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A little while back, I wrote that I thought Twitter was ultimately going to become the most important social network. I got some flack for that, especially among those who believed that the richer canvas of Facebook would ultimately prevail. I argued that Twitter's inherent mobile nature set it up to be on the same course as culture in general and that people would simply trend toward Twitter because it just seemed to make sense.
Well, I am happy to report that I seem to be in the right on this one. I'm happy to report this because some of my other predictions ("We already have Yahoo, why do we need another search engine?") have not worked out as well.
Being a San Franciscan, I definitely watched the Super Bowl last week. And, yes, I'm feeling better, but it will still take some time to get back to feeling really good again. At least we still have the World Series champions. But I also had my phone with me the whole time. In fact, I was upstairs, checking on dinner when the most spectacular moment of all occurred: the lights went out in the Superdome.
It was spectacular because I found out what was happening from Twitter. Actually, I thought "lights out" referred to the Niners' secondary. It wasn't long until I realized, like the rest of the country (this is still the biggest shared TV moment in the U.S.), to tune away from the TV and toward social media.
The reason is clear: we are rapidly becoming two-screen people and that second screen is tending to be Twitter. I think the reason is simple: watching television is the destination. The two-screen dynamic is not that people have TV in the background while they tweet; people are tweeting about what they are watching. One could even argue that a handful of people watch TV so they have something to tweet about.
And Twitter is just simply not a destination. If you want to join a conversation about a TV show on Facebook, you must go directly to that show's Facebook page. Often, you have to like it to participate. With Twitter, you simply add a hashtag to a tweet or a search and you are in. It takes some technical skill to set up a Facebook page. It takes no effort to invent a hashtag. Twitter is better suited as a second screen companion simply because it respects the other screen's dominance. In fact, I think we should refer not to "two screens" but rather a primary screen (usually the TV) and the secondary screen (the computer/phone/tablet).
Most of the advertisers in the Super Bowl seem to have gotten the message: the predominant online call-to-action was a hashtag, followed by a Facebook page ("visit our site" has been sent packing). But even more important is that Twitter has gotten it: it just made its biggest acquisition, ever, with BlueFin Technologies, an analytics firm that provides social data about TV shows.
Clearly Twitter gets it: TV is here to stay. We may watch shows time-shifted and on-demand. Shows may be developed not by big studios but by small startups and scrappy players. But TV is here, all 60 inches of it in your living room.
By acquiring BlueFin, as well as hiring a head of TV last year, Twitter is ready to help advertisers do the thing they really want to do: create ads. Let's be honest: interactive sites are amazing and have evolved the nature of communications between brands and consumers. Apps are cool and provide great functionality, as do lots of other innovations. But TV ads, those :30 spots, are not going away. We tried to kill them 16 different ways, but here they are.
That means Twitter is going to win, at least with advertisers, because Twitter has shown its commitment to working within that world. It is committed to making the advertising world better, using the odd little technology that it invented and people seem to love. This is the big realization that we all need to make: advertising is growing and changing. Nothing is a hulking dinosaur waiting to keel over. We are a media-hungry culture and everything seems to be additive. There's a great book about advertising called "Life After the 30-Second Spot" that describes the myriad new ways that brands can build value. Today, that book should be reissued with a slight change to the title: "Life With a Whole Bunch of Other Stuff as Well as the Thirty Second Spot."
Honestly, it's cool with me. I think the broadcast commercial format is pretty cool. You can do a lot inside of it. At work, just yesterday, I heard a pitch for a spot that I thought was amazing. I want to see that built.
And I want to surround it with things like Twitter, to make sure it is successful.
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In part one a few weeks ago, we discussed what brand TLDs (top level domains) are, which brands are applying for them and why they might be important. Today, we'll take an in-depth look at the potential benefits for brands, and explore the challenges brand TLDs could help solve.
Hashtags continue to lose in this year's Super Bowl
According to a report, references to hashtags appeared in just 30% of Super Bowl 51's commercials this year, down from 45% a year ago.
How to build a successful digital video strategy in 2017
The explosive growth of video in 2016 makes 2017 an important year for video content and as more publishers are tempted to use it, it's useful to consider the best strategies to maximise its effectiveness.


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Which Joe gave his name to 'sloppy joes'? We look at five interesting sandwiches and their lexical origins.
nounmass noun
An alcoholic drink made from yeast-fermented malt flavoured with hops.'a pint of beer'as modifier 'a beer bottle'count noun 'he ordered a beer'
ale, beverage, brewView synonyms
- 'We walked and chatted all the way to the pub and went in, ordered some beers and found a table.'
- 'Mark and John had just sat down with the beers when Chick and Brian caught up.'
- 'We are now in Melbourne getting ready to go meet Andy and Tim for more beers.'
- 'The girls sat down next to us, we ordered a couple of beers and were given a book each with a list of songs.'
- 'We all had a few beers at Peters, and Simon invited us all be his guest at his club that night.'
- 'But rather than party into the night, he says he had one or two beers and went home.'
- 'Kip got some beers out of the fridge, but only dad, uncle, granddad and himself drunk them.'
- 'We drank a few beers and Bill loved it: Ada was exactly as he thought my friends would be.'
- 'As the other guys sat back with their beers whilst I had that traditional English drink.'
- 'All my friends stumbled into the lounge whilst Daisy and me went to the kitchen to get the beers and wine.'
- 'Coolers were laid near the sink, where kegs and bottles of beers were thrown in melted ice.'
- 'Too many beers later and God knows how many shots of whisky, the club was beginning to empty.'
- 'All I really want when I venture out at weekends is a few quiet beers, a bit of mickey taking and a few laughs.'
- 'Brewers use several methods to produce beers and lagers with low alcohol content.'
- 'So what I'll be doing for the rest of the evening is lying back on my couch with a couple of beers listening to music.'
- 'Woke up slowly and ambled down in the pouring rain to the pub for lunch and beers with various people.'
- 'They decide to spend a long weekend, catching up and drinking beers, as snow falls.'
- 'One of them asked me if I wanted one of his beers when I was enjoying a burger outside a fast food restaurant.'
- 'We both are quite fond of football and enjoy the lime and a couple of beers as well when we go there to watch.'
- 'He walked back to the bar and asked for two plates of fish and chips, and two beers.'
beer and skittles
often with negativeAmusement or enjoyment.'life isn't all beer and skittles'
pleasure, entertainment, enjoyment, amusement, excitement, gratificationView synonyms
- 'As we well know, the writer's life is all beer and skittles, answerable to no-one, making vast fortunes from every stroke of the pen… and those fabulous public engagements, reading to masses of adoring fans.'
- 'So there you go, it's not all beer and skittles being top dog.'
- 'For the elderly, the late life period is not one of beer and skittles, but rather one of transition and adjustment to loss.'
- 'Long term intense relationships have parts where you have to work at it - it ain't all beer and skittles.'
- 'And as he said, working in China isn't all beer and skittles.'
Old English bēor, of West Germanic origin, based on monastic Latin biber 'a drink', from Latin bibere 'to drink'; related to Dutch bier and German Bier.
Further reading
6 punctuation marks you might be using incorrectlyRead more
We take a look at several popular, though confusing, punctuation marks.
An A-Z of country name originsRead more
From Afghanistan to Zimbabwe, discover surprising and intriguing language facts from around the globe.
How brothers became buddies and brosRead more
The definitions of 'buddy' and 'bro' in the OED have recently been revised. We explore their history and increase in popularity.

