels
by Eddie Collins from Glossop Caravans
SERENE RELAXING TIMEPosted Aug 24, 2014 on Hotels
Having stayed at the Severnside B & B on business for five nights, we found it to be a very tranquil stay and truly relaxing, certainly after a hard days work. The scenery is second to none. Both Jane and Chris are very hospitable and made us feel very welcome. Would I recommend this B & B to my friends? absolutely.
for Couplesby A verified traveler from Warwickshire
Severnside B&B is exactly what is says!Posted Aug 18, 2014
Pros: Friendliness of Jane and Chris. Easy come and go arrangement.
Cons: Apart from sound-proofing the rooms, nothing!
Location: A little hamlet right out in the country but with easy access to Worcester, Upton on Severn and of course Malvern.
In a hamlet near to Malvern is a B&B where you receive tea and cake on arrival, get a bus timetable printed for you in 5 minutes and have a lovely choice of breakfast including home-made yoghurt. The ground/garden right beside the river Severn boasts a small orchard with fallen apples and plums to munch into. It was a very enjoyable stay and is a base for exploring not only the Malvern Hills but other Worcestershire places of interest.


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Apprenticeships simplified
Apprenticeships are essentially designed to develop your skills and knowledge for a specific career.
An apprenticeship is a real job in which you will learn specialist skills, work towards nationally-recognised qualifications and gain that all important experience.
Apprenticeships are essentially designed to develop your skills and knowledge for a specific career.
As an apprentice you will:
- Work alongside industry experts
- Gain essential real-life experience
- Work towards nationally-recognised qualifications
- Attend training sessions or complete e-learning tasks
- Earn a weekly wage
- Be entitled to company benefits (such as paid holidays).
There are three levels of apprenticeships available
Still wondering why you should become a Vision Apprentice? Here are 10 great reasons to follow the apprenticeship route with us:
- 1
You'll earn a weekly salary
- 2
Benefit from one-to-one training
- 3
Achieve a package of qualifications once you finish
- 4
Real work experience
- 5
Skills and knowledge that employers want
- 6
No student loans or debts to worry about
- 7
Holidays and benefits like other employees
- 8
Train with a reputable employer
- 9
Support throughout your apprenticeship
- 10
Progression opportunities – Advanced and Higher Apprenticeships, Foundation Degrees and more
- 1
- 2
- 3
- 4
- 5
- 6
- 7
- 8
- 9
- 10
Our frameworks
Commonly asked questions
What support will I receive?
You will be fully supported throughout your apprenticeship. Our specialist recruitment team will be on hand to support and guide you from the word go. Our team will make sure you're fully prepared for any roles we match you with, and we'll never recommend an apprenticeship that isn't right for you.
Am I guaranteed a job?
Although a permanent job isn't guaranteed, over 85% of our apprentices stay in employment following their apprenticeship. It is up to the employer to decide whether they can offer you a permanent position with the company once you have completed your apprenticeship. Some of our apprentices also choose to go onto Further or Higher Education.
How much will I earn?
As an apprentice you will be paid a weekly wage, set by your new employer. Rates vary depending on the level of apprenticeship you're studying, the industry you're in, the company you work for and the type of work you will be doing. Apprentice wages are monitored like all other employees. There is a national minimum wage for apprentices set each year by the government. For more information visit www.gov.uk.
How long do apprenticeships last?
Generally, your first apprenticeship should last between 15 and 18 months, then you can choose to progress to a higher-level course.
The length of your apprenticeship will vary depending on your skill level, the level of apprenticeship you're studying and the employer you're working for.
Am I eligible for an apprenticeship?
Apprenticeships are available to anyone living in England over the age of 16


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ED Magedson – Founder
Ripoff Report
DEALER FUNDINGP. O. Box 888759 Atlanta, Ga. 30356 Atlanta, Georgia United States of America
Does your business have a bad reputation?
Fix it the right way.
