text:
We are defined by what we do,
The choices we make,
The way we react
To the situations that present themselves to us.
All we do can have a profound effect
On to our path that guides our future.
The choices once made
Can never be taken back,
Mistakes can be learned from,
Experience gained,
But we will always be defined by the choices
Good or bad.
The choices that make us,
The choices that destroy us,
And all that remains
Is the shell.
The shell of who we are,
The shell of who we were always meant to be.
If you enjoyed this poem and would like to read more of my work I currently have two books available that can be obtained through the links below.
Velvet Death – The Dark Poetry Anthologyhttp://www.lulu.com/content/177683 Velvet Paradox – The 2nd Dark Poetry Anthologyhttp://www.lulu.com/content/254973
© Copyright 2017 Byron Quinn. All rights reserved.
Status: Finished
Genre: Poetry
Poem / Fantasy
Poem / Fantasy
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Marc Anthony says working with Jennifer Lopez is like second nature, which is good since the exes are uniting for one night in concert.
Jennifer Lopez, 'American Idol' Judging Deal Is Done
By: Reuters on August 1, 2011 in Fall TV Preview 2011, TV News
By Tim Kenneally LOS ANGELES (TheWrap.com) – Fear not, Jennifer Lopez fans: the singer is returning to "American Idol" to judge for at least another year, TheWrap has learned through two individuals familiar with the deal. The pop star will be returning to the television singing competition with a contract with the Fox Broadcasting Network […]
J.Lo and Marc Anthony Team Up For New Show
By: Associated Press on April 27, 2011 in American Idol, Celebrities
By NEKESA MUMBI MOODY NEW YORK — Jennifer Lopez is teaming up with "American Idol" creator Simon Fuller for a new TV show – and she's bringing husband Marc Anthony into the fold. The trio announced Wednesday that they are creating "Q'Viva! The Chosen." The show would feature the superstar couple as they travel 21 […]

subset: commoncrawl


text:
Published on
At Ci&T, we believe that in the not-so-distant future, everyone will not only have a smartphone, but truly use it as a mobile computer and require unique software to maximize their experience. Already imperative, engaging, innovative user experience will only continue to grow in importance in appealing to such demanding users.
Needless to say, at Ci&T, we go beyond just mobile app development. We manage the entire mobile strategy lifecycle from ideation; to Lean UX development (which rolls art, agile development and UX design into one process); to metrics and support; we keep the process tight and light to ensure you achieve your business goals - fast. We use the most innovative approaches to accelerate product validation by real users, while minimizing investments.
Ci&T offers a full range of mobile services across all major available platforms. We focus on the following three areas, each of which requires a unique design approach, enabling us to fully maximize value to your business:
Digital Marketing:
To enhance, and later support, our clients' market strategies, we build and maintain highly engaging mobile web apps and native apps that connect users to our clients' brands.
Consumerization fast became a reality and as a result, your employees are both driving adoption and business innovation. Eager to access internal systems on their smartphones and tablets, enterprise apps are inspiring motivation in and improving the productivity of your employees – not a bad combination. Whether you need to integrate a CRM, SAP or BI system, just to name a few, we can connect those dots and make things work.
We have extensive experience working with start-ups and understand the need to move quickly, especially under uncertain circumstances. Accelerate, our product engineering service, turns innovative ideas into marketable products by applying the Lean Startup model, managing the customer development process, employing Lean UX concepts and managing low burn-rate. So it is clear that we don't just talk the talk, we… well, you know.


text:
Our ACS is an outstanding professional society with a passion for professionalism, chemistry in the broadest sense, diversity and inclusion, and being all it can be for members. The Society is a highly valued and effective force for the chemistry enterprise. As a volunteer driven organization, chartered by Congress, we have many responsibilities and have achieved so much in 140 years of existence. Going forward, we must build on our strengths and achievements in publications, scientific information management, chemical education, and member career development. My vision is that the ACS of the 21st century will be the first choice professional home for all individuals employed in chemistry and the chemical related sciences.
Becoming an ACS Fellow (2014)


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Encanto Paseo del Sol
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- Suites
- Shuttle bus service
- Beach
- Free High-Speed Internet
- Swimming pool
- Fitness/Health center
Photos of Encanto Paseo del Sol
- Beach
- Swimming pool
- Bar/lounge
- Fitness/Health center
- Free High-Speed Internet
- Wheelchair accessibility
- Kitchenette
- Free parking
- Suites
- Shuttle bus service
TripAdvisor Reviews Encanto Paseo del Sol Playa del Carmen
Travel Blogs from Playa del Carmen
The final countdown, Playa Del Carmen
... was the European equivalent of the Spanish/ Italian riviera.
We set off down the street for our first meal and it was like we had been transported back in time, the restaurants all had enthusiastic waiters cajoling sunburnt foreigners into their taverns. We were enticed into a particularly lively establishment and proceeded to have the best Mexican food since Las Vegas! It is not without irony that the food was more akin to what you would receive in the UK than ...
Mexico 5: playa del Carmen 2
... but once disturbed it looks murky and blurry as the two liquids mix. It can be visually disorientating for the diver as he suddenly finds himself a bit blind. Furthermore salt water has a different density versus fresh water, so you have to adjust your buoyancy accordingly. It's normally a quick affair, but sometimes the halocline zones can be long so you end up swimming into this weird dreamlike mess for some time. You need to remain calm and not freak ...
... days somewhere and can sort that out when breakfast isn't included in our accomodation. We sleep on the way after none of us having solid sleep, Another long bus ride of loud Spanish movies. Wish english subtitles were played to keep me amused. Get to Coba, grab our tickets and head into the jungle. I read online that you can hire bikes through here as there is a lot of walking. The best 40 pesos spent so far. It was a good solid Km between each major ruin and it was hot and muggy. ...
That Was a First!
... cool water and some great people watching. The resort has a small zoo practically, tons of little capybara and some raccoon-like animals, along with the expected iguanas and some apparently invisible monkeys that were warned not to feed. Having such unique animals around is really a treat, kind of like a petting zoo! It's great waking up to all the bird songs in a beautiful "jungle" setting. ...
Day Quatro - When it rains it pours
... in front of me. Anyway, throughout the dinner I am feeling worse and worse, my nose is now like a faucet, I go through an entire tissue package, and my sinuses are hurting so much it is giving me a headache. After dinner, we stop by the shop where I get a nasal spray to help with my condition, and Sabrina insists I get a Vick's Vaporub inhaler - which I haven't seen since the 80s in Canada. Why don't we make these things anymore, it is ...


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Wearable Terminal Only - Wrist Mount and Scanners not included
Enhance worker efficiency and productivity with rugged, high-performance, hands-free mobile computing and scanning
The WT4000 Series Wearable Terminal from Motorola empowers warehouse and package handling workers to achieve new levels of efficiency, productivity and accuracy by bringing the most advanced computing and scanning technology to their fingertips in an ergonomic, wearable design. The hands-free system offers maximum flexibility, with headset support for voice picking, as well as mix and match connectors on either side of the device enabling the right- or left-handed scanning. The robust system delivers the kind of dependability you need for every-day all-day operation, offering the latest in mobile operating systems and processing power in a rugged design to endure the most demanding environments from the dusty warehouse floor to the cooler and freezer, or to a postal sorting area. Connectivity to virtually any wireless LAN provides a critical always-on connection to your business systems. Accuracy is increased in picking operations since workers can receive instant feedback if the wrong item is selected, reducing the costs associated with erroneous shipments.
Physical and Environmental Characteristics
Dimensions: 142 mm L x 93 mm W x 26 mm H
Weight: Terminal w/ Standard Battery: 320.3 g (238.1 g + 82.2 g)
Holder: 70.9 g (inc. straps and cushion)
Keyboard: 23 key alpha-numeric
Display Type: Backlit color TFT
Display Resolution: QVGA in landscape mode (320 x 240)
Power: Rechargeable Li-ion battery 2,330 mAh @ 3.7 VDC
Performance Characteristics
Processor: Intel XScale PXA270 @ 520 MHz
Operating System: Microsoft Windows CE 5.0
Memory: 64 MB/128 MB RAM
Application Dev: Microsoft IDEs and Motorola SDKs
Data Capture Options: Wearable scanner RS309 and RS409 are optional accessories
Voice Recognition: Can run voice recognition engines and text-to-speech engines for voice picking applications
Communications: Over charging connector - USB (Host and Client)
Audio Options: AC97 Compatible Codec to play audio over speaker or headset
User Environment
Operating Temp.: -20° to 50° C
Storage Temp.: -40° to 70° C
Humidity: 5% to 95% non-condensing
Drop Spec: Multiple drops to concrete: 1.2 m across the operating temperature range
Tumble Spec: 500 one-half meter tumbles at room temperature (1,000 drops)
Sealing: IP54 Category 2
Electrostatic Discharge (ESD): EN61000-4-2 15kV Air, 8kV contact, 8kV indirect
Wireless Data Communications
WLAN: IEEE 802.11a/b/g
Output Power: 100 mW U.S. and international
Data Rate: 802.11a: up to 54 Mbps; 802.11b: up to 11 Mbps; 802.11g: up to 54 Mbps
Antenna: Internal
Frequency Range: All country dependent; typically 2.4 to 2.5 GHz for 802.11b & g; 5.0 GHz for 802.11a
WPAN: Bluetooth 1.2
Electrical Safety: Certified to UL60950-1, CSA C22.2 No. 60950-1, EN60950/IEC 60950-1 plus all national deviations
EMC: FCC Part 15 Subpart B, ICES-003 Class B, EN 60601-1-2, EN 61000-3-2, EN 61000-3-3, CISPR 22 Class B, CISPR 24
RF: FCC Parts 15.247, 15.407, 15.205, 15.207, 15.209, 15.203, EN 300 328, EN301 893, RSS-100, RSS-210, ARIB STD-66 & 33, ARIB STD-T70 & 71
Audio & Video Cables
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Headphones & Mic
- View all categories...
- View all categories...
Remotes & Presenters
TV & Audio Devices


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2-Phenylethanol is used also as an aromatic essence and preservative in pharmaceutics and perfumery. It is also used as odor agents, air care products, cleaning and furnishing care products, laundry and dishwashing products, personal care products.
Etschmann, M.; Bluemke, W.; Sell, D.; Schrader, J. Biotechnological production of 2-phenylethanol. Applied Microbiology and Biotechnology. 2002, 59, (1), 1-8.
Kenji Hanabusa.; Yukinobu Maesaka.; Mutsumi Kimura.; Hirofusa Shirai. New gelators based on 2-amino-2-phenylethanol: Close gelator-chiral structure relationship. Tetrahedron Letters. 1999, 40, (12), 2385-2388.
Gefahrenhinweise (EU): H311-H302-H315-H319
Toxic in contact with skin. Harmful if swallowed. Causes skin irritation. Causes serious eye irritation.
Sicherheitshinweise: P280-P305+P351+P338-P361-P362-P301+P312-P312-P363-P321-P405-P501a
Wear protective gloves/protective clothing/eye protection/face protection. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Remove/Take off immediately all contaminated clothing. Take off contaminated clothing and wash before reuse. IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell. Wash contaminated clothing before reuse. Specific treatment (see label). Store locked up. Dispose of contents/container in accordance with local/regional/national/international regulations.