subset: commoncrawl


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More Money Spent Updating Southampton Port
April 27, 2015
The Mayflower cruise terminal in Southampton has been given a facelift. Work has now been completed after a 6 million pound renovation effort. The terminal now features a larger passenger lounge, 36 check in desks and extra security lanes. The […]
Read More »
Chairman Says P&O Aren't Worried About Anthem!
March 10, 2015
The UK is known for having a booming cruise industry and according to Carnival Corporation's chief executive Arnold Donald, the country has enough room for two brand new cruise ships. In just over five week's time on April 20th, Anthem […]
Read More »


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My Reviews
My favorite vitamin/herb
Posted by Reviewer1587782 on Aug 19, 2016
I have been taking these for 5 years and these pills have helped me lead a nearly painless life where my knees are concerned. I had to go off them for 5 days and I was really hurting on the 3rd day. I couldn't wait fast enough for this tests to be over and I could restart them again. It's Boswella ( Frrankenscence in the Bible). It takes about 5 weeks in your system to really take effect.
For the Liver
Posted by Reviewer1587782 on Dec 11, 2016
When my brother in law got liver cancer, his cancer doctor told him to take milk thistle and it had to be European. He said everyone should be on it. It helps to detoxify. I've taken them for 7 years- am and pm and all my liver and kidneys stats are excellent. I am 73. When I moved to Fl, my new Doctor was very happy I was on it. He said it will keep your liver heathy.
prostrate helper
Posted by Reviewer1587782 on May 10, 2016
My husband hasn't started it yet. I gave it a 5* because of the Dr. Best name.
helps with toxins.
apple pectin helps with rid of toxins and taked in the cholesterol we just ate. Besure and use 20-30 min. before a meal with alot of water.
Great for Blood Pressure
Posted by Reviewer1587782 on May 10, 2016
This has lowered from pressure to 130/78. I am elated. It acts like a ace inhibor. If you also take it with celery- a big drop. I can't because celery messes up your Thyroid numbers.
Posted by Reviewer1587782 on Jun 26, 2010
No need for over the counter sinus meds now. My sinus's are clear- clear- clear.
Great for your heart and cholesterol. I would not be without these anytime. Learned about these through a cardiologist.
A must have
Posted by Reviewer1587782 on Jan 08, 2011
To keep my glucose in control. It's Boswellia- Frankenscense in the Bible.
greatest product ever.
5-loxin ( boswellia- alka Frankincense alka 3 kings gave to Jesus)) best product ever made. I have arthritis, bad back and knees with alot of inflammation. This product is fabulous and rids that inflammation where the pain goes away and ables me to walk. I take 1 am and 1 pm. My 15 yr maltase came down with such severe srthritis that her quality of life was in question. She would moan getting up and we always knew when she was in pain- she would chew the fur off her feet. I decided to get her off metcap ( like celebrex to a dog) and put her on my5-loxin. What a difference it made. She gets 1/3 cap am and pm. She hasn't been in pain for 2 weeks now and started her morning walk again- slow but walking. The moans are gone and appetite is back. She is a changed dog and the vet was shocked. She told us she thought she was going to have to put her down. She took down my food receipe we feed her and the amount we give her for inflammation, to give to another patient. I also take grape seed/skin,pycnogel, vit c, ginger-all for inflammation.
great pomegranate.
Posted by Reviewer1587782 on May 15, 2016
This has all the parts of the pomegranate.
love it
Posted by Reviewer1587782 on Jan 08, 2011
Cleans my hair- lathers well and leaves thick with no oily residue. I also use the conditioner .
Posted by Reviewer1587782 on Dec 11, 2016
My alc ( blood sugar) was high so I am trying these herbs to bring it down. Berberin, befotiamine, mulberry and cinnulin. My next test is in March.


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Added On: 2014-08-25 02:50:37 • Views: 178 • Duration: 13:47
Categories: Bath
Description: Nebraska Coeds - Since 2001, we embarked this site out of our college dormitory room here in Lincoln, NE. Weve become professionals at partying, and hottest of all, weve gotten truly good at finding damsels that are willing to get naked for our cameras. T


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Victim was taken to Mercy Health St. Vincent Medical Center for serious injuries.
Department posted images from surveillance video on its Facebook page.
Ben Brewton, 20, was found with a gunshot wound to the chest.
Councilman Webb said city needs officers to keep up with retirements.
Airman about to celebrate 21st birthday assigned to the 180th Fighter Wing.
Lucas County commissioners agreed Tuesday to buy a new life squad vehicle for $229,729. The commissioners voted 2-0 in favor of buying the vehicle, a 2017 McCoy Miller 170 Module, from Burgess Hearse & Ambulance Sales Inc. of Loudonville, Ohio,
Scheduled to be arraigned today in Defiance Municipal Court.
Both were taken to the University of Toledo Medical Center.
The victim's name and condition were not immediately available.
Current deputy chief will take over when Chief Kowalski retires next month.