Corporate Advocacy Program™
This report was posted on Ripoff Report on 11/28/2012 10:31 AM and is a permanent record located here: http://www.ripoffreport.com/reports/dealer-funding/atlanta-georgia-30067/dealer-funding-lying-about-changing-due-date-atlanta-georgia-974957. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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Category:Bois Blanc Island Lighthouse and Blockhouse National Historic Site
|This category is for a cultural heritage site in Canada, number 16144 in the Canadian Register of Historic Places.
This category has only the following subcategory.
- ► Bois Blanc Island Lighthouse (4 F)
Media in category "Bois Blanc Island Lighthouse and Blockhouse National Historic Site"
The following 6 files are in this category, out of 6 total.
- Bois Blanc Island Blockhouse.jpg 2.56 MB


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Tutoring Sessions!Mature
I gave a small nod, and hid my grin with my hand that wasn't scratching my head in frustration at the ridiculously high-level questions on my mini-test.
Then, boom, it was time to mark them.
'Swap your papers, guys.' Mr Stevans ordered us.
I reluctantly handed my quiz to Kaya, who took it with a grim look on her face. No one handed me their test, as usual. Due to my utter lack on concentration which would lead to their test score lacking because I hadn't marked it.
I kept on glancing at the clock nervously, maybe I could grab my test; tell sir I needed the loo and toss it in the bin, then arrive; only to act perfectly nonchalant towards him.
'Pass the tests to the front,' Mr Stevans said in a monosyllabic, bored tone.
'Shoot.' I snatched my paper from Kaya who gave me a look which gave me the impression she was trying her hardest not to laugh her head off at me. I crumpled it up into a ball and shoved it into my blazer pocket. Mr Stevans eyes fixed onto me and I flushed, he smiled encouragingly and dismissed the class.
'Roi, you coming?' Kaya called from the doorway.
'Um, no...'
'Um, because--'
'No, I'm afraid I'll have to keep her for an hour or two. Roi needs tutoring sessions if she has a hope in hell of passing any test to come.' Sir said sharply.
'I'll be at the dorms later. Not hungry.' I smiled and waved her off, not making eye contact with him.
'Huh?!' I gasped as he wrapped his arms around my waist and extracted my test paper from my pocket. I pouted and looked down.
'Why would you hide it? We need to see what you can.. work... on... Roi, do you even listen in class at all?' He asked sharply, staring at my feeble score of two. I'd even got some of the few questions I did wrong. How embarrassing.
'Then what's with this score?'
'Dunno.' I scuffed my shoes on the floor, refusing to look into his face; until he tilted up my head so it was just inches from his, 'I'm just bad at lessons.'
He frowned and sighed, his coffee-breath filling up my lungs in a pleasant way.
'What is it?'
'Sorry.' I mumbled. He smiled faintly and pulled me into a hug, not like the ones my brother gives me, but a real hug. A really tight one. Only Kaya had ever given me those.
I accepted it gladly, 'I'm still giving you tutoring sessions.'
'Umm...' I grunted uncertainly.
'What? It means we can be together without anyone being suspicious!'
'Oh yeah! That's clever!' I squealed, sounding idiotic.
He kissed me lightly on his cheek, 'but that doesn't mean I'm not teaching you anything!' He said grimly. My heart sunk.


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Space Station Live: Veggie Harvest Shows Promise
Space Station Live commentator Brandi Dean talks with Dr. Gioia Massa, Veggie science team lead, about the validation test of the Veggie compact greenhouse aboard the International Space Station.
Job search
Welcome to this Futures workbook on finding work. It is split into several sections describing different techniques for finding work. You can dip in and out of the different sections; however it is not wise to rely on only one mechanism for finding work. It is a competitive market for final year students and new graduates and so you should try many alternate ways to find work.
Who Are the Poor?
In this assignment, we will perform a simple analysis of poor households in the United States, considering whether certain kinds of households are more likely to be poor and what sorts of households make up the largest proportion of the poor. Students will be looking at three characteristics that may be associated with poverty: race, age, and family type.