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October 31st, 2014
Episode 169 of 192 episodes
Thane Prince was the preserves judge on BBC Two's The Big Allotment Challenge, spent 11 years as the Telegraph's Weekend Cook and is the author of 12 books. Her latest book is Perfect Preserves and she joined Jenni to cook the perfect Raspberry Jam.
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Welcome to the daily audio workout for your head.


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Dope Springs
What's better than looking good? How about feeling good doing it. The DOPE Show (Designers Organized with PurposE) is a fashion-meets-fundraising event that showcases styles, both upcoming and upscale, while raising money to benefit #HashTagLunchbagPhx, an organization that works to end hunger. This 18-and-over event will roll out the red carpet for one night only and feature runway fashion, a fashion showroom, DJs, a hookah patio, cash bar, raffles, visual art, performance art, and live fashion art by Babe & Butter. There also will be contests for the best dressed and best walk, as well as a model search.
The Dope Show kicks off at 5 p.m. Sunday, April 27, at Monarch Theatre, 122 East Washington Street. General admission is $15 online in advance or $20 if purchased the day of at any Zia Records locations. For details and to get your tickets ahead of time, visit www.thedopeshow.us or call 619-344-1849.
Sun., April 27, 5-9 p.m., 2014
Related Location
122 E. Washington St.
Phoenix, AZ 85004
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Maxies Bar is located in Soi 16 off Walking Street Pattaya, right next door to the entrance to the X-Zone and Club Boesche gogo bars. For those
who don't know Soi 16 is at the Bali Hai Pier end of Walking street, in other words at the far end if entering from Beach Road. It was
previously under the same ownership as Wildcats Bar just around the corner in Soho Square (see
It is an open air beer bar but is larger than average with plenty of seating both at the bar and away from it.
There is a central area within the bar itself with three chrome poles for dancers.
It's nice to know that "Old School" Beer Bars still exist in Pattaya, At Maxies Bar you will be shown Friendly
Thai Hospitality in comfortable, cool surroundings.
Maxies Bar offers a full range of Beers and Spirits served up with lots of Sexy Young Ladies who are eager to
entertain and please.
Music is played at a level where people can still converse, and party at the same time. All LIVE sports are
A Quality American Pool table completes the Bar.
Beers Start from 70 baht and Spirits Start from 60 baht. Lady drinks are 100 baht all night.
There is a great Daily Happy Hour from 6.00pm till 8.00pm where beers are ONLY 50 baht!!
Open 6.00pm till Late Everyday
No food available in the bar itself but there are plenty of food stalls around and we can order in pizza and
other foods from Nics Pizza.
Listed On
Pattaya Bar Girls
Pattaya Resort


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CU Soon is a series of new videos released every other week counting down to Orientation that will help you learn more about all the exciting things waiting for you when you come to CUA this August.
Episode 1:
Episode 2:
Episode 3:
Episode 4:
Episode 5:
Episode 6:
Can't wait for the newest episode? Check out past written issues of the Newsletter on the right!


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Top Definition
When in the proccess of getting G-checked, a gangsta can pass the check so blatantly gangsta-like that the check ends up getting reversed onto the original checker
Steve: Yo this dude tired to g-check me by ganking my wad, so i faced pushed him, put half a clip in his dome, and made his old girl my bitch...
Darren: Damn bro, guess that nigga got g-ciggaD-checked.
Darren: Damn bro, guess that nigga got g-ciggaD-checked.
by Smokey and the Bandezzy April 04, 2008
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Note: Select a region before finding a country.
Winter Storm Watch
Gregory-Brule-Aurora-Charles Mix-Douglas- 337 ...
California's stormy winter continued early this week as another potent storm swept through Northern California with heavy rain and gusty winds.
With bathing suit season on the horizon, many are desperate to lose the excess weight they've put on during the dark, dreary days of winter.
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Your prayer needs are very important to us. The Prayer Line Ministry will keep you in their prayers.
Your contribution to our Church will be an enduring symbol of your faith and devotion, and will benefit the St. John Baptist Church now, at its inception, and for years to come.
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|Omaha, Neb.
|High School:
|Millard West
2009-10 Season Outlook
Kaitlin Arntz will be one of Nebraska's top threats in the breaststroke and IM this year after an excellent 2008-09 season. Arntz, along with fellow senior Hailey Dean, will anchor the breaststroke events for the team, and hopes to finish in the top 10 in both the 100- and 200-yard breaststroke at the conference meet for the fourth consecutive year. Arntz will also contribute in the 200-yard IM for NU, where she finished 11th in last year's Big 12 Championships. After a strong finish to the 2008-09 season, Arntz is primed to make a leap into the Big 12 elite in her senior campaign.
2008-09 (Junior)
Arntz had an outstanding junior season, as she was consistently one of Nebraska's top swimmers in the breaststroke and IM. Arntz had 19 top-five finishes during the season, including a fifth-place mark in the 200-yard breaststroke at the 2009 Big 12 Championships. Arntz had the team's fastest 100- and 200-yard breaststroke time, along with the fastest 200-yard IM time. Arntz also had an NCAA B-Qualifying time in the 200-yard breaststroke with a time of 2:15.12 at the Last Chance Meet in Columbia, Mo. Arntz capped off the year by being named team MVP by the coaching staff.
2007-08 (Sophomore)
Arntz followed an exceptional freshman season with a fantastic sophomore campaign, finishing the year with NU's top times in the 200-yard breaststroke and the 200-yard individual medley. Arntz earned 14 top-20 finishes, including a win in the 200 breaststroke at the Husker Invitational. Arntz had identical eighth- and 10th-place finishes in the 200- and 100-yard breaststrokes, respectively, at the 2008 Big 12 Championships after earning those same spots at the 2007 Big 12 meet as a freshman.
2006-07 (Freshman)
Following an excellent regular season that included 20 top-three finishes, Arntz ended her first year with the Huskers by qualifying for the finals of the 200-yard breaststroke at the Big 12 Championships. She swam the team's fastest time in 2006-07 (2:18.87) and became just the second Husker since 2001 to reach the top eight in the event at the conference meet. Prior to her February success, Arntz's regular season in the 200-yard breaststroke was highlighted by a seventh-place finish at the Minnesota Invitational, a win against Arkansas and runner-up finishes against four Big 12 teams, including nationally ranked Texas and Texas A&M. In addition to her eighth-place finish at the Big 12 Championships, she also finished 10th in the 100-yard breaststroke and 18th in the 200-yard individual medley, an event where she held the Huskers' No. 1 time (2:05.97).
Arntz earned high school All-America honors 16 times while competing for Millard West High School. She set seven school records and was a five-time Nebraska state champion. Arntz ended her high school career by capturing the Class A 100-yard breaststroke title for the second year in a row after winning the 100-yard butterfly gold medal as a sophomore in 2004. Arntz was part of two state champion relay teams, including the 2003 state record 200-yard medley relay (1:46.35) squad that included former Husker C.J. Bray.
Kaitlin is the daughter of Dan and Allison Arntz and was born on Aug. 23, 1988. She has two brothers, Bryan and Colin. Kaitlin is majoring in advertising.


text:
Top Definition
Stubble grown by dark haired men between the time in the morning they shaved, and the the time at night (usually around 5:00 PM) when it is noticeably growing back.
John looks like a mess, but it is mostly because of his five o'clock shadow.
by TangClock April 16, 2009
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> Terra Nova is still sitting on it with their mouths gagged, despite
> promises to get back to me with photos of it in bloom last October.
I sent an email to Terra Nova about the coneflower this weekend and they
answered me back.
They said that the blooms last year were 'not sufficient to evaluate the
plant'; I'm not sure what that means. At any rate, they are growing it
to the next bloom cycle and we'll see what happens there!
I've also asked them if they're interested in the Vinca sport that I
found in my backyard, attached to the straight species:
To sign-off this list, send email to firstname.lastname@example.com with the
message text UNSUBSCRIBE GARDENCHAT


text:
Whisk together in a small bowl salt, sugar, ground pepper, Dijon mustard, and balsamic vinegar. Then take the olive oil and slowly add it to the mixture whisking quickly. Once all the ingredients are mixed well you will have nice thick balsamic vinaigrette. Enjoy!
This video is a creation of naturalfusionvideos. You can visit naturalfusionvideos for complete recipes, and more videos.
Serving size: Complete recipe
Calories 1089 Calories from Fat 986
% Daily Value*
Total Fat 112 g171.6%
Saturated Fat 15.2 g75.8%
Trans Fat 0 g
Cholesterol 0 mg
Sodium 1956.8 mg81.5%
Total Carbohydrates 21 g7%
Dietary Fiber 2.4 g9.6%
Sugars 14 g
Protein 3 g6.4%
Vitamin A 0.3%
Vitamin C 1.8%
Calcium 4%
Iron 13.9%
*Based on a 2000 Calorie diet