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Upper: Featuring superior microfiber construction to provide weather protection in a light, supple, quite fabric that doesn't sacrifice breathability, durability, or comfort.
Midsole: Dynamic viscous fluid Hydroflow for heel and forefoot enhance midsole cushioning with industry-leading dampening and comfort.
Outsole: HPR Plus a higher durability, abrasion-resistant rubber outsole compound wears longer. Forefoot Hyper Pod Construction components are engineered to set the foot in an efficient balanced position from heel strike to toe-off, offering exceptional flexibility and unrestricted forefoot ride.
Available in width D (Medium).
|Weight :
|6.1 Ounces


text:
This is the full research report for 188.8.131.52, which is an IP address.
BSROU651 Rouen Bloc 2
Wanadoo France Technical Role
48 rue Camille Desmoulins, 92791 ISSY LES MOULINEAUX CEDEX
Telephone: 33 1 58 88 50 00
Wanadoo France Technical Role
48 rue Camille Desmoulins, 92791 ISSY LES MOULINEAUX CEDEX
Telephone: 33 1 58 88 50 00
18.104.22.168 is the IP address you have a ran a report for on February, 24, 2017.
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- Member since: January 2016
- Recent Delivery: about 1 month
- Avg. Response Time: 4 Hrs.
luminointernet's Gigs
luminointernet's Reviews
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Excellent seller A+++++
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Wednesday, January 29, 2003
Some teams thrive at home, while others respond on the road. Then there's the teams that perform well no matter what the surroundings.
The HRV wrestling team is one of those go-anywhere, win-everything teams. And, after another successful weekend, the Eagles are starting to raise a few eyebrows in the Intermountain Conference.
Six wrestlers won both their matches over the weekend at Redmond (36-29) and Bend (55-17) to help HRV raise its IMC dual meet record to 4-1 — good enough for first place in the standings.
"Coming to the IMC is the best thing that could have happened to us," said junior 140-pounder Rocky Level, who won both his weekend matches, along with Zach Bohince (103), Trent Shelton (152),
Jacobe Krizman (171), Nate Dethman (189) and Nigel Bond (215).
"We get to wrestle the best guys in the state every night, and that's really helped us reach a new level. If we had stayed in the Mt. Hood, we wouldn't be as prepared to get where we want to be," he said.
With just three matches remaining before the Feb. 14-15 district meet, Level and his teammates are now beginning to focus on the bigger picture.
"I'm going to go out with a vengeance at districts this year," said Bohince, a 103-pound sophomore.
"Last year was a little shaky and I felt a bit intimidated at times. But I've learned a lot since then and my teammates have been behind me the entire way. Win or lose, this is the closest-knit team I've ever been a part of," Bohince said.
Win or lose, that team unity has been evident in every match this season. The Eagles have lost just two matches decisively (Hermiston on Dec. 12 and Southridge on Dec. 19), and continue to add to their strength with every dual meet and every tournament.
"Just like we said at the beginning of the season, we're a tournament team," Level said. "The way I see it, if we can wrestle this well in dual meets, we should be able to do even better at tournaments like district and state.
"Individually, I know I'm not going to be satisfied with anything less than a district title. There are a lot of other guys who are looking to take it as well. And if a bunch of us advance to the late rounds, we'll be right in the running," he said.
Level has reason to be confident, too. He has lost just one match all year — a 14-7 decision to Jason Appleton of Southridge — and is gaining momentum with every match.
Likewise, Krizman continues to roll toward district supremacy, with his only loss of the season coming against Mountain View on Dec. 19.
"You just have to be ready to wrestle for three rounds every night," he said. "Conditioning is everything this late in the season, and that's where our team has an advantage. You just have to be better than your guy every match, and a lot of us feel like we are at this point."
The next big challenge for the upstart Eagles is a home rematch with the Crook County Cowboys, who defeated them 33-31 at the Jan. 11-12 Oregon Classic in Redmond.
"Crook County is definitely our focus right now," said senior heavyweight Tommy Owyen. "We want to get them back because that was a match we should have won. There's a lot at stake, both for the team and for individuals."
Because district seeding depends on head-to-head records, the Eagles will be thinking about more than just revenge on Friday. Every IMC loss counts, and if a wrestler loses a match he should have won, that could make for a far more difficult road at the district tournament.
"This match is going to be personal," said assistant coach Jeff MacKay. "Every one of the kids wants to get revenge over Crook County. But they're also thinking ahead to district as individuals, which should help the team achieve its goals."
HRV will conclude the IMC dual meet season on Saturday during the Elks Invitational, when they face off against last-place Summit. The annual Elks tournament will be bracketed by weight class, but separate points will be kept for the Summit match.
"We have the potential to win 10 of the 13 weight classes," MacKay said. "But we won't worry about the Elks until we beat Crook County."
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Latest video:
Parkdale third graders sing "12 Disaster Days of Christmas"
Welcome to your sing-able Christmas gift list. What follows is an emergency rendition of "12 Days of Christmas" – for outfitting your home or car in case of snow storm, earthquake, flood or other emergency. Read it as a simple list, or sing it to the tune of "12 Days" – you know, as in " … and a partridge in a pear tree…" Not to make light of it, but the song is a familiar framework for a set of gift ideas that you could consider gathering together, even if the recipient already owns items such as a bunch of coats, tire chains and flashlights. Stores throughout the Gorge are stocked up on all these items. Buying all 12 days might be prohibitive, but here are three ideas for checking any of the dozen off your list (notations follow, 1-12.) The gift items needed to stay warm, dry and safe are also coded to suggest items in your abode (A) in your car (C) or both (B). 12 Gallons of Water (A) 11 Family meals (B) 10 Cans of propane (A) 9 Hygiene bags (B) 8 Packs of batteries (A) 7 Spare coats (B) 6 Bright red flares (C) 5 Cozy blankets (B) 4 Tire chains (C) 3 Flashlights (B) 2 cell phone chargers (B) 1 And a crush-proof first aid kit (B) Price ranges? Here's a few quotes for days Three, Two, Four and Nine: n A family gift of flashlights (three will run $15-30, Hood River Supply, Tum-A-Lum) n Cell phone chargers (two will run $30-60) n Tire chains (basic set, $30, Les Schwab, returnable if unused for the winter) n Family meals ($100 or so should cover the basics for three or four reasonably well-fed days) n The home kit should be kept in a handy place near an exit, and remember that water needs to be replenished every few months. If you have a solid first aid kit already, switch out the gift idea with "and-a-sto-o-u-t- tub-for it-all …" Otherwise, it's a case of assembling your home or car kits and making sure all members of the family know what the resources are and how to use them (ie flares and propane). Emergency situations are at worst life-threatening, at best deeply uncomfortable if you and your family are left without power for an extended period, or traveling and find yourself in a situation where you need to wait out a storm, lengthy traffic delay, or other crisis. Notes on the 12 gift ideas: 12 – Gallons of water: that's one per person in a four-member family to last for three days, the recommended minimum to be prepared for utility outages. 11 – Easy-open packaged goods, energy bars, dried food and nuts are good things to include for nutrition. Think of what your family of four needs for three days to stay fortified and hydrated (see number 12). Can-opener also recommended 10 – If you have a propane camping stove, keep extra fuel handy. 9 – Hygiene bags: put packaged moistened towelettes, toilet paper, and plastic ties in large garbage bags (for personal sanitation) Resource list courtesy of Hood River County Emergency Management, Barbara Ayers, manager/ 541-386-1213. The county also reminds residents to Get a Kit, Make A Plan to connect your family if separated, and Stay Informed. See www.co.hood-river.or.us to opt-in for citizen alerts. Enlarge