6.003 Signals and Systems (MIT)
6.003 covers the fundamentals of signal and system analysis, focusing on representations of discrete-time and continuous-time signals (singularity functions, complex exponentials and geometrics, Fourier representations, Laplace and Z transforms, sampling) and representations of linear, time-invariant systems (difference and differential equations, block diagrams, system functions, poles and zeros, convolution, impulse and step responses, frequency responses). Applications are drawn broadly from
VUCast Extra: Vanderbilt advances 'organ-on-a-chip' research
A team of Vanderbilt researchers is working on a radical new way to test drugs and toxins. It all starts with an "organ on a chip."
Lecture notes
Lecture notes - UNSPECIFIED Keywords:uniform continuity
Patricia Johnston Sings "I See the Sun"
This beautifully done video sings about the sun, moon, stars, ...Some of the lyrics include "I see the sun-a big round sun. I see the moon-a big round moon. I see the stars-the little, little stars---smiling just for me". These videos by Patricia Johnston were designed for young French children to begin to learn English. This is a great teaching resource for early childhood learners and/or special education students and would work well in conjunction with a unit on Earth and to help build backgr
Lecture 23 - 11/15/2010
Lecture 23
Digital Signal Processing
The course treats: the discrete Fourier Transform (DFT), the Fast Fourier Transform (FFT), their application in OFDM and DSL; elements of estimation theory and their application in communications; linear prediction, parametric methods, the Yule-Walker equations, the Levinson algorithm, the Schur algorithm; detection and estimation filters; non-parametric estimation; selective filtering, application to beamforming. Study Goals: You will have acquired insight in how signal processing mathematics i
Biomedical Engineering Design
This course presents a design philosophy and a design approach, dedicated to rehabilitation technology. This field was selected because of human-machine interaction is inherent and vital. Illustrative examples will be discussed by their entire design proces. Study Goals: The student must be able to: 1. Make a Problem Analysis of a given assignment in the field of medical or rehabilitation technology; identify the underlying cause of the problem as presented in the assignment; translate the resul
Genetic Analysis - Question Sets
The Genetic Analysis SBLi is a set of scenarios designed to support an undergraduate course in Genetics. In the scenarios the student plays the role of a genetics researcher, and performs simulated experiments to reinforce concepts taught in lectures. This project is part of the the Higher Education Academy's UK Centre for Bioscience Open Educational Resources programme. This package contains question sets extracted from the Genetic Analysis Scenarios so that these can be easily used seperately.
Engineering and Policy Analysis
In contemporary society, many of the most vexing issues we face cannot be solved by technology alone. Engineering excellence must be coupled with insight into societal needs and the mastery of project and process management tools. At the intersection of technology and public policy TU Delft's MSc Programme in Engineering and Policy Analysis (EPA) is an international interdisciplinary programme, designed to provide its graduates with a unique combination of technical, analytical and management
Policy Analysis of Multi-Actor Systems
This course is about solving complex problems. Our favorite problems are not just technically complex but also characterized by the presence of many different social actors that hold conflicting interests, objectives, and perceptions and act strategically to get the best out of a problem situation. This course offers guidance for policy analysts who want to assess if and how their analysis could be of help, based on the premise that problem formulation is the cornerstone in addressing complex pr
Cavitation on Ship Propellers
Summary: Cavitation is the transition of a fluid into vapour due to local reduction of pressure which is generated by high local flow velocities. The transition of a fluid into vapour also occurs during cooking of water by an increase of the local temperature. The term cavitation is generally reserved for conditions in which the temperature of the bulk fluid is not changed. Although cavitation can occur in many situations this course focuses on ship hydrodynamics and ship propellers. The course
Acoustic Remote Sensing and Sea Floor Mapping
The course treats the following topics: - Relevant physical oceanography - Elements of marine geology (seafloor topography, acoustical properties of sediments and rocks) - Underwater sound propagation (ray acoustics, ocean noise) - Interaction of sound with the seafloor (reflection, scattering) - Principles of sonar (beamforming) - Underwater acoustic mapping systems (single beam echo sounding, multi-beam echo sounding, sidescan sonar) - Data analysis (refraction corrections, digital terrain mo
Checklist - Study Placement
This is a resource released as part of the E-Portfolio Toolkit based on experience of developing the "Year Abroad E-Portfolio", undertaken by the School of Languages at Leeds Metropolitan University.