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The 5-0 thumping will remain fresh in the hosts' minds, but both sides have no shortage of motivation going into Saturday's clash
|By GOAL.COM
Ferreira, Leijer, Gallagher, Traore
Finkler, Milligan, FloresRojas, Thompson, Nabbout
Franjic, Smith, Jurman, Stefanutto
Murdocca, Paartalu, BroichHalloran, Berisha, Henrique
Second-placed Adelaide United increased their advantage over third-placed Victory with a 4-2 win at Hindmarsh Stadium last Friday, while Brisbane Roar missed the chance to climb to fourth following a 1-0 loss at Western Sydney Wanderers on Sundayand instead slipped to ninth - equal on points with bottom-placed Sydney FC.
Victory, however, have been one of the form teams of the competition since their humiliating defeat at the hands of Brisbane in Week2, with five wins in eight matches establishing Ange Postecoglou's men as genuine title contenders in his first season at AAMI Park.
Roar, on the other hand, have lost five matches since dismantling Victory at Suncorp Stadium to leave the defending champions plummeting towards the bottom of the ladder.
But coach Rado Vidosic believes Brisbane have been unlucky since he took over from Postecoglou and is confident the side will improve.
Postecoglou's former No.2 will take confidence in Victory's problems in defence, where they have conceded an alarming 20 goals this campaign.
For all the goals the hosts have scored this season, their goal difference is still in the negative. Only last-placed Sydney FC has conceded more.
But Vidosic's men have been reduced to a shadow of the side that won the past two grand finals and seem to have lost the belief and desire that fuelled their success under Postecoglou.
Both receive key men back from international duty with Australia - Victory regain Mark Milligan and Archie Thompson, while captain Matt Smith and Ivan Franjic return for the Roar. Smith's return is particularly timely, with James Donachie - having served in his captain's absence in defence - out with a calf injury.
Adama Traore, in turn, further bolsters Victory's defence.
Thompson turned 34 this year, but looks more than comfortable alongside younger team-mates Marco Rojas [21] and Andrew Nabbout [19] and provides a wealth of experience as one of the A-League's most prolific forwards.
Thomas Broich, meanwhile, sets the tone for Brisbane, and the German's form is always a good barometer for how the Roar are tracking.
Just as his team have not been travelling particularly well so far in 2012-13, Broich is yet to truly hit his best.
Ange Postecoglou knows all too well he can be a match winner and will be praying it does not happen on Friday.


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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
My wife and I are currently separated and have been so for a year, we are still legally married, and there hasn't been court determining full custody so as far as I know I still have parental rights to my kids.
She left the state with them for one year, during which I paid child support, and returned this year in August of '09 which is when she gave Power of Attorney to my father and step-mom. I had no option to take responsibility or I would have, and I also had no knowledge of this until several months after. She did this with the condition that I may not leave with the children, but that I can visit them there. However there is no legal or court ordered reason, current or past, that I should not be able to spend alone time with my children. These are just 'her' terms.
What can I do?
go to court and request custody and/or visitation on terms that suit you and are best for your kids. However, think about why your ex trusts your father and stepmom but not you. Perhaps you should discuss this with your dad.
The response given is not intended to create, nor does it create an ongoing duty to respond to questions. The response does not form an attorney-client relationship, nor is it intended to be anything other than the educated opinion of the author. It should not be relied upon as legal advice. The response given is based upon the limited facts provided by the person asking the question. To the extent additional or different facts exist, the response might possibly change.
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text:
+44 12 93 51 80 80
South Terminal, Crawley Crawley Gatwick RH6 0LL United Kingdom
Location This airport hotel is directly linked to the southern terminal of London's Gatwick Airport; it takes only 5 minutes to reach the terminal building via a covered walkway. Links to the public transport network lie 700 m away, whilst shops may be found within 100 m of the hotel. The south coast of England may be reached by taking the M23 and M24 motorways or guests may alternatively make use of the train service. Facilities The hotel was built in 1981. Travellers can feel at home in one of the 821 rooms. Most storeys are accessible by lift. Amenities include a safe. Wireless internet access is available to guests in the public areas. The establishment offers a number of facilities for guests with disabilities. Various gastronomic options are available, including a restaurant, a café and a bar. Shopping facilities are also available. Additional facilities at the accommodation include a newspaper stand. Those arriving in their own vehicles can leave them in the car park of the hotel. Available services and facilities include room service and a laundry service. In addition, a shuttle service is offered. Rooms Each of the rooms is appointed with air conditioning, central heating and a bathroom. The carpeted accommodation units feature a double bed. There is also a safe and, for a fee, a minibar. A fridge, a mini fridge and a tea/coffee station ensure a comfortable stay. An ironing set is also available for travellers' convenience. Internet access, a telephone, a TV, a radio and a stereo system add to the comfort of the holiday. Rooms suitable for wheelchair access are bookable. The bathrooms are fitted with a shower and a bathtub. A hairdryer is also available. The establishment offers non-smoking rooms. Sports/Entertainment A refreshing dip in the swimming pool is a great way to cool off on hot days. The hot tub is the perfect place to relax. A range of options are available, including a gym, a sauna, a steam bath and a solarium. Additional services include an entertainment programme for children, offering numerous activities. Meals Bed and breakfast is bookable. Breakfast, lunch and dinner are available. Staff are also happy to provide special dietary cuisine. Payment The following credit cards are accepted at the accommodation: American Express, VISA, Diners Club and MasterCard.


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- Darkeyedgirl
- Reviews: 48
Twin Knobs Campground: The ONLY Campground you need...
Twin Knobs has been VERY well kept and updated. Recently - late 2011 - they demolished the bathhouses / restrooms and those will be upgraded as well.

The campground is set up in 'loops'; from A through J (I believe?)... some of the loops are reservation only; others are first come, first serve; some are both. You have to go to the reservation site to see which campsites are reservable (I HIGHLY recommend reserving your campsite!).

The sites are clearly marked; A for Ash, B for Birch, C for Cedar, etc.... they are all named after trees, which is really cool, cuz who doesn't love trees. =-D

Each Loop has it's own restroom / bathhouse facility. It is easy to reach (every numbered campsite within the Loop has it's own path worn to the bathhouse; so you can easily find your way and not worry about poison ivy or anything). The restrooms are VERY clean, with flushable toilets (this is important due to how many people share the things!), and private showers. Private meaning, there is a door to shut, it's not just a spout hanging next to the toilet or anything crazy like that.

The Loops also each have a potable water spout (within easy reach of each numbered campsite), for washing dishes and the like. And, there are very large, covered garbage cans for you to put your trash when you are done. Every night, the employees in charge of their Loop will come by on golf carts and clean up the area (bathhouses, trash duty, etc.).

Each Campsite itself is clearly numbered; and, if you reserve it, your last name is written very clearly and boldly on the stake in the ground. Each campsite has it's own driveway to park in (back in), and can fit two cars or one very large RV. Your car will be literally right next to your gravel area where you pitch your tent; so it's very easy to get things in and out of your car (or just leave it in there til ya need it).

The tents are set up onto a gravel flat area; it is VERY easy to drive stakes into the gravel to pull the tents taut. Also - the sites are VERY heavily wooded - which is EXCEPTIONAL due to keeping the sun off of you or keeping the RAIN off while you set up your tent! We got there on a Friday night, while it was raining (very odd for September, but hey, KY has seen some crazy weather lately), and had zero problems setting up the tent in the rain (it dries out, ya know).

Anyone who has been camping knows that it's not "private" (you can't run around naked or anything like that), I mean you DO have neighbors in the Loop; but it is NOT right on top of one another. You can't hear conversations, but you can feed their dogs when they wander over. =-D

Also, there will be the occasional "talker", who just kinda wanders over while you're making burgers on the grill... and horns in on your business. There are easy ways to chase people off (Jeff talks in German and I just smile and nod; they think we're foreign and go away), or you can bring up weird subjects to scare them off. =-D

The campsites each have their own firepit; set into the ground, very safe. BRING YOUR OWN FIREWOOD! The campground does sell firewood - but it is EXPENSIVE. We knew this ahead of time, so we brought our own. However - there is a Wal Mart nearby (I hate to admit that, but oh well), and you can buy firewood there, if you have a small car and can't fit the wood...

The only time we actually felt like we weren't totally alone (besides the "Talker" fellow invading our site), was very very very late at night while sitting around the fire... I had um, let out a rather large belch, and the group of manly fishing dudes at the site next to us responded in kind. (I won, of course, so it ended there).

One more thing: cost! It's CHEAP! Around $18.00 per day. If you want to have a peep-meet and have friends meet up with you - it's an extra few bucks for the car.
The campsites each also have two tall metal poles with hooks on them; you can hang your trashbags up here (most people do) just in case critters come around looking for food. Or, hang a lantern, radio, etc...