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Hotfrog Canada provides information regarding Booster Juice in Edmonton AB. Booster Juice is located at 6136 28 Ave NW, Edmonton AB T6L 6N4 and provides Social services. Contact them on (780) 490-7036 or by visiting them on their website http://www.boosterjuice.com.
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Springbok beauty
- Posted May 18, 2012 by Elainett Viewed 1014 times
- This is a migrated legacy post. Image resolution is low. Info
2 There are 2 comments, add yours!
2012.05.19 Edited
They are so beautiful, love the buck.
2012.05.18 Edited
Go Bokke!!! They are so dainty and graceful and beautiful, no wonder they are the national animal...
I do love the eyelashes!


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Last week the Helsinki District Court decided that Peter Sunde, one of the co-founders of file-sharing portal The Pirate Bay, owes quite a bit of money to the music industry. Specifically, he owes a coalition of Sony Music Entertainment, EMI, Universal Music and Warner Music $US400,000 ($533,429), as reported by Torrent Freak. His response? No, screw you.
Tagged With music industry
From thinking they don't need licensing deals, to "aggressively courting" the big record labels, Amazon's change of heart on the Cloud Player storing/streaming issue will likely cost them dear. After all, it's not like the labels will be responding nicely to their calls, after effectively setting up a streaming service to rival their own (in Sony Music's case, anyway.)
Selling individual tracks online is not the only thing Pink Floyd has been raging against recently. The band has removed several albums from iTunes, Amazon, et al, due to EMI's contract expiring at the end of June.
It's a lousy time to be a record label. Profits are tanking, bands are angry - OK Go just ditched EMI - and YouTube and BitTorrent changed the game. Still, some labels are transforming themselves to help musicians in the digital age.


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Day 2 at the RNC Jul 20, 2016 - 12:07 AM EST Thirteen months after launching an improbable bid for the White House, Donald Trump has captured his party's nomination, having seen off 16 party rivals and provoked controversy at every turn.
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Newsmaker with Preet Bharara Jul 13, 2016
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Advanced Post Nov 10, 2016 REUTERS/Mark Kauzlarich The cavalcade of Republican luminaries continues in and out of Trump Tower today.On today's bill:Rick Perry, former...
North Korea spy agency runs arms operation out of Malaysia, U.N. says
It is in Kuala Lumpur's "Little India" neighborhood, behind an unmarked door on the second floor of a rundown building, where a military equipment company called Glocom says it has its office.


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iPhone apps
Get Any Girl: Free Lite Edition (Dating Guide)
Have you ever wondered about the secret to getting those attractive women who seem out of reach? Well the answer is not a secret at all. You know why? Because you were born with the answer. Despite how complex the world has seemed to become, the….. Read more
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If you love deals and surprises, then you have to fly on over to CMP favorite Darlybird and snatch up one of their very limited and very discounted Bags O' Goodies.
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Royal Dutch Shell declined to provide details about its plans to resume offshore oil and natural gas exploration in the Arctic, after it encountered several drilling setbacks in the region in 2012. "Our future plans for offshore Alaska will depend on a number of factors, including the readiness of our rigs and our confidence that lessons learned from the 2012 operating season have been fully incorporated," said Curtis Smith, a spokesman for the company.
Full Story:Los Angeles Times (tiered subscription model)


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Holland Study Abroad Program
Travel to Holland! Explore the numerous museums of Holland, learn about Vincent van Gogh and Ann Frank, and travel down the canals of Amsterdam. For more information, please click here to contact us.
Our Travel Programs
Holland Travel Guide
This document is to be used as a reference for both frequent fliers and for those who have never traveled outside of the country. The guide will help to equip you with the essential information for travel to Holland, what to prepare for, and what you will need while in the country. This guide is complete with a checklist for travel as well as all traveling restrictions and guidelines for international travel. Additional information on Holland can be found in the important information on Holland page of this guide.


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Kevin L. Hoag
Skill Development for Engineers: An Innovative Model for Advanced Learning in the Workplace
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· OZON.ru 2569р. [Проверить наличие]
ISBN: 0852969791Издательство: IEE Publishing
Organizations operate in an increasingly competitive environment, which drives a need for continuous employee skill development. The rapid pace of technological change requires everyone to continue learning throughout life. This book questions the effectiveness of current employee learning in most organizations and presents an alternative framework model -- which is less costly and more successful. Many practical examples of successful approaches are given in a highly readable format, with many diagrams. The author has many years of practical experience as a Training Manager in a large North American engineering company, and his work on skills development there won him and his company the Glenn Martin Award as the Best Training Organization at the 1998 World Conference on Continuing Engineering Education.
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Personalized Golf Tees
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Rihanna For Harper's Bazaar August 2012
Get More: Fabolous, Music News
Fabolous says, "Loso's Way 2," will drop in the early Fall.
You can trust Gucci to put a luxurious spin on a stalwart style, and this re-worked varsity jacket is a great example of the brand's aesthetic. Featuring a fine suede front and cotton-jersey sleeves and back, this cover-up makes a bold statement. Layer yours over a button-down collar shirt and knitted tie for a dose of all-American cool.
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Pikeville is located in Pike county and has a population of 6,295.
The average in-state tuition for full time undergraduate students in practical nursing schools in Pikeville during the 2009 - 2010 school year was $14,535.00. The average tuition was $14,535.00 for non-residents studying in Kentucky.
The cost, on average, for books and supplies needed for practical nursing schools in Pikeville is $2,000.00. The average cost for room and board for students living on campus at practical nursing schools in Pikeville was $6,000.00. The average cost for students living at home was $5,000.00 for the 2009 - 2010 school year. There is one practical nursing college in Pikeville for students to choose from. 20 students were reported to have graduated from practical nursing programs in Pikeville in the 2008 - 2009 school year.
|Average Salary
|Number Employed in City
|Licensed Practical and Licensed Vocational Nurses