Engaging students in ethical debates
This case study outlines an investigation into the acceptance of a new pedagogical paradigm aiming to engage and inspire students in ethical and entrepreneurial activity
Badminton : Backhand en netdrop De leerlingen krijgen in deze les allerhande oefeningen op de backhand. Ze leren te verdedigen met aandacht voor: In een tweede …
De leerlingen krijgen in deze les allerhande oefeningen op de backhand. Ze leren te verdedigen met aandacht voor:
In een tweede …
John Hanks provides live accompaniment for American Dance Festival at Duke University
John Hanks, Staff Associate with the Duke Dance Program, provides live accompaniment for American Dance Festival classes at Duke University, whether the dancers need a slow warm-up piece, a waltz, or a "full-tilt boogie." He's an improvisational one-man band! More information: http://americandancefestival.org Website: http://duke.edu YouTube: http://youtube.com/Duke Google+: http://plus.google.com/+dukeuniversity/posts Facebook: http://facebook.com/DukeUniv Twitter: http://twitter.com/DukeU In
Virtual Maths, Shapes Space and Measure, Surface of semi cylinder
Interactive simulation demonstrating how to calculate the surface area of a cover for an astro turf pitch including the ends by using a semi (half) cylinder.


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quick find after no success finding micro in the woods.
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|Publication number
|Publication type
|Application number
|Publication date
|Jul 11, 1995
|Filing date
|Nov 9, 1993
|Priority date
|Nov 9, 1993
|Fee status
|Publication number
|Joseph Yourno
|Original Assignee
|Yourno; Joseph
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (11), Non-Patent Citations (8), Referenced by (40), Classifications (23), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to a method and apparatus for recovery of blood cells from dried blood on filter paper for diagnostic and research tests.
The testing of blood samples in mass screening programs (e.g., testing performed on newborns and individuals remote from the testing center) has introduced a need for greater preservation time of blood samples in a more efficient manner before many types of lab tests can be performed. Currently, blood is preserved through two methods for microscopic or cytometric examination of cells or formed elements. In the first method, blood is preserved in a dry form by spotting on glass slides. The second method includes preservation of wet blood in packages (e.g. in test tubes) having anticoagulant therein. The difficulty with preserving dry blood on glass slides is the blood spot on each glass slide is generally limited to a single testing procedure. Once the blood is spotted on the slide, it cannot be transferred to another slide to perform further microscopic examinations. When a large number of individuals are being tested, this complicates the testing in that each slide needs to be carefully labelled and arranged for indexing. Also, the weight for storage and shipping becomes costly for a large number of glass slides.
The main difficulty with wet blood-in anticoagulant is the limited preservation time before the blood cells begin to fall apart (e.g., through hemolysis or pyknosis). The preservation time of wet blood is generally twenty-four (24) hours for microscopic testing. In mass central screening programs a greater time span than twenty-four hours is desirable for shipping and storage. For example, if a United States lab was being used for mass central screening of blood from Africa or Southeast Asia for Acquired Immunodeficiency Syndrome (AIDS), cells in wet blood samples would fall apart and be unusable for microscopic cell-based testing by the time they reached the lab. Another area with similar concerns in which mass screening of preserved blood is used is with newborns for detection of inherited conditions/disorders.