The critter issue... the ONLY critters we saw were dogs! We are dog folk ourselves so it wasn't an issue when the very friendly old dog of or neighbors came over for a bite to eat; however ---- there is a RULE in the campground - dogs need to be leashed at all times, and during rest times, either in a crate / tent or a tie-out stake. If we brought our dogs, for example - our little'un Barney wouldn't wander far (out of sheer terror), but Ripley would have to be constantly on the tie-out, because she's nuts. =-D

The main reason we adore Twin Knobs / Cave Run Lake so much is of course, the aforementioned SUNSETS. Hubby is a photographer, a great one at that, and besides us going oooh and aaah while enjoying a few cold ones on those flat rocks, he got some beauty pics of the sunsets.
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The USPTO's Patent Trial and Appeal Board has ruled in favor of the Broad Institute of MIT and Harvard retaining intellectual property rights covered by its patents for CRISPR gene-editing technology.
Medical Marijuana for Kids?
July 17, 2013|
The drug has brought relief to children suffering from cancer and other serious ailments, but getting access is often limited by considerable regulatory hurdles.


text:
You gotta love a gal who loves fries right? And getting naked - Check out nutty_nick's PV profile ...
Here is the second set. Same as before a litle fun on a weekend away. What do you think?
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Results 1 to 4 of 4
- 03-31-2011, 08:51 PM Thread Author #1
Has anyone had issues trying to set up your emails?
I'm trying to set up my pacbell.net and sbcglobal.net accounts. But every time I do this. I get
Warning "Cannot connect to the mail server to verify your account information. Your server is not responding.
Anyone else have this issue.
I've tried doing this via wifi and over the vzw network, with the same results.
- Posts
- 3,148 Posts
- Global Posts
- 3,645 Global Posts
- Mentioned
- 0 Post(s)
- Tagged
- 0 Thread(s)
- Carrier
- Latest OTA Official ROM
If you are unsure of this information either go to their webpage and research the mail settings or call them to find out.
- 04-01-2011, 04:14 PM Thread Author #3
Thanks Frank. My lazyass should have just gone to their site to find out the settings. Here are the settings.
Address POP server SMTP server
@sbcglobal.net pop.sbcglobal.yahoo.com smtp.sbcglobal.yahoo.com
@swbell.net pop.swbell.yahoo.com smtp.swbell.yahoo.com
@pacbell.net pop.pacbell.yahoo.com smtp.pacbell.yahoo.com
@nvbell.net pop.nvbell.yahoo.com smtp.nvbell.yahoo.com
@snet.net pop.snet.yahoo.com smtp.snet.yahoo.com
@ameritech.net pop.ameritech.yahoo.com smtp.ameritech.yahoo.com
@prodigy.net pop.prodigy.yahoo.com smtp.prodigy.yahoo.com
@wans.net pop.wans.yahoo.com smtp.wans.yahoo.com
@flash.net pop.flash.yahoo.com smtp.flash.yahoo.com
- 04-01-2011, 04:15 PM Thread Author #4


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University of Rochester Medical CenterSchool of Medicine and Dentistry601 Elmwood Ave, Box 626Rochester, NY 14642
Office: (585) 275-8748
Lab: (585) 275-1739
Administrative: (585) 275-3191
Fax: (585) 273-3637
Dr. Miyamoto's primary research interest includes molecular biology of steroid hormone receptors in genitourinary tumors. Specifically, he has been investigating the role of androgen receptor signals in the development and progression of bladder cancer. Epidemiological and clinical data indicate that men have a substantially higher risk of bladder cancer, and the underlying mechanisms of how androgens regulate bladder tumorigenesis and tumor outgrowth will offer explanations for this gender-specific difference in cancer incidence. He has also assessed the effects of new classes of androgen receptor antagonists on prostate cancer progression and has characterized novel androgen receptor co-regulators in prostate cancer cells.
Showing the 5 most recent journal articles.
163 available »


text: Buy Houseplants That Won't Kill Your Petstrappleton5/14/14 6:25amFiled to: petsplantssafety153EditPromoteShare to KinjaGo to permalink Keeping houseplants around the home and office can have all sorts of benefits, but pet owners need to make sure they're not also buying something that could make their cats and dogs extremely ill — or worse. Why are some plants toxic to our animal companions? Sometimes it's because their bodies can't handle substances that we can, like chocolate, but often the problem is that our pets are more likely to start chewing on houseplants than we are.Some of the worst offenders are also among the most commonly sold, including lilies, tulips, chrysanthemums, baby's breath, and aloe vera, to name a few. Not every plant is off the list, though. While mother-in-law's tongue should be avoided, its two companions, areca palm and money plants, are safe in any pet-owner's home. Those two will go a long way toward giving you more breathable air. Some pet stores even sell "cat grass," foliage expressly designed to be nibbled on by felines. While this isn't a dietary requirement in any sense, it's probably not doing any harm. There's no one reason why our carnivorous friends are occasionally wont to munch at the salad bar, but it's easier to change our habits than theirs. The ASPCA has an expansive database of plants that are toxic not only to cats and dogs, but horses, as well. Make sure to check it before your next trip to the garden center! Photo by Lindsey TurnerReply15 repliesLeave a reply


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Hello Loïc,
Thanks for the feedback on CLOWN PANTS. I'm glad it's done so well for you and that you've gotten to try so many interesting crosses with it.
I had tried it with tetraploids several years ago and didn't have much luck, but I'd always used it as the pod parent. A recent e-mail conversation with Harald Mathes, makes me think I should have tried it the other way too. Maybe you'll show that way to be more productive.
I think you'll appreciate the diversity of seedlings, that I'm sure you'll get from these crosses. Best of luck to you! I look forward to hearing about them!
loïc wrote:
Since you bring up the subject, here are the pods that took from Clown Pants's pollen here in Holland:
bangles X clown pants
baubles & beads X clown pants
bushwacked X clown pants
calico cat X clown pants
chelsea turner X clown pants
fair aldis X clown pants
marginal way X clown pants
merit X clown pants
MM06-01a(=bumble beeXregina) X clown pants
steffie X clown pants
As you can see i have also crosses with tetra (Bushwacked and Calico Cat (that is usually so hard to pollinate...!)). We'll see what happens to them with time.
Clown Pants has grown like mad, and had 2 stalks!
when you think it crossed the ocean last October, it shows how tought and good it is!
Thanks again for this wonderful gift!
I will keep you posted on the progress of these pods.


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Volunteers inspect damage after vandals hit YAM Building
Submitted by crecord2010 on
Volunteers at Yorktown Assistance Ministry were greeted with busted sign, broken windows, graffiti and a lot of clean-up when reporting for duty on Wednesday, Aug. 20 at the old City Hall building on North Eckhardt Street. Vandals had attacked the building not once, but twice, in one week.
Workers tiptoed gingerly over glass shards and sweated through the morning heat pouring in through the busted windows while helping clients on Wednesday, while YAM officials tried to decide what to do about the damages. According to Kathy Frels, city officials had been notified and city workers were due to repair the broken windows and doors.
"It's a shame," Frels said, pointing to the broken windows. "This place is here to help the needy in the community, and then this happens."
Frels noted vandals struck sometime during Sunday night and the damage was discovered on Monday morning by someone leaving a donation at the site. Workers spent Tuesday cleaning up the debris only to discover more vandalism had occurred again on Tuesday night, leaving another mess to clean up on Wednesday morning.
A brief inspection of the facility on Wednesday morning revealed a damaged YAM sign, broken windows all the way around the structure, damaged doors and graffiti. In addition, vandals left behind a broken big screen TV which had been left as a donation, its screen completely shattered. Despite efforts by YAM volunteers, shards of glass still lay everywhere, waiting to be cleaned up.
"They could have sold that TV. Someone could have used it," a volunteer said, pointing to the now-broken big screen TV standing next to the donation box outside.
Joseph Jendrzey, another YAM volunteer, was equally upset with the vandalism as he pointed out broken glass and damages. "It's just sad," Jendrzey said, shaking his head.
A stack of rocks, apparently used by the vandals, sat in a corner on the porch of the building. A large paver was also used to cause the destruction.
"Who knows the mind of an idiot?" longtime YAM volunteer Larry Odom said of the vandalism. Odom was busy working in the building's food pantry Wednesday morning while his wife, Sue, worked on client paperwork in the office. Sue Odom fanned herself with papers, pointing out the broken window behind her. She voiced her displeasure at the vandalism, stating she wished the public would realize the importance of YAM. "We just try to serve the public. We help a lot of senior citizens, women with children. We're run totally by volunteers."
Sue Odom said YAM is a Christian ministry supported through churches and individual donations.
"We try to keep our overhead low, basically the electric bill." She pointed to a used computer donated to the facility, stating "YAM doesn't have a phone. We all use our own cell phones," referring to YAM volunteers.
Frels pointed out the organization has a partnership with the Food Bank of the Golden Crescent, where food is purchased for 19 cents per pound for local residents.
YAM raises money through its resale shop, of which proceeds are used toward utilities and the purchase of food. Donations of usable clothing and items are accepted from the public in the drop box located outside the facility. The big screen TV was left next to the donation box and wound up being destroyed by the vandals, a loss for the center as well as those local residents who would have benefitted through the sale of the TV through food purchases from the food bank.
YAM has purchased a lot at the corner of North Church and Fifth Street and is currently raising money to build a new facility at the location. YAM supporter Bill Tapp is heading up this project. Donations to this endeavor are greatly appreciated. For more information, contact YAM director Sharon Metting at 361-564-3260.
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text:
Posted by Tommy on Thursday, February 7, 2008 at 11:42pm.
Use the Z score formula:
Z = (X - ƒÊ)/SD
After finding the appropriate Z scores in a table in the back of your statistics text called something like "areas under the normal distribution," insert that Z value into the formula to find the value of your score.
Some problems need you to determine the Z score before consulting this table to get a proprotion.
I hope this helps. Thanks for asking.


text: 6 inch Swirl Glitter Christmas Onion Ornament (set of 6): Red. Alternating glossy and glittered swirls wind around this onion-shaped ornament to create a decoration that glistens in the light cast by your Christmas tree. This ornament features a classic shape to types of sparkling glitter. Their 6-inch height makes them ideal for use on trees or to fill glass apothecary jars.