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Winner: 2012, 2013, 2014, 2015
PEPS - Program for Early Parent Support
PEPS Groups bring parents together to share the joys and challenges of parenthood and develop confidence in their own abilities. PEPS was born in Seattle more than 30 years ago and for many parents, a PEPS Group becomes a supportive community that lasts a lifetime. PEPS stands apart from other new parent groups with a focus on creating connections and community – factors that we know help strengthens families, increase family wellness, and prepare families to cope with life stresses. One parent said, "At PEPS, it's okay to ask any and all 'is this normal??!!' questions!" Groups provide ongoing resources and peer education, facilitated by trained volunteers. PEPS Newborn Groups start when babies are 0-4 months old and meet for 12 weeks facilitated by a trained volunteer. Register for an evening or daytime group in your neighborhood. Create Your Village with PEPS.


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Use of ex vitro composite plants to study the interaction of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides
- Karolina E Mellor1,
- Ava M Hoffman1 and
- Michael P Timko1Email author
© Mellor et al.; licensee BioMed Central Ltd. 2012
Received: 16 May 2012
Accepted: 21 June 2012
Published: 28 June 2012
Cowpea (Vigna unguiculata L.) is an important grain and forage legume grown throughout sub-Saharan Africa primarily by subsistence farmers on poor, drought prone soils. Genetic improvement of the crop is being actively pursued and numerous functional genomics studies are underway aimed at characterizing gene controlling key agronomic characteristics for disease and pest resistances. Unfortunately, similar to other legumes, efficient plant transformation technology is a rate-limiting step in analysis of gene function in cowpea.
Here we describe an optimized protocol for the rapid generation of transformed hairy roots on ex vitro composite plants of cowpea using Agrobacterium rhizogenes. We further demonstrate the applicability of cowpea composite plants to study gene expression involved in the resistance response of the plant roots to attack by the root parasitic weed, Striga gesnerioides. The utility of the new system and critical parameters of the method are described and discussed herein.
Cowpea composite plants offer a rapid alternative to methods requiring stable transformation and whole plant regeneration for studying gene expression in resistance or susceptibility responses to parasitic weeds. Their use can likely be readily adapted to look at the effects of both ectopic gene overexpression as well as gene knockdown of root associated defense responses and to the study of a broader range of root associated physiological and aphysiological processes including root growth and differentiation as well as interactions with other root pests, parasites, and symbionts.
KeywordsAgrobacterium rhizogenes Composite plants Hairy roots Resistance Striga gesnerioides Witchweed
Cowpea (Vigna unguiculata L. Walp.) is the most important grain legume grown in sub-Saharan Africa [1, 2]. Approximately 12.5 million tons of cowpea grains are produced worldwide each year with a majority (over 64%) of the production taking place on low-input, subsistence farms in West and Central Africa [3]. Two characteristics contribute to its agronomic and economic importance. The plant is generally drought tolerant and can provide some yield even under harsh conditions, and it fixes nitrogen symbiotically thereby enhancing soil fertility especially when used in rotation with cereals [4, 5]. Throughout the Sahel cowpea is referred to as "poor man's meat" because of its high protein content (20-25%) and good nutritional value [6]. The pods and seeds are consumed at all stages of growth (e.g., green pods, fresh or dry seeds) and the young leaves are often used for soups and stews [7]. In addition to its value as human food, cowpea hay is an important source of animal fodder [8]. Improvement of cowpea as a multifunctional crop is a key breeding concern and significant efforts are currently aimed at its genetic improvement [2].
Like most crops, cowpea growth and grain yields are greatly reduced by a variety of biotic pests (e.g., bacterial, fungal, and viral diseases, insects, nematodes, and herbivores) and abiotic stresses (severe drought, salinity, and heat) [2]. Among the major biotic constraints is parasitism by Striga gesnerioides (L.) Walp. (Orobanchaceae) commonly referred to as witchweed. Witchweeds are noxious and persistent pests in farmers' fields and yield losses due to S. gesnerioides parasitism are extensive in the Sudano-Sahelian belt of West and Central Africa [9]. Control of the parasite is difficult because it produces thousands of seeds per generation that remain in the seed bank for years and most of the damage to its host plant occurs prior to its emergence from the ground [10]. The damaging effects of Striga in this region are further compounded by poor soils and drought [11].
While most cowpea plants are susceptible to Striga parasitism, some local landraces and wild accessions have been identified that are resistant to the parasite, and in most reports resistance is a dominant characteristic, inherited in a monogenic manner [2, 12]. Complicating the identification of Striga-resistant germplasm is the variable nature of the parasite with at least seven distinct races of S. gesnerioides (designated SG1 (Burkina Faso), SG2 (Mali), SG3 (Nigeria and Niger), SG4 (Benin), SG4z (Zakpota region of Benin), SG5 (Cameroon), and SG6 (Sénégal)) now identified throughout West Africa [13–15]. Analysis of several advanced populations segregating for resistance to one or more of the different races of S. gesnerioides has resulted in the genetic mapping of several race-specific resistance (R) genes within the cowpea genome and the development of molecular markers liked to these genes [16]. Using a positional cloning approach, Li and Timko [17] isolated and characterized a gene (designated RSG3-301) capable of conferring resistance to S. gesnerioides race 3 (SG3). RSG3-301 encodes an R protein homolog containing a coiled-coil (CC) protein-protein interaction domain at the N-terminus, a nucleotide binding site (NBS), and a leucine-rich repeat domain at the C-terminus. Silencing of RSG3-301 in the resistant cultivar B301 leads to susceptibility to race SG3, but does not affect resistance to other races of the parasite, underscoring the specificity of the resistance response [17].
Resistant cowpea genotypes exhibit two different response mechanisms to Striga attack. When challenged by a known race, cultivars carrying the appropriate race specific resistance gene exhibit a rapid and robust hypersensitive response typified by a browning and necrosis at the site of parasite attachment, and subsequent rapid death of the parasite within 3–4 days [15, 18, 19]. In host plants lacking the appropriate resistance gene, the parasite rapidly penetrates the host root cortex, forms connections to the host vascular system, swells to form a tubercle, and expands its cotyledons leading to subsequent above ground growth and flowering.