The present invention solves the difficulties of the prior art by providing a method and apparatus for analysis of dried blood collected from blood spots on filter paper (e.g., Guthrie cards) (See Guthrie R., Blood Screening for phenylketonuria, JAMA 1961;178:863, which is hereby incorporated by reference. Preserving blood cells on filter paper provides an advantage over glass slides in that filter paper is lighter and less bulky for indexing, storage and transportation. Also, the dried blood on a single spot on the filter paper may be made into a plurality of slides after the filter paper has been degraded. The present invention also overcomes the problems with wet blood in that the dried blood on filter paper increases the shelf life of the preserved blood.
The present invention provides a method and apparatus for molecular diagnostics of blood cells on filter paper, such as the polymerase chain reaction (PCR) for use in applications such as genetic screening (See Seltzer, et al., Screening for cystic fibrosis: Feasibility of molecular genetic analysis of dried blood specimens, Biochem Med and Metab Biol 1991;46:105 which is hereby incorporated by reference) and for infectious diseases screening. (See Cassol et al., Diagnosis of vertical HIV-1 transmission using the polymerase chain reaction and dried blood spot specimens, J Acquir Immune Def Syndr 1992;5:113; Comeau et al., Detection of HIV in specimens from newborn screening programs, N Engl J Med 1992;320:1703.6; and Yourno et al., A novel polymerase chain reaction method for detection of human immunodeficiency virus in dried blood spots on filter paper, J Clin Microbiol 1992; 30:2a887, which are all hereby incorporated by reference.). More generally, similar analysis of pediatric or adult blood collected far afield from the testing site may be expedited when specimens are dried on filter paper. Microscopic examination of formed elements recovered from dried blood has not been attempted in the past, presumably because these elements have been considered beyond salvage, whether due to irreversible degradation in-situ or binding to the paper matrix. Contrary to this presumption, mummified white blood cells (WBC) can be recovered from dried blood spots on filter paper such as Guthrie cards.
The present invention is expected to have great utility and application in development of tests on white blood cells (WBCs) such as in-situ molecular analyses; morphologic or other type of enumeration of cells; and recovery of red blood cells and platelets from dried blood on filter paper for microscopic tests. In general, its applicability is expected to include molecular, quantitative, and enumertative hematology. Some specific projected applications include:
(1) detection of infectious agents in newborn, pediatric, or adult WBC by in-situ hybridization or PCR (e.g. Human Immunodeficiency Virus (HIV), Human T-cell Lymphotropic Virus (HTLV), Cytomeglovirus (CMV)) .
(2) detection of leukemic WBC or tumor cells in blood or other tissues by in-situ molecular techniques.
(3) WBC count and differential counts for numerous infectious diseases, inflammatory states, leukocytosis or leukocytopenias.
(4) detection of maternal WBC in cord blood or newborn blood by in-situ molecular techniques.
In summary, the invention is expected to be applicable to the same range of microscopic testing or cytometric examination that wet blood is applicable to.
FIG. 1 represents Wright's stained slide preparations of partially rehydrated, mummified WBCs recovered from dried blood spots from whole blood source with 5700 WBC/μL using Kodak Pan-X, ASA100 at 100� magnification. See Table I for further details.
FIG. 2 is substantially the same as FIG. 1 except that the dried blood spots are from whole blood source with 8300 WBC/μL.
FIG. 3 is substantially the same as FIG. 1 except that the dried blood spots are from WBC concentrate with 51,600 WBC/μL.
The mummified formed elements of dried blood on filter paper are first fixed in-situ to maintain the bodily integrity of the WBC through storage and subsequent isolation. Red blood cells (RBC) are next laked in-situ to reduce RBC background, leaving the fixed WBC and RBC ghosts adherent to the paper matrix. Last, the paper matrix is itself digested away with cellulase in acid buffer, releasing the partially rehydrated, mummified WBC and other cellular materials into suspension for collection and examination.