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|Property type
|Minimum Stay
|3 nights
This is our beautiful 2 bedroom 2 bath beachfront condo located near the heart of Jacksonville Beach, Florida. Brand new in 2006 The LARGE balcony sits atop the Atlantic Ocean and can be accessed from both the living room and master bedroom. It has a very well equipped kitchen with stainless appliances and granite counter-tops. The dining room table can seat 8 (2 office chairs and additional folding chairs in garage) The living room and 2nd bedroom has a large flat screen tv. Master has a smaller flat screen tv. The building has a full-time resident manager on site. There is a fitness center with showers, sauna steam room, and 2 pools (an ocean-side pool and a pool on the back upper deck), both pools have hot tubs attached. We have chairs, umbrellas, bikes, surfboard, boogie board, beach towels, beach bags, kids plastic beach toys and more for your use stored in our one car garage.. I have just installed beautiful new wood floors throughout and painted so repeat renters will see a big change. Both bedrooms now have king sized beds (the master is actually 2 twin adjustable beds with remote controls that make up to look like a king (see photos) Everyone has loved them so far!
This is our Primary home (we rent it out when we are traveling) and we have it furnished and supplied it in that manner. (even China and crystal :) for your use. Our new Sealy Posturpedic memory foam bed (with dual controls) has been a big hit with vacationers; but the double ocean front balcony is their favorite. The entire unit has just had beautiful "real wood" wide plank floors installed Jan. 2016. So there is no carpet in the unit.
Carol O'Donoghue purchased this condo in 2006
Wonderful large oceanfront balcony with access from family room and from the Master Bedroom. Garage full of bikes, ocean toys, beach chairs and umbrellas for your use. Strictly Enforced - NO PETS are ever Allowed, and this is a strictly Non- Smoking Unit and complex. (no smoking on the balcony either) We are not young child/baby proof and this is a luxury condo with a majority of full time residents. Only a few units are rented out . We ask that 'if you have children' ....please ensure that they are always with you and carefully watched.
2 pools, 2 hot-tubs (one oceanside), work out facility, sauna and steam room. The ocean views from just about anywhere in the condo are great; but just hanging out on our balcony is my favorite spot.
I would highly recommend this property.
Beautiful 2 bedroom condo right on the beach with two swimming pools, fitness center and a garage full of bikes and beach stuff. Beautifully, decorated with all the amenities anyone could want for a short or long term stay at the beach.
Lovely condo with impressive sunrise views on the beach. It's close to Neptune beach restaurants and has access to beach bikes beach toys and beach chairs that comes free with the condo so no need to rent anything. Management bent over backwards to be accommodating. Furnished great and also has comfy outdoor furniture on the large balcony overlooking ocean.
Carol and company are outstanding. Everyone should experience their hospitality and graciousness. Need a place at the beach? Look no further, you've found it.
We have travelled to Jacksonville every summer for the past 6 years and have stayed at a condo on the beach every time. We stayed in this same building last year so we were already familiar with the location and layout. We enjoy the view and the 2 pools especially the one on the 3rd floor. This could be a nice unit if there were simple things taken care of. It just seemed every time we turned around something else was not up to par. First off the owners live in this unit so I would think they would be aware of all the little things that need attention. First off don't even try showing in the master bathroom. It is impossible. The shower head is ridiculous. They have 6 bicycles in the garage for you to enjoy but only one of them is operable. My husband did find some tools in the garage and fixed 1 of them so at least 2 out of 5 of us could ride. There is no beach buggy which is a must when going down to the beach. So we had to go buy one $40. Especially since the private gate access to the beach was broken. Which no one seemed to have told anyone. After our 4th trip to the beach we called and got a call back that yeah it was broke. 3 days later there was a sign on the gate. So it never did get fixed while we were there for a whole week. Such a huge inconvenience. There is no storage in the kitchen. There needs to be at least a pantry we could use. There is no storage in either bathroom. The tv was not working properly had to call owner to find out how to fix it. It continually messed up day after day. The tabs in the bedrooms are not worth turning on. The left sink in the master bathroom drains slow. Could not use. Couldn't get the washer door open. The bathroom at the 3rd floor pool was not useable for at least three days it looked like someone had puked or something.
You just couldn't get any better than this. The condo was exactly as described. The balcony was huge, with very nice furniture, and we enjoyed all of our time out there, looking at the beach-goers and the ocean.
The condo building had two very nice pools and the location was near several great restaurants. All in all, it was perfect and we would certainly recommend it to our friends (and we will try to stay there again on our next trip to Jacksonville Beach).
Walk or bike to all restaurants, bars and shops!
|Rate Period
|Weekend Night
|Monthly *
My Standard Rate
3 night minimum stay
|Refundable damage deposit
|Tax Rate
Renters will received a full refund including all fees if Owner has to cancel their reservation for any reason (items that are out of the owner's control such as Painting of condo building, acts of god, etc.
* Approximate monthly rate. Actual rate will depend on the days of the month you stay.


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Water heaters have a long lifespan nevertheless they need to be replaced eventually. As much as water heater installation can be involved, it is not a complex process with an experienced handyman and is easily completed in a brief time period of time. However, it is very important ensure that all the connections are produced correctly and there are no errors.
Cruising you need to keep in mind when you are thinking of installing a fresh water heater on your own is which you will want the right tools to ensure that every one of the connections are made in the right manner. You won't get past an acceptable limit without the right tools.
The very first thing you need to do is to detach the present water heater. Before you start detaching, you should shut off all the connections including water collection, gas connection or electrical connection with regards to the type of heater you might have in your place. Keep in mind that hot water heaters are heavy and you have to be prepared to handle the body weight to prevent it from falling down and cause injury.
Now, you should connect the hose for the drain valve with the old heater. This drain valve can be found near the bottom of the water heater. The hose needs to be placed outside. Now you must to unscrew the Inlet and also outlet fittings. They are on the top of the heater along with a pipe wrench will be essential for this. You will also must unscrew the gas line fitting.
All things considered the connections have already been disconnected, you can safely take away the old heater. You should dispose of the old heater as reported by the regulations and rules in your location. You can now safely squeeze new water heater rather than the old one and level it.
You should ensure that various controls from the heater are accessible in the position you are going to use it. Once heater is within place, you can connect the outlet as well as inlet pipe. Again, you will need a pipe wrench to screw the inlet and outlet pipes. You should open the water line and permit the tank fill completely.
When the tank has been filled, it's time to connect the gas line with the aid of the pipe wrench or you have chosen an electric heater, it's switch on the mains. This is all there is to setting up water heater.
However, you should engage in this activity only when you are sure about handling this all. If you have any doubts or you are not good with all the tools, you should hire the services of an expert technician to do the water heater installation at a reasonable cost.