Like most legumes, the genetic transformation and regeneration of cowpea has proven to be difficult and challenging [20]. Cowpea is susceptible to genetic transformation by Agrobacterium, and a number of reports have appeared in the literature describing stably integrated and heritable transgenes following A. tumefaciens-mediated transformation [21–28]. However, in vitro regeneration of shots/seedlings from various seedling explants (including primary leaves [29], epicotyl [30], mature cotyledon [31], cotyledonary node [32, 33] and nodal thin cell layer [34]) appears to be highly dependent on cultivar genotype with even the most promising genotypes giving very low regeneration frequency and numbers of regenerants. Coupled to transformation, the efficiency of recovery of fertile transgenic cowpea plants ranges from 0.1% to a 1-2% [25–28]. Given this limitation, traditional transgenic approaches for gene functional characterization in cowpea are difficult.
Ex vitro composite plants consist of a wild-type shoot with transgenic roots induced by transformation with A. rhizogenes[35–37]. The use of ex vitro composite plants has proven to be a successful alternative strategy for candidate gene analysis and genetic pathway dissection in plants where transformation and regeneration is difficult, time consuming and infrequent. The technique has been applied to the study of root nutrient uptake, hormone transport, interactions with root nodulating bacteria and mycorrhizal symbiotic, and parasitic nematodes [35, 36, 38]. Here, we present a protocol for the rapid development of transformed hairy roots on composite plants of cowpea using A. rhizogenes induced root formation. We further demonstrate the applicability of this approach to studying gene function involved in the interaction of cowpea with the root parasitic angiosperm, S. gesnerioides.
An optimized protocol for the generation of ex vitro composite cowpea plants
At multiple points in the protocol we empirically determined the optimal conditions for recovery of composite cowpea plants. Since composite plants potentially generate a mixture of transgenic and non-transgenic roots, in order to optimize transformation efficiency (defined as the percentage of plants exhibiting one or more transgenic roots) and increasing the portion of transgenic versus untransformed roots, we used high level constitutive expression of green-fluorescent protein (GFP) as a biomarker for transformation. In these studies, plasmid pCambia1300-Gmubi3 [39], containing the soybean polyubiquitin 3 promoter fused to the coding region of GFP, was introduced into A. rhizogenes R1000 strain and used to determine the best conditions for cowpea transformation.
Among the factors that significantly enhance transformation efficiency, we found that the temperature at which co-cultivation with A. rhizogenes was carried out and the length of the dehydration stress period imposed following co-cultivation were most important. As shown in Figure 3 plants grown at 21°C showed the highest transformation rate. Over 80% of plants infected with Agrobacterium harboring pCambia1300-Gmubi3 binary vector underwent a stable transformation (Figure 3C). Plant transformation efficiency was significantly lower when co-cultivated with A. rhizogenes at 23°C. Less than 40% of plants expressed GFP in their roots. Temperature also had an impact on the percentage of transgenic versus non-transgenic roots formed on the composite plants, with plants transformed at 21°C exhibiting a significantly higher percentage of stably transformed roots (~25% of total roots generated) compared with 23°C (~10% transgenic) and 25°C (5% transgenic), respectively (Figure 3D).
Use of composite plants to study striga-host interactions
We have previously demonstrated that resistance in cowpea to parasitism by S. gesnerioides is conferred by monogenically inherited resistance genes that function in a race-specific manner [16, 17]. The cowpea cultivar Blackeye is known to be susceptible to all races of S. gesnerioides identified in West Africa [15]. Having established conditions for the efficient generation of cowpea composite plants, we next explored whether our optimized protocol could be used for the study of root associated activities, in particular the interaction of cowpea cultivars with S. gesnerioides. To this end the cowpea RSG3-301 gene, known to confer resistance to S. gesnerioides race SG3, was placed under the control of the constitutive CaMV 35S promoter in plasmid pK7WG2 and pK7WG2D yielding pKM0-RSG3-301 and pKMG-RSG3-301, respectively. pKM0-RSG3-301 allows for the overexpression of RSG3-301 alone, whereas pKMG-RSG3-301 expresses both RSG3-301 and GFP. The two plasmids were independently introduced into A. rhizogenes R1000 and used to generate composite plants using the optimized protocol described above. Composite plants were then used to determine whether (i) transformed roots of composite cowpea plants responded similarly to wild-type roots with respect to the resistance/susceptibility phenotype to attempted parasitism by S. gesnerioides, and (ii) whether introduction of a race-specific resistance gene (RSG3-301) into a susceptible cowpea genetic background (i.e., the Blackeye cultivar) confers race-specific resistance demonstrable in composite plants roots.
Tabular data of relative RSG3-301 transcript levels in transgenic and non-transgenic roots of cowpea composite plants
Sample name
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Average relative transcript levels
Discussion and conclusions
The recalcitrance of cowpea to efficient transformation and regeneration has been a bottleneck in functional genomics analysis and bioengineering for genetic improvement of the crop. The current study addresses one of these fundamental limitations by providing a fast and relatively easy method to rapidly generate and evaluate multiple independent transformants for screening gene expression associated with root localized plant functions. The transformation efficiency we have achieve (up to 80% plants with at least one transgenic root; and 25% of all roots formed being transgenic) with our optimized protocol is comparable to that obtained by Collier et al [36] for soybean who reported between 2 and 4 transformed roots per transformed plant with a transformation efficiency of ~ 80%. It is also similar to that reported for Medicago Phaseolus and pea [35, 37, 38] although direct comparisons are more difficult because of the manner in which transformation rates were measured. Nonetheless, the recovery of transgenic events we achieve are significantly higher than the efficiency of recovery of fertile transgenic cowpea plants using traditional A. tumefaciens –based transformation and regeneration which range from 0.1% to a 1-2% [25–28]. It should be noted that the limitations of the composite system are that although these are stable transformations they do not allow recovery of stable fertile offspring and therefore analysis is constrained to only the initial generation.