Blood spot specimens were prepared on Guthrie cards (See Yourno et al, supra, which is hereby incorporated by reference) from a normal donor WBC concentrate (buffy coat) anticoagulated in acid-citrate-dextrose and from seven normal donor whole bloods anticoagulated in ethylenediaminetetraacetic acid. WBC counts were performed by hemacytometer on 10� dilutions of each anticoagulated blood source in 2% acetic acid (Nelson, D, Basic examination of the blood, In: Henry JB, ed. Clinical diagnosis and management by laboratory methods, 18th edition, Philadelphia: W. B. Saunders Co., 1991:564, which is hereby incorporated by reference) (duplicate grids, 2�0.1 mm3 fields counted in each). Counts ranged from low to high normal in whole bloods to markedly elevated in the WBC concentrate (reference interval, 4500-11000 WBC/μL (See Nelson, supra)) (See Table I). WBC differential counts were made on Wright's stained blood smears (Quick Stain procedure, 100-200 cell counts). All specimens yielded differential counts within the reference interval (See Nelson supra) (granulocytes, 58-78%; lymphocytes, 21-40%; monocytes, 2-10%). Blood spots of 1/2 in diameter (equivalent to 50 μL blood) were prepared dropwise onto Guthrie cards with a serological pipette and air-dried 2-4 h at room temperature. The cards were stored in plastic bags at 4�-8� C.
After 24-h storage in each case, the filter paper strips with dried blood spots �5 were cut from the cards, immersed in 60 mL absolute acetone for 10 min at room temperature, air-dried for 10-20 min at room temperature, and stored over drierite in a 1-L roller bottle at 4�-8� C. After storage for 24 h (normal whole bloods) to 51/2 months (WBC concentrate), the treated strips were immersed lengthwise in an upright centrifuge tube containing 55 mL 2% acetic acid for 3 h at room temperature to lake the RBC. The RBC is burst, releasing the hemoglobin and leaving only the empty red cell membrane on the paper (RBC's contain no nucleus, only cytoplasm rich in hemoglobin which is enclosed in a cell membrane. The hemoglobin released from the disrupted RBC formed a loose sediment at the tube bottom over this time, leaving each blood spot residue a light reddish-brown. The doubly-treated strips were then air dried for 2-4 h at room temperature and stored as above.
TABLE 1__________________________________________________________________________Recovery of WBC from dried blood spots on Guthrie cards; hemacytometercounts.Anticoagulated source blood Dried filter paper specimenstarting WBC WBC recovered WBC leftWBC count/μLa WBC count/12 μL WBC countb % WBC countc %X � S.D. X X � S.D. X � C.V. X X__________________________________________________________________________Whole blood(1) 5729 � 426 68748 A 57420 � 9811 84 � 14 183 0.3 B 56100 � 9714 82 � 14 183 0.3(2) 5804 � 668 69648 A 69300 � 11016 100 � 16 B 66825 � 8775 96 � 13(3) 6583 � 920 78996 A 73838 � 19478 93 � 26 B 58163 � 13299 74 � 23(4) 6559 � 421 78708 A 54038 � 20625 69 � 26 B 51975 � 14190 66 � 18(5) 8316 � 459 99792 A 94600 � 31900 95 � 32 367 0.4 B 84150 � 17886 84 � 18 550 0.6(6) 4673 � 535 56076 A 38775 � 3160a 69 � 6 550 1.0 B 41250 � 19503a 74 � 35 367 0.7(7) 5603 � 405 67236 A 53213 � 16208 79 � 24 B 51563 � 10276 77 � 15WBC concentrate51577 � 2445 618924 A 532950 � 39278a 86 � 6 2750 0.4 B 505730 � 13303a 82 � 2 4216 0.7 Overall X � C.V. X 82 � 10 0.4__________________________________________________________________________ a 2 � 0.1 mm3 fields each counted in duplicate grids, b 4 � 0.1 mm3 fields each counted in duplicate grids except as noted. c Count over entire grid.