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The expression of the Flo11 flocculin in Saccharomyces cerevisiae offers the cell a wide range of phenotypes, depending on the strain and the environmental conditions. The most important are pseudohyphae development, invasive growth and flocculation. The mechanism of cellular adhesion mediated by Flo11p is not well understood. Therefore, the N-terminal domain of Flo11p was purified and studied. Although its amino acid sequence shows less similarity with the other flocculins, Flo11p belongs to the flocculin family. However, the N-terminal domain contains the 'Flo11-domain' (PF10181), but not the mannose-binding PA14 domain, which is present in the other flocculins (Flo1p, Flo5p, Flo9p and Flo10p). Structural and binding properties of the N-terminal domain of Flo11p were studied. It is shown that this domain is O-glycosylated and is structurally composed mainly of β-sheets, which is typical for the members of the flocculin family. Furthermore, fluorescence spectroscopy binding studies revealed that N-Flo11p does not bind mannose, which is in contrast to the other Flo proteins. However, surface plasmon resonance analysis showed that N-Flo11p self-interacts and explains the cell–cell interaction capacity of FLO11-expressing cells.
Saccharomyces cerevisiae shows different phenotypes depending on its genetic background and its environmental condition. The yeast has been commonly studied as a single-cell organism living freely in suspension. However, in its natural environment, the cells frequently switch over from a planktonic life style to complex multicellular structures such as flocs, filaments, mats and flors (Palkova, 2004). These morphological transitions have been suggested as a mechanism for this nonmotile species to permit foraging for nutrients (Gimeno et al., 1992; Iserentant, 1996; Cullen & Sprague, 2000; Dickinson, 2008).
For example, haploid cells become adherent and invade the surface of a semi-solid agar medium, so that they do not wash off. This is called invasive growth, and this morphological switch is caused by glucose depletion around the growing cells (Roberts & Fink, 1994; Cullen & Sprague, 2000). Diploid cells display a similar filamentous transition by adhering to each other after cell division, when sensing a low nitrogen concentration. In diploid pseudohyphal growth, the cells adopt an elongated shape and form long filaments that grow out from the colony edge (Gimeno et al., 1992; Kron et al., 1994; Mosch, 2000). Pseudohyphal development of S. cerevisiae has features in common with dimorphism of pathogenic fungi, where the dimorphic transition is often correlated with pathogenicity. Thus, the molecular models drawn from studies of pseudohyphal development of S. cerevisiae build a valuable basis for studying dimorphism in pathogenic fungi that are experimentally less accessible and therefore offer important insights into the mechanism and control of fungal disease (Mosch, 2000).
Yeast cells exhibit these morphological transitions when a class of genes, encoding flocculins, is present in their genome. More specifically, the adhesion of the cells to substrates, the pseudohyphal and the invasive growth are S. cerevisiae phenotypes resulting from the expression of the FLO11 (also known as MUC1) gene (Lambrechts et al., 1996; Lo & Dranginis, 1998; Rupp et al., 1999). Other phenotypes of S. cerevisiae associated with the FLO11 expression are the formation of mats, complex colony-like structures on low-density semi-solid medium (Reynolds & Fink, 2001; Reynolds et al., 2008); the air–liquid interfacial cellular aggregation in the process of sherry-like wine fermentations, called a flor or velum (Zara et al., 2005; Ishigami et al., 2006); and the adherence to a range of solid surfaces, such as glass, stainless steel, agar and plastics, possibly leading to the development of biofilms (Reynolds & Fink, 2001; Mortensen et al., 2007). Biofilm formation is a severe problem in the medical sector as they offer protection to the cells, for example, by conferring resistance to antifungal drugs (Bryers, 2007).
Furthermore, it was observed that the expression of the FLO11 gene also controls cell–cell adhesion, which leads to the aggregation of the yeast cells into clumps that sediment to the bottom of the fermentor (Lo & Dranginis, 1996; Bayly et al., 2005; Douglas et al., 2007). This phenomenon occurs at the end of the beer fermentation, when almost all fermentable sugars are converted into ethanol and carbon dioxide and is called flocculation.
Remarkably, the flocculation phenotype caused by Flo11p was observed only for Saccharomyces cerevisiae var. diastaticus (Lo & Dranginis, 1996; Bayly et al., 2005; Douglas et al., 2007), and also microscopic flocs consisting of 6–30 cells could be observed under the microscope for the laboratory strains BY4741 and BY4742 (S288C) when Flo11p was overexpressed (Purevdorj-Gage et al., 2007; Van Mulders et al., 2009). The other phenotypes, such as invasive growth, pseudohyphal growth, development of mats and biofilm formation, are mainly associated with S. cerevisiae strain Σ1278b, expressing Flo11p (Lo & Dranginis, 1998; Cullen & Sprague, 2000; Douglas et al., 2007). However, when overexpressed in S. cerevisiae strain Σ1278b, a Flo11p-dependent, but calcium-independent flocculation behaviour was observed (Guo et al., 2000), indicating that an identical adhesin expressed in different yeast strains can exhibit different FLO11-dependent properties. Clearly, other factors must be involved in these phenomena.
The FLO11 gene in S. cerevisiae encodes the Flo11 protein, which is a member of the flocculin family. However, Flo11p is more distantly related to the other flocculins in this family. Indeed, the predicted protein product of FLO11 is 37% similar to the product of FLO1 (26% identical) (Lo & Dranginis, 1996), while the other members of the flocculin family show a higher degree of similarity (Jin & Speers, 1998). The FLO11 gene contains 4101 base pairs and is located on chromosome IX. It encodes a 1367 amino acid cell wall protein assembled by three major domains: a carboxy (C)-terminal domain possessing a glycosylphosphatidylinositol (GPI) remnant to anchor the protein in the cell wall, a central domain containing a highly repeated threonine- and serine-rich sequence and finally an amino (N)-terminal domain located outside the cell wall (Lambrechts et al., 1996; Lo & Dranginis, 1996, 1998).
The wide range of phenotypes that are directly attributed to the FLO11-encoded glycoprotein are based on adhesion events, and therefore, the binding properties of Flo11p were investigated in this study. As Flo11p is an adhesin from the Flo family, where the adhesive properties are located in the N-terminus of the proteins, only the N-terminal domain was examined. In the Pfam database, a domain is associated with this part of Flo11p, which is called the 'Flo11-domain' (PF10181), covering residues 42–195. This domain is conserved in some related species within the Saccharomycotina (Linder & Gustafsson, 2008). Moreover, it is suggested that Flo11p shows a lectin activity that is related to amino acids present in the N-terminus, in particular the WQWGT motif (Bayly et al., 2005; Douglas et al., 2007). The N-terminal domain of Flo11p was expressed in S. cerevisiae, purified and studied. The secondary structure of the domain was evaluated as well as the presence of glycans on the protein. Our results show that N-Flo11p is not a mannose-binding protein but can make homotypic interactions.
Materials and methods
Construction of N-FLO11 expression plasmid
The determination of the N-terminal domain of FLO11 was based on the results of Lambrechts et al. (1996). It was proposed that the N-terminal domain stops where the tandem repeats start (at aa 210). Two oligonucleotides [Flo11_3, 5′-(TCCTTAGTCAAAAGGTTTCCAACTGCACTAGTTCCAAGA)-3′; and Flo11_4, 5′-(GGAGATCGGA ATTCGTCAGTGATGGTGATGGTGATGCTTCGTACCGCCACAATTATTGTCA)-3′] were designed to amplify the N-terminal domain of Flo11p coding sequence from the genomic DNA of S. cerevisiae strain S288C. Primer Flo11_4 contains a sequence for 6 histidines to add a histidine tag to the C-terminal part of the protein, which will allow purifying the protein using a Ni-NTA column. The predicted molecular weight of the expressed protein is 22 kDa. The yeast-Escherichia coli shuttle vector pYEX-S1 was used for cloning of the N-terminal part of the FLO11 gene. Cloning was performed using the In-Fusion™ method (Clontech, Mountain View, CA), which is based on the recombination between identical sequences of the vector and the amplified gene (Benoit et al., 2006; Zhu et al., 2007). The construction of the expression plasmid was confirmed by a combination of PCR and DNA sequence analysis (Genetic Service Facility, VIB, Belgium). In the pYEX-S1 vector, the cloned gene is regulated by the strong constitutive phosphoglycerate kinase (PGK) promoter, and this vector includes the β-lactamase gene for selection in E. coli and the yeast-selectable marker URA3. The Flo11p secretion sequence (aa 1–21) was not included during cloning, as the pYEX-S1 vector contains the full-length leader sequence from Kluyveromyces lactis to direct proteins through the secretory pathway for secretion into the growth medium. Transformation into E. coli strain DH5α was carried out using a heat shock as described previously (Sambrook & Russel, 2001). The S. cerevisiae strain S288C BY4741 was transformed using the lithium acetate procedure (Ito et al., 1983; Gietz & Schiestl, 2007).
Expression and purification of the N-terminal domain of Flo11p
Transformed S. cerevisiae cells were cultivated in shake flasks. The cells were grown in yeast extract-peptone-dextrose (YPD) medium containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) d-glucose. Overnight cultures were grown by inoculating 90 mL YPD medium with one pYEX-S1-containing S. cerevisiae colony. Cultures were shaken and incubated at 30 °C. Subsequently, the overnight cultures were diluted into 2 L of YPD medium and allowed to grow to an OD600nm of 10 by incubation at 30 °C for 64 h while shaking (110 r.p.m.). The cells were separated from the medium by centrifugation (12 000 g, 20 min, 4 °C), and the resulting supernatant was adjusted to pH 7.2 to protonate the histidines of the His-tag. The N-terminal domain of Flo11p was captured from the growth medium by affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) Sepharose column (6 mL) and was eluted with 1 M imidazole in phosphate-buffered saline. To further purify N-Flo11p, the elution fractions were pooled, concentrated to 2 mL (using an Amicon centrifugal filter unit with MWCO of 3000 Da; Millipore, Billerica, MA), and subjected to gel filtration chromatography (Superdex 75 HR 10/30; GE Healthcare) in 20 mM Tris, pH 7.5 and 150 mM NaCl. The eluate of the Ni-column and the purified proteins from gel filtration were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of pure N-Flo11p was estimated from the absorption at 280 nm.
Detection of (glyco) proteins
After purifying the N-terminal domain of Flo11p, SDS-PAGE was performed to visualize the protein and to estimate its molecular mass. The gel (15% resolving gel) was stained with Coomassie staining [1 g L−1 Coomassie Brilliant Blue R250, 50% (v/v) methanol, 10% (v/v) acetic acid] for 30 min and destained with 50% (v/v) methanol and 10% (v/v) acetic acid. To visualize glycans, the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain kit (Invitrogen) was used.
Enzymatic deglycosylation
To remove the N-glycans, the protein was treated with endo-β-N-acetylglucosaminidase H (Endo H; New England Biolabs) at a concentration of 100 U μg−1 protein, in 20 mM Na-acetate buffer (pH 5.0). The mixture was incubated at 37 °C for 4 h.
Circular dichroism
Circular dichroism (CD) measurements were performed with a J715 spectropolarimeter (Jasco) in the far-UV (200–240 nm) region, using a cell with a path length of 0.1 cm. The protein was diluted in 400 μL phosphate buffer (50 mM at pH 7.5) to obtain a final concentration of 0.3 mg mL−1. Data were acquired with a scanning speed of 50 nm min−1, a 2-s integration time and a 1-nm bandwidth at a constant temperature of 25 °C. Six spectra were measured and averaged. The mean residual ellipticity, [θ], was calculated as follows: [θ] = (100 × θm) (cL)−1, where θ is the observed ellipticity, m is the mean residual weight, c is the concentration (mg mL−1) and L is the path length (cm). A quantitative estimation of the secondary structure composition was facilitated by applying the K2D2 method and using the selcon3, continll and cdsstr programs (Provencher & Glockner, 1981; Andrade et al., 1993; Johnson, 1999; Sreerama et al., 1999; Perez-Iratxeta & Andrade-Navarro, 2008).
Fluorescence spectroscopy
The binding of d-(+)-mannose to N-Flo11p was studied by fluorescence spectroscopy. The measurements were performed at 20 °C, using a luminescence spectrometer (model LS55; PerkinElmer, MA). An excitation wavelength of 277 nm, a scan speed of 100 nm min−1 and an excitation and emission slit width of 5 nm were used during the experiments. The initial sample volume was 500 μL, containing 2 μM N-Flo11p in flocculation buffer (50 mM sodium acetate, pH 4.5, 100 mM NaCl and 7 mM CaCl2). One hundred millimolars of mannose from a concentrated stock solution containing 2 M sugar was added to the protein solution, then the sample was mixed and the fluorescence was measured immediately. As a positive control, 100 mM d-(+)-mannose was added to N-Flo1p, under exactly the same conditions. Emission spectra were collected from 300 to 450 nm and corrected relative to a buffer blank. Averages obtained from three independent experiments were displayed on the graph.
Surface plasmon resonance
The interaction of the N-terminal domain of Flo11p to another N-Flo11p was studied by surface plasmon resonance (SPR) using a Biacore 3000TM instrument (GE Healthcare, Upsalla, Sweden) at 25 °C. The covalent immobilization of N-Flo11p in flow cell 2 (FC 2) on the CM5 BIAsensor chip was accomplished via amine coupling chemistry, using an amino coupling kit (BIAcore; GE Healthcare) with 20 mM sodium acetate pH 4.0 as immobilization buffer. Glycosylated N-Flo11p was immobilized to a response around 95 resonance units (RU). Flow cell 1 (FC 1) was treated in the same way as FC 2 except that it was blocked immediately after activation and was used as the reference surface to subtract the RU as a result of the refractive index of the protein solution and any instrument noise.
Increasing concentrations ranging from 11 nM to 90 μM of glycosylated N-Flo11p were allowed to flow across both flow cells FC 1 and FC 2 of the CM5 chip containing N-Flo11p. The experiment was carried out in HBS buffer [20 mM Hepes pH 7.4, 150 mM NaCl, 10 mM CaCl2 and 0.005% (v/v) Tween 20]. No binding was detected in the control flow cells. Complete dissociation of N-Flo11p was performed with HBS buffer before starting a new binding cycle. The association time was 3 min, and the dissociation time was 7 min. Additionally, a zero concentration cycle of N-Flo11p (injection of running buffer) was run to determine the effect of the buffer. All measurements were performed at a flow rate of 20 μL min−1 in HBS buffer, and the proteins were dialysed to exactly the same buffer (Spectra/Por® Dialysis membrane, 3500 Da MWCO; Spectrum Laboratories, Inc., CA). The buffer was filtered through a 0.45-μm filter (MF-Millipore™ membrane filter) and degassed before usage.