The great advantage of the composite system is the utility for studying root associated parameters such as parasitism by weedy parasitic angiosperm and other root pests and pathogens. In this regard, the use of composite plants make it possible to look at the effects of both ectopic gene overexpression as well as gene knockdown on root associated defense responses. As demonstrated in our above studies, the composite plant system was capable of recapitulating what is found in non-transgenic plants with regard to the resistance or susceptibility response to attempted Striga parasitism. Overexpression of a race-specific resistance gene (e.g., RSG3-301) in a susceptible genotype resulted in acquisition of the resistance phenotype similar to what is observed in a naturally occurring resistant cultivar such as B301. This observation indicates that the process of transformation does not fundamentally change the nature of the signal transduction apparatus involved in defense in the transgenic roots. This might have been the case since the introduction of ROL genes by A. rhizogenes and generation of the hairy-root phenotype is known to be associated with alterations in phytohormonal concentrations [40]. Clearly, this is not significantly impacting the defense processes (or lack thereof) in the plant. This result opens up the potential for using the composite plant approach to evaluate various candidate genes that might be involved in cowpea resistance or susceptibility to S. gesnerioides, as well as other root parasitic angiosperms of economic significance such as Alectra vogelii. The utility as noted by Collier et al [36] lies in the fact that candidate gene constructs identified by screening in composite plants can be moved immediately without further manipulation to A. tumefaciens for the production of stable transgenic plants. In the case of cowpea, where the efficiency of traditional A. tumefaciens –based transformation and regeneration is low, this would result in a significant economy of time and effort.
The cowpea composite plant system also has applicability to studying gene expression in other physiological processes such as root development, growth and differentiation, and aphysiological conditions such as bacterial, fungal or viral induced diseases and interactions of roots with parasitic nematodes, nodulation by symbiotic bacteria, and colonization by mychorrizal fungi.
Materials and methods
Plant materials and growth
Descriptions of the cowpea accession used in this study can be found in [17]. B301 is a Striga multi-race resistant cultivar from Botswana; Blackeye is a Striga susceptible commercial variety. Seeds of the cowpea cultivars B301 and Blackeye were surface-sterilized with 1% Metricide (Metrex, Romulus, MI) for 5 min [41]. They were then placed between two sheets of moist glass fiber filter paper (GF/A Whatman; Piscataway, NJ), held between two blocks of moistened rockwool (Grodan Inc., Milton, ON) for 10 days until leaves have fully developed. Plants were grown at 30°C in a growth chamber to ensure moist environment. The chamber was covered with a transparent dome which light passage.
Vector construction and generation of Agrobacterium rhizogenes strains
The structure of the pCambia1300-Gmubi3 vector has been previously reported [39] and contains green fluorescent protein (GFP) coding region under the control of the soybean polyubiquitin 3 promoter. Gateway compatible destination vectors pK7WG2 and pK7WG2D [42] were obtained commercially (VIB, Brussels, Belgium). For overexpression of the cowpea RSG3-301 resistance gene [17], the RSG3-301 coding region was mobilized into the pENTR/D-TOPO intermediate vector (Invitrogen, Grand Island, NY) and pENTR/D-TOPO-RSG3-301 recombined with pK7WG2D using the LR recombination reaction in the Gateway LR Clonase Enzyme mix (Invitrogen, Grand Island, NY). The resulting plasmid is designated, pKMG-RSG3-301. The RSG3-301 coding region in pENTR/D-TOPO-RSG3-301 was similarly recombined with pK7WG2 generating pKM0-RSG3-301. The relevant portions of pKMG-RSG3-301 and pKM0-RSG3-301 were sequenced to confirm the integrity of the introduced gene.
The various plasmids (pCambia1300-Gmubi3, pKMG-RSG3-301 and pKM0-RSG3-301) were transformed into Agrobacterium rhizogenes R1000 competent cells by electroporation and transformed colonies were selected by growth for two days on solid YEB medium containing 0.1 mM KAN. Independent positive transformants were picked and inoculated into liquid YEB medium containing 0.1 mM KAN and grown at 28°C until saturation. Cells were collected and plasmid DNA was prepared using the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Glen Burnie, MD). The presence of the introduced plasmid into the Ti plasmid was verified by restriction digestion using BsrG1 enzyme (New England Biolabs, Ipswitch, MI). Colonies of Agrobacteria confirmed to be transformed with the various constructs were selected, grown in liquid culture, and stored at −80°C in 50% glycerol solution until ready to use.
Plant transformation
Two days prior to plant transformation, A. rhizogenes carrying pKMG-RSG3-301, pKM0-RSG-301, pK7WG2D and pK7WG2 plasmids were thawed and streaked out on solid YEB medium containing 0.1 mM (50 mg/l) KAN, 0.4 mM acetosyringone and grown at 28°C. Individual colonies were picked and inoculated into 10 ml liquid YEB media with antibiotic and supplement as mentioned above and grown overnight until OD600 = 0.8 was reached (OD600 of 1.0 = 5 x 108 cells/ml).
On the day of plant transformation, bacterial suspension was spun down at 3,500 rpm and resuspended in 15 ml 20% MS media with vitamins (Caisson labs, Logan, UT) supplemented with 0.4 mM acetosyringone. Bacteria were grown for another several hours until OD600 = 0.8 was reached and then 6 ml of the bacterial suspension was pipetted onto a 1 cm3 rockwool cube (Grodan Inc., Milton, ON) so that each cube was saturated.
Ten day-old cowpea plants were taken out of growth chamber, and the roots excised approximately 3 cm below the cotyledons using a sterile razor blade. The plant was then inserted into the bacteria-saturated rockwool cube and placed into a darkened growth chamber at 21°C to ensure high transformation efficiency. On the second day, a lid from the growth chamber was removed which allowed plants to dry. Applying drought stress appeared a crucial factor for increasing amount of transformed roots. Plants were rewatered after leaves became obviously wilted. This procedure was repeated two days in the row. On the fourth day plants were transferred into a 22°C growth room and allowed to grow in cubes. First transformed roots emerged from the cube 12–14 days after transformation. Plants were then taken from growth room and rockwool surrounding emerging roots was gently removed using forceps. Cowpea was then transferred to 24 cm x 24 cm x 3 cm growth chamber containing rockwool with a 100 μm mesh separating the cowpea roots from the rockwool [17]. Cowpea seedlings were grown for another 7 days in a controlled environment growth room under a 12 H light–dark photoperiod at 30°C. After a week, plants infected with pKMG-RSG3-301 were tested for GFP present in their roots using Zeiss Stereo Discovery V20 with Axciocam MRc High resolution camera. Several roots (transformed and non-transformed) were then collected and stored in −80°C for qRT-PCR to assess RSG3-301 transcript levels in the tissue. Due to the lack of GFP, plants infected with pKM0-RSG3-301 were only collected for qRT-PCR.