After storage for 24 h, filter circles were punched from the blood spots with a 1/4 inch paper punch and duplicate circles from each blood spot specimen were transferred each into a 1.5-mL gasketed screw-cap tube (Sarstedt). A solution of Penicillium cellulase (Sigma) was freshly prepared at a concentration of 10 mg/mL of isotonic phosphate buffered saline, pH 5 (PBS.5). To prepare PBS.5, a 25-mL solution of standard PBS, pH 7, was brought to pH 5 by addition of 2.0 g KH2 PO4, then restored to isotonicity by 4� dilution with distilled water. The fresh cellulase preparation was filtered through a 22-μm nitrocellulose filter to remove insoluble debris and 1.0-mL aliquots of the clear, light yellow solution were added to each digestion tube containing a filter circle. The filter circles were then digested for 2 h at 37� at 100-150 rpm on a continuous action vortexer (Vortex Genie II). The end point of the digestion was evidenced by complete disintegration of the filter circle into a coarse, fluffy suspension.
The digests were allowed to settle 5-10 min at room temperature. The pale yellow supernatant and loose reddish-green upper layer of sediment were aspirated from the compacted white paper underlayer and transferred to a graduated 15-mL screw-cap test tube. The digest pellet was twice resuspended in 1.0 mL PBS.5, and resedimented as above. Aspirates from the resedimentations were combined with the original in the 15-mL tube. This suspension (3 mL) was centrifuged at 1250�g for 10 min at room temperature in a swinging-bucket centrifuge (International CRU-5000). The clear supernatant was aspirated off to the 0.3 mL mark and the pellet resuspended in residual buffer. A 100-μL aliquot of this suspension was mixed with 10 μL of Wright's stain (Quick Stain solution 3) for cell counts and for preparation of slide buttons on glass slides. All fractions including the paper residue were stored at 4� C.
After 24-h storage of WBC suspensions at 4� C., direct hemacytometer counts of each were made with duplicate grids, where four fields each were scored. The calculated recovery of WBCs ranged from 66 to 100%, based on an equivalent of 12 μL anticoagulated source blood per filter circle (See Table I). The dilute suspensions from whole blood (equivalent to 12 μL blood in 330 μL final volume, i.e., 27.5� dilution) yielded expectedly high coefficients of variation ranging from 6 to 35%). The suspensions from the WBC concentrate gave less variation at equivalent dilution (2 and 6%). An aliquot of the paper residue from selected preparations was likewise examined by hemacytometer scan as a wet squash mount. Rare WBC, fewer than 1% of the calculated starting count in each case, remained within the mass of paper fibers (See Table I).
Morphologically, the wet hemacytometer mounts showed mummified WBC with moderately stained basophilic nuclei, surrounded by a thin, lightly staining, clear to granular rim of shrunken cytoplasm and cell membrane, which was visible more by phase contrast than by staining (400�).
The recovered WBC ranged from an estimated 7 to 15 μm in diameter. Nuclear detail of the differentiation of granulocytes from mononuclear cells is expected to be greatly enhanced. Occasional WBC with outstretched, segmented nuclei were reminiscent of granulocytes. As seen in FIGS. 1-3, mummified WBC with moderate to deep stained nuclei are visualized against a background of granular RBC debris. The thin rim of lightly stained, shrunken cytoplasm and cell membrane around the WBC nucleus is not seen. The arrow in FIG. 1 shows apparent granulocytes, each with outstretched segmented nucleus. The arrow in FIG. 2 shows cell fragments. The arrow in FIG. 3 indicates occasional cellulose (paper) fiber. Basophilic cell fragments an estimated 2-4 μm in diameter, not counted as WBC, comprised up to 5% of the WBC count in about half the preparations; in the remaining these ranged from 7-15% of the WBC count. Some cells showed outstretched nuclear lobes connected by a thin bridge. These are a likely origin of the cell fragments, reflecting some pyknotic change during isolation. A background of lightly staining RBC debris was present in each wet mount, as well as occasional residual paper fibers. These did not interfere with cell counts and morphologic examination.