The results were analysed with the BIAevaluation software version 4.1 (GE Healthcare). Binding was observed by measuring the increase in RU when injecting different concentrations of the analyte and after subtracting the background response.
Expression and purification of N-Flo11p
N-Flo11p was captured from the growth medium using a nickel column and was eluted with imidazole. The eluate was analysed by SDS-PAGE, and staining of the proteins revealed two bands with different molecular masses (Fig. 1a). N-terminal sequencing and mass spectrometric analysis of the protein bands revealed that the upper band corresponds to N-Flo11p. Four peptides belonging to N-Flo11p were identified after trypsin digestion (FPTALVPRG, KENIDLKY, KYLWSLKI and KIIGVTGPKG). To further purify N-Flo11p, the elution fractions were subjected to gel filtration chromatography. The purified protein was visualized with SDS-PAGE (Fig. 1b), and the concentration was estimated from the absorption at 280 nm. The yield obtained with this purification protocol was about 0.5 mg L−1 culture.
The N-terminal domain of Flo11p is O-glycosylated
The calculated molecular mass of the N-terminal domain of Flo11p without glycosylation is 22 kDa. On the SDS gel (Fig. 1b), it is observed that N-Flo11p corresponds to this molecular mass, which indicates that the protein is not N-glycosylated. This observation is in accordance with the prediction of N-glycosylation sites based on the amino acid sequence of the N-terminal domain of Flo11p (data not shown). To confirm the absence of N-glycans on N-Flo11p, the protein was treated with Endo H and visualized with SDS-PAGE (Fig. 1c). No decrease in molecular mass of N-Flo11p is observed. Hence, it is concluded that no N-glycans are present on the N-terminal domain of Flo11p.
O-glycosylation often occurs in a region of the protein that contains a high proportion of serine and threonine, with neighbouring proline residues (Lehle & Bause, 1984). In the amino acid sequence of the N-terminal domain of Flo11p, several threonine (18), serine (14) and proline (7) residues are found. To verify whether N-Flo11p contains O-glycans, the SDS gel containing N-Flo11p was stained with the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain kit (Fig. 1d). This staining procedure allows the visualization of glycans. As N-Flo11p can be visualized on the SDS gel, it is concluded that N-Flo11p is O-glycosylated.
N-Flo11p is mainly composed of β-sheets
A CD spectrum of N-Flo11p was recorded in the far-UV spectrum to investigate its secondary structure (Fig. 2). The observed spectrum is typical for folded proteins, and its shape, exhibiting one minimum around 215 nm, indicates a prevalence of β-sheets. The fitting of the CD spectrum, using the method of Perez-Iratxeta and Andrade-Navarro, indicates a β-sheet content of 48% and an α-helix content of 4% (Perez-Iratxeta & Andrade-Navarro, 2008). In addition, secondary structure content deconvolution of the spectrum, using the selcon3, continll and cdsstr programs, suggested that the β-sheet content was 31%, 40.5% and 42.8%, respectively. The α-helix content was also evaluated with the same programs. Values of 2%, 3% and 2.8% were found for the α-helix content using selcon3, continll and cdsstr, respectively. On average, the secondary structure of N-Flo11p contains about 40.6% β-sheets and 3.0% α-helices.
N-Flo11p does not bind mannose
A pentapeptide in the N-terminal ligand-binding domain of several yeast adhesins was identified to be involved in sugar recognition (Kobayashi et al., 1998; Zupancic et al., 2008). In the case of the epithelian adhesins proteins from Candida glabrata, this pentapeptide is the EYDGA motif (Zupancic et al., 2008). In the flocculin family from S. cerevisiae, the amino acids interacting with the carbohydrate are found in the VSWGT motif (Kobayashi et al., 1998). This pentapeptide is well conserved among the members of the flocculin family (Bayly et al., 2005; Van Mulders et al., 2009; Veelders et al., 2010). The tryptophan residue at position 228, which is present in all the studied adhesins with the exception of Lg-Flo1, was shown to be involved in mannose recognition through binding with the C-2 hydroxyl group of mannose (Kobayashi et al., 1998; Veelders et al., 2010). Aligning the binding region of the flocculins shows convincingly that Flo11p also contains the crucial tryptophan residue (Fig. 3). In binding studies using fluorescence spectroscopy, the quenching of this residue upon binding with mannose can be followed by measuring the intensity of the emission peak at 350 nm (Goossens et al., 2011). For N-Flo1p – a known mannose-binding protein – the fluorescence intensity decreases to approximately 50% of the initial value upon the addition of 100 mM mannose (dashed line in Fig. 4). In contrast, no change in the intensity of the emission peak at 350 nm was observed upon addition of 100 mM mannose to N-Flo11p (Fig. 4). This result indicates that mannose does not interact with the N-terminal domain of Flo11p, in contrast to the other flocculins (Flo1p, Flo5p, Flo9p and Flo10p).
N-Flo11p molecules self-interact
Recently, a homotypic adhesive mechanism has been detected for Flo11p (Douglas et al., 2007). The whole Flo11 protein was purified and covalently attached to microscopic beads. The Flo11p-coated beads were able to bind S. cerevisiae var. diastaticus expressing Flo11p, but not to cells that do not express Flo11p. Therefore, Flo11p itself was considered as an important adhesive target on the cell wall for Flo11p, if not the only target (Douglas et al., 2007).
In our study, we have assessed the homotypic interaction of the N-terminal domain of Flo11p. SPR was used to determine qualitatively the binding between N-Flo11 proteins. Therefore, N-Flo11p was coupled to a BIAsensor chip, and increasing concentrations of N-Flo11p (11 nM, 88 nM, 350 nM, 5.6 μM, 11.2 μM, 22.5 μM, 45 μM and 90 μM) were flown over that chip allowing the proteins to interact. The clear concentration-dependent increase in signal indicates that N-Flo11p is able to bind to N-Flo11p, which is called a homotypic interaction (Fig. 5).
The flocculin encoded by the FLO11 gene is responsible for a wide variety of phenotypes exhibited by S. cerevisiae cells expressing this Flo11p. These include haploid invasive growth (Roberts & Fink, 1994; Cullen & Sprague, 2000), development of pseudohyphae (Gimeno et al., 1992; Kron et al., 1994; Mosch, 2000), biofilm formation (Reynolds & Fink, 2001), mat formation (Reynolds & Fink, 2001; Reynolds et al., 2008), flor formation (Zara et al., 2005; Ishigami et al., 2006), adhesion to surfaces (Lo & Dranginis, 1998; Reynolds & Fink, 2001; Mortensen et al., 2007) and flocculation (Lo & Dranginis, 1996; Bayly et al., 2005). These cellular adhesion phenomena are mediated by Flo11p, but the mechanism is not well understood at the molecular level and very little is known about the structure and function of the protein. Therefore, in this study, this versatile flocculin was purified and studied.
The members of the flocculin family are composed of three domains, and the binding activity is located in the N-terminal part (Bony et al., 1997; Kobayashi et al., 1998; Goossens et al., 2011). Other adhesins from both pro- and eukaryotic origin also possess their binding domain at the distal tip of the protein. For example, FimH is the lectin domain at the tip of type 1 pili from E. coli and recognizes terminal mannose units of uroplakin Ia, a membrane glycoprotein that is abundantly expressed on superficial epithelial umbrella cells of the urinary tract (Zhou et al., 2001); the N-terminal part of Als1p from Candida albicans interacts with extracellular matrix proteins, epithelial and endothelial cells (Loza et al., 2004; Sheppard et al., 2004; Donohue et al., 2011). Also, it was suggested that Flo11p shows a lectin activity that is related to amino acids present in the N-terminus (Bayly et al., 2005; Douglas et al., 2007). For these reasons, only the N-terminal domain of Flo11p was cloned, expressed and purified from S. cerevisiae to study its structural and binding properties.
In this study, the purified N-Flo11p was analysed by SDS-PAGE, and it was found that this domain is decorated with O-glycans and does not contain N-glycans. Moreover, the secondary structure was evaluated by CD. By analysing the spectrum, we calculated that the secondary structure of N-Flo11p contains about 40.6% β-sheets and 3.0% α-helices. These values are comparable with the percentages found for N-Flo1p (37.7% β-sheets and 6.5% α-helices) (Goossens et al., 2011). The prevalence of β-sheets in the N-terminal domain is typical for flocculins. This part of most of the flocculins has been assigned to a new domain family (PF07691) in Pfam (Finn et al., 2010) (http://pfam.sanger.ac.uk), called the PA14 domain (Rigden et al., 2004). The PA14 domain is a conserved domain that has been discovered in a wide variety of bacterial and eukaryotic proteins, which include many glycosidases, glycosyltransferases, proteases, amidases, bacterial toxins, such as anthrax protective antigen (PA), and also yeast adhesins. Most of the experimentally characterized PA14-containing proteins are involved in carbohydrate binding and/or metabolism. Therefore, the N-terminal domains of the member proteins of the flocculin family were considered as one of the many PA14 domain variants (Rigden et al., 2004). The anthrax toxin PA also contains a PA14 domain, and the crystal structure of this toxin revealed that this domain consists of a series of antiparallel β-strands (Petosa et al., 1997). Although a PA14 domain was not predicted for Flo11p (Goossens & Willaert, 2010), the secondary fold of the N-terminal part of the protein is also dominated by β-sheets.
The fact that the Flo11 flocculin does not contain this PA14 domain indicates that no lectin activity was predicted. Indeed, by fluorescence spectroscopy, it was shown that N-Flo11p is not able to bind mannose (Fig. 4). Glycan array screening for N-Flo11p confirmed this result (Consortium for Functional Glycomics, www.functionalglycomics.org, request 2078 and 1224, supplying investigator: Lars-Oliver Essen, Department of Chemistry/Biochemistry, Philipps-Universität Marburg, Germany). The Flo11 protein was tested for its interaction with 465 different glycan structures (version 4.1) printed on an array (request 2078) and 377 glycan structures (version 3.1) (request 1224), but no conclusive data were obtained for glycan interaction analysis of N-Flo11p with and without the GST tag, N-Flo11p with a His-tag and after dimerization of N-Flo11p. No binding to mannose or to any other glycans from the array was detected. The detection was based on fluorescence as the adhesin was directly labelled with the fluorophore 'Alexa 488'. Other members of the flocculin family, however, are able to bind mannose, and their N-terminal domains correspond to the PA14 domain (Rigden et al., 2004). Indeed, a clear lectin activity was demonstrated for N-Flo1p and N-Flo5p (Veelders et al., 2010; Goossens et al., 2011).
In contrast, Douglas et al. (2007) indirectly detected mannose-binding activity for Flo11p. The whole Flo11 protein was purified from the growth medium after overexpression in S. cerevisiae var. diastaticus or in S. cerevisiae Σ1278b. It was observed that beads coated with Flo11p were able to bind S. cerevisiae var. diastaticus cells expressing Flo11p. This interaction was inhibited by the addition of a high (1 M) concentration of mannose, which suggests that Flo11p is a member of the Flo1-type flocculins and functions as a lectin in S. cerevisiae var. diastaticus (Douglas et al., 2007). Moreover, by studying the interaction between the coated beads and yeast cells, it was put forward that Flo11p shows a homotypic interaction (Douglas et al., 2007). Indeed, the beads could only interact with yeast cells that express Flo11p, suggesting that Flo11p itself is an important part of the adhesive target on the cell wall, if not the only target.
In this study, the homotypic binding of Flo11 proteins was confirmed by SPR as a clear and concentration-dependent interaction of N-Flo11p molecules was observed (Fig. 5). The mechanism of this interaction cannot be based on a lectin–carbohydrate interaction. The protein–protein interaction could be based on electrostatic and hydrophobic interactions, and influenced by the presence of the O-glycans (Fig. 1). Also, yeast cells expressing Flo11p have a highly hydrophobic character (Reynolds & Fink, 2001; Van Mulders et al., 2009), although the link between the hydrophobicity of the cell wall and the presence of expressed Flo11p on the cell wall is not yet clear. Recently, it was suggested that the whole Flo11 protein has a hydrophilic character (Karunanithi et al., 2010).
Recently, it has been demonstrated that many adhesins from budding yeasts contain amyloid-forming sequences that can play a role in cell–cell interactions (Otoo et al., 2008; Alsteens et al., 2010; Frank et al., 2010; Nobbs et al., 2010; Ramsook et al., 2010; Garcia et al., 2011). These adhesins contain sequences with high β-aggregation potential; VVSTTV and VTTAVTTTVV were indicated for the full Flo11p (Ramsook et al., 2010) (these sequences are, however, not present in the N-terminal part of Flo11p). Experimental results indicate that amyloid formation leads to the bundling of adhesins into nanodomains, which can greatly increase the intercellular binding strength by increasing the avidity (Douglas et al., 2007; Ramsook et al., 2010) and, consequently, can reinforce the Flo11p homotypic interactions between cells. It has been shown that an applied force can induce the formation of these nanodomains (Alsteens et al., 2010) and that they can be regulated by exogenous peptides (Garcia et al., 2011). Experimental results also suggest that amyloids are formed between adhesion molecules on contacting cells (Rauceo et al., 2004; Ramsook et al., 2010). Such intercellular amyloids would also strengthen the protein–protein intercellular adhesive bonds.
In conclusion, this study describes for the first time the purification and subsequent characterization of the N-terminal domain of Flo11p, a flocculin exhibiting a plethora of phenotypes. SPR analysis showed a homotypic interaction. However, in contrast to the other Flo proteins, N-Flo11p does not bind mannose. The exact mechanism of this protein–protein interaction and the involvement of the O-glycans have still to be revealed.
We thank Prof. J. E. Edwards (Division of Infectious Diseases, Medicine Deptartment, Harbor-UCLA, CA, USA) for providing us with the pYEX-S1 vector. We also acknowledge Msc. Catherine Stassen and Prof. Dr. Bart Devreese (L-Probe, Ghent University) for the mass spectrometry analysis. This work was supported by the European Space Agency (Prodex program), the Institute for the promotion of Innovation by Science and Technology in Flanders (IWT) and the Research Council of the VUB.