Assessing Striga – host interactions
S. gesnerioides is a federally regulated noxious weed. All work involving viable S. gesnerioides seeds, developing parasites, and all analysis of host-parasite interactions, were performed in our APHIS approved quarantine facility at UVA (Facility Number 669; Permit No. P526P-11-03310). Seeds of the various S. gesnerioides races were collected in the field as reported previously [15]. Seeds of S. gesnerioides race SG3 were surface sterilized, pre-conditioned for 9 days [15], and germination triggered using root exudates from cowpea cultivar B301. Pre-germinated seeds (50 mg equivalent to ~7500-10,000 seeds, with an average germination of 75% at time of infestation) were gently transferred on developed cowpea roots (pKMG-RSG3-301 fluorescent and non-fluorescent; pK7WG2D fluorescent and non-fluorescent) using a paint brush. Growth chambers were closed and left for another 8 days for infection to occur. After 8 dpi, infection rate was assessed. Number of events (tubercle formation and hypersensitive response) was counted on plants infected with pKMG-RSG3-301 for both fluorescent and non-fluorescent roots as well as plants infected with pK7WG2D. Number obtained from 14 individual plants per treatment were averaged and presented ± SE.
Verification of RSG3-301 transcript levels using qRT-PCR
qRT-PCR was used to validate RSG3-301 transcript levels of RNA samples prepared from Blackeye root tissues subject to the various treatments described above. Total RNA was extracted as described in [43] with minor modifications and quantified using a Nanodrop instrument (Thermo Scientific, Wilmington, DE). cDNA was synthesized from 2 μg of DNAse- treated (Roche, Indianapolis, IN) total RNA using an Invitrogen Thermoscript Kit (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. The reaction was carried out in triplicate.
For qRT-PCR reactions, gene-specific primers were designed against the RSG3-301 gene coding region [17] that give a 147 bp amplicon and an actin reference gene (XM_003521168; GI: 356504867) giving a 132 bp amplicon. The primers used are as follows: RSG3-301-F: 5′- AGTAAGGGATGTTGGAAGCAA -3′and RSG3-301-R: 5′- AATTACATCAGACTCGGGAAT -3′); Actin-F: 5′- CGAGCAGGAATTGGAAAC-3′and Actin-R: 5′-ATCATGGATGGCTGGAAC-3′. Amplification reactions (25 μl) containing 15 ng of cDNA were carried out in Multiplate PCR 96-well plate formats (Bio-Rad, Hercules, CA), using the iQ SYBR Green supermix kit (Bio-Rad, Hercules, CA) in an iCycler Optical Module PCR instrument according to the manufacturer's instructions quantification of relative transcript abundance was performed described by Schmittgen and Livak [44] by calibrating threshold cycles of the RSG3-301 transcripts with that of the actin reference gene. The equation 2(−ΔΔCq) was used to calculated relative expression (difference in concentration between samples based on normalization with a reference gene), in which Cq is the cycle number of the threshold point at which the fluorescence is detectable. A total of 5 independent biological replicates were used and mean and standard error (SE) were determined.
- dpi:
days post- inoculation
Green fluorescent protein
Hypersensitive response
Luria bertoni
Murashige and skoog
Optical density
- qRT-PCR:
quantitative reverse transcriptase polymerase chain reaction
- rpm:
Revolutions per minute
Yeast extract and beef.
We thank Kan Huang for helpful comments on various aspects of this work. This work was supported in part by grants from the Kirkhouse Trust and Natural Science Foundation (IBN-0322420 and IOS-07-01748; CR-20006-477823) awarded to MPT.
Authors' Affiliations
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United Kingdom
Hotel rooms in Crawfordsburn
There are not so many hotel rooms in Crawfordsburn, just 2, still everyone can find an offer to suit his choice. The most common offers in this city are lodges, they count 1 out of 2, and their prevalent location is central district. According to our practice, it is the most profitable to book a hotel in the west of Crawfordsburn, as it is the location of the hotels with optimal cost and quality offers along with the cheap hotels. Taking into account the small size of the city, there is no need to dwell hotel reservation on the location: the road to the city center would take less then half an hour, so one can book a hotel in any district of Crawfordsburn. The most populated with hotels district of Crawfordsburn is the north, 50% of cheap hotel rooms are concentrated here. Crawfordsburn is an ideal place for couple and solo, such travellers rate hotels there 8.8 out of 10 on average. Apart from all these, hotels in Crawfordsburn offer their customers laundry service, gym, business centre and bar. The most appreciated amenities in Crawfordsburn hotels are service and breakfast. According to the visitors reviews, hotels in Crawfordsburn has tasty breakfast, great location and delicious food. Take a look at our selection of the cheap hotel rooms in Crawfordsburn to make a well-informed choice for your reservation!
Best hotels in Crawfordsburn
Hotels in Crawfordsburn are spread around the city within the distance of 2.6 kilometers, while 1 hotels are located directly in the city center. Those who are keen on antique should definitely fall in love with
The Old Inn
, the oldest hotel of Crawfordsburn, constructed in 1614. This hotel is located 0.3 km away from the city center. The most popular accommodations in Crawfordsburn is
The Old Inn
, 4 stars hotel located in a 0.3 kilometers from the city center.
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Join Frederick Van Johnson, Alex Lindsay, and friends each week as they discuss photography, camera technique, technology and news. With in-depth guest interviews and discussion around everything from taking family photos to understanding how state-of-the-art cameras work — you'll hear it all on This Week in Photo.
May 7th, 2015
Episode 410 of 499 episodes
TWiP guest Joe Giordano is abused by Baltimore police while covering the protests and riot. Is the Apple watch a photographer's best friend? Photofocus' Doug Daulton thinks so. Join Frederick Van Johnson and his guests Martin Bailey, Jeffrey Totaro and J.M. Giordano.
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