From the same suspensions used for cell counts and wet mounts, 5-μL aliquots of each specimen were spotted onto a glass slide, air-dried, refixed in methanol for 1-2 min, stained in eosinophilic stain for 60 min, and then in Wright's stain for 60 min (Quick Stain solutions 1,2, and 3 respectively). Prolonged staining was found better to visualize the partially rehydrated, mummified WBC. Microscopic examination of these slide button preparations showed a picture similar to that of wet mounts, but the thin rim of shrunken cytoplasm and cell membrane surrounding the WBC nucleus was not evident, even under oil immersion (1000�). (See FIGS. 1-3).
The method described for recovery of partially rehydrated, mummified WBCs from dried blood spots on Guthrie cards has been developed empirically. When unfixed filter circles containing dried WBC concentrate were rehydrated and teased apart in Hank's medium, pH 7, only rare free WBC were recovered, but Wright's stained buttons of the paper macerates on glass slides, encouragingly, revealed abundant WBC remnants trapped in the paper fibers. To release the WBC from the paper, the strategy was adopted to destroy the paper substratum with cellulase. Fungal cellulase showed poor activity when filter circles were treated at physiological pH in Hank's medium. The cellulase, which has a pH optimum around pH 5, readily digested the paper matrix at pH 5, however, and released the trapped WBC into suspension. For improved preservation of WBC through storage and isolation from filter circles, dried blood spots were fixed in-situ on the filter paper. Several fixatives were assessed. On the one hand, "hard" fixatives such as methanol yield sharply stained, well preserved WBC mummies, but also cause troublesome clumping of WBC and RBC. On the other hand, "softer" fixatives such as acetone yield WBC which are more hydrated and lightly staining, but clumping is largely eliminated. Unfixed blood spots on Guthrie cards, stored at 4�-8� C. without drying agent and warmed to room temperature numerous times, showed acceptable WBC preservation over a 4 month period from preparation. When next checked at 18 months, however, significant degeneration was evident. Acetone-fixed blood spots have shown good WBC preservation up to 9 months of storage over drierite. Longevity of these fixed preparations is probably much greater than this last measured time span.
Several methods for RBC lysis were tried to eliminate the overwhelming RBC background in cellulase digests of filters. A simple solution of 2% acetic acid produces excellent results. While the protocol which has been devised gives efficient recovery of partially rehydrated, mummified WBC, cell morphology is not sufficiently preserved for differential counts. It is not yet clear whether this is the irreversible result of blood desiccation on the filter paper (instead of on a flat slide) or of the nonphysiological isolation procedure itself. When WBC suspensions produced by cellulase digestion of filter circles were brought back to pH 7 in PBS, lysis of WBC resulted. It remains to be determined whether accurate differential counts can be developed for recovered WBC, by morphologic or other criteria. Further modifications of the procedure are expected to improve cellular integrity and morphology, viz., use of purified cellulase, gentler agitation and/or ambient temperature for digestion. A few neutral cellulases have been described (See Yamane et al., Cellulases of Psuedomonas fluorescens var. cellulos, In: WA Wood and ST Kellogg, eds. Methods Enzymol, Vol. 160. San Diego: Academic Press, 1988:200, which is hereby incorporated by reference) which may permit recovery of WBC in unmodified physiological buffers. This prototype study was based on workup of anticoagulated bloods. As such it is directly applicable to screening procedures using this starting material. It should be extended to bloods prepared from skin punctures directly onto filter paper, possibly impregnated with anticoagulant as an easy way to deal with potential blood clotting problems.
These results are expected to have application to diverse tests on WBC recovered from dried blood spots, from WBC counts to more complex analyses such as in-situ hybridization and PCR. Finally, the results encourage evaluation of variations on the basic procedure to retrieve the other formed elements (e.g., red blood cells and platelets) from dried blood.
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