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Web Agency Brandography Sees Client Win Dot Award for Website Redesign
Minneapolis web marketing agency Brandography is happy to announce one of its clients' continued success.
Minneapolis, MN, April 30, 2014 - (PressReleasePoint) - Minneapolis web marketing agency Brandography is happy to announce one of its clients' continued success. Oak Meadows, a senior living community that won a free digital makeover from the online advertising and marketing company, recently received a Dot.Org Award for Website Redesign at the 2014 Nonprofit Technology and Communications Conference.
The Dot.Org Awards, run by the Minnesota Council of Nonprofits and MAP for Nonprofits, are given annually to nonprofit websites recognized for creative and inspiring use of technology and communications. Oak Meadows took the award for Website Redesign due to its user-friendly features and valuable resources.
As a winner, Oak Meadows was invited to attend the 2014 Nonprofit Technology and Communications Conference, where they had space in the Exhibit Hall and were formally given their Dot.Org Award. Jason Dailey, owner of Brandography, attended the awards ceremony with Kim Prayfrock, Director of Community Relations at Oak Meadows.
In Spring 2014, Oak Meadows was voted "the worst" in the "Doggone Worst Website" contest for bad website design co-hosted by Brandography and the Saint Paul Area Chamber of Commerce (SPACC) – winning Oak Meadows $10,000 worth of graphic design, logo creation, custom photography and video, SEO tips, and other website services. Later that year, Oak Meadows was selected as the Judges' Choice for "Best Website" in the MOMBA Awards (Mature Online Marketing Business Achievement), an international competition by RetirementHomes.com.
Brandography continually strives to help its clients succeed in their industries, within professional organizations, and with customers. Explore the Minneapolis marketing agency's capabilities and past projects online at Brandography.com, or schedule a free consultation by contacting the online marketing specialists at (612) 460-0016, firstname.lastname@example.com, or through the agency's online contact form.
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Brandography is a comprehensive Internet marketing and web company providing a full spectrum of services performed with the highest level of integrity. Offering advanced website development, graphic design, online email marketing, mobile app design, search engine optimization (SEO) and more, Brandography is renowned for imaginative brand creation, successful online marketing and advertising techniques, and the very best customer service and support, bar none. Contact them at 612-460-0016 or email@example.com.
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PC Classes Online On Shark Tank
PC Classes Online is a subscription based service in which you pay
a full year in advance to become a member. To have access too the entire video library of past subjects covered as we'll as all the upcoming live classes costs only $199/year.
Some of the classes taught online may seem a little basic to some people such as setting up a FaceBook page or learning Windows 7, but they also have more technological classes that cover subjects such as learning the importance of iCloud or building your own web site. By the looks of the partial list of subjects covered, they have a class for just about everything you can think of concerning technology. David Cox, the owner, confirms they will continue to add new classes as technology evolves ensuring you will always be learning the most up to date information available.
Are You A Great Teacher? PC Classes Online May Hire YouOn the website they are actively looking for new teachers that can teach their (usually older) student's new tricks Thur interactive video. I would imagine more then a few entertaining teachers will watch this episode and who knows, you very well may be working for one of the Sharks in the near future.
Getting A Clearer View Of The PC Classes Online
How Smart Is The Owner of PC Classes Online?"If your going to be teaching an old dog a new trick, you'd better be smarter then the old dog". We'll I don't think David Cox will have a problem convincing the Sharks that he is the Real Deal and can teach you anything you want to learn. David clearly has the credentials to run an online classroom. In fact I found a few learning books Cox wrote over 15 years ago that even the titles make you go, Hugh?.
You don't believe me? Hows this for a title of a book?
"Primes of the Form x + ny: Fermat, Class Field Theory, and Complex Multiplication (Pure and Applied Mathematics: A Wiley Series of Texts, Monographs and Tracts"
By David A. Cox (May 8, 1997) available on his Amazon page for $139.00
He has at least a half dozen more books in print just like this.
Is The Shark Tank Investors smart enough to invest in David Cox and PC Classes Online?
My money says that yes, at least one Shark will invest in PC Classes Online or Tech Talk America.
Visit PC Classes Online Website
Visit Tech Talk America


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Great Deal!!!
For a week or until we run out if that is before a week is up, Orbitnutrition.com is running a huge sale on Cyanostane Rx.
We are selling it for $31.99 for a week or when inventory runs out.
Get your bottle here: IRONMAGLABS :: Cyanostane Rx
Great Deal!!!
Is this compound good for bulking? Or is it more of a hardener?
Cyanostane is more of a hardener/cutter, but this product also contains 10mg of Dymethazine per cap, so its still a good mass builder.