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Located in the Exclusive town of Coronado at the Coronado Country Club Towers, right on the beach-front of the Pacific Ocean.
This fantastic, large condo is located on a lower floor in tower 300. The residence is light and spacious, with 245m2 of living space, offers 5 bedrooms and 4 bathrooms, a gourmet kitchen, living/dining room, laundry and, a maid quarters w/full bath behind the kitchen with the laundry room.
This beautiful condo has a large, private terrace overlooking the gorgeous swimming pool, garden areas, and the Pacific Ocean. This home has air conditioning throughout and many upgrades such as tinted windows, ceiling fans, marble designed wall, and more.
The condo could have 'two master suites' each on the ocean side or, as the current owners are set up, the 2nd master is converted to a TV/Family room. This could also be dedicated as an office. A true 5 bedroom condo is a rare and special find here on the Pacific.
Monthly maintenance fee of $370.00 provides water, gas, trash collection, immaculately kept grounds, the 2nd largest swimming pool in Latin America, 5 hot tubs, two tennis courts, 24-hour manned security property-wide, and direct access to the beach, right in front of your new home. The property is offered with air-conditioning, lights, ceiling fans, appliances. Furnishings are negotiable.
Move in today and begin experiencing the ultimate in beach-front living, where life and luxury meet at the crest of a wave. Located in the exclusive resort town of Coronado, just an hour's drive to Panama City, the Coronado Country Club Towers is a spectacular beach-front condominium residence with breathtaking views of the Pacific Ocean.
For more information on this property, please call 6205-5782


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Hi everyone! Each month I always make a card and tag set with my monthly kit from the Flying Unicorn…..I just love the 7 Dots Lost and Found Collection in this months kit.
So many fun tags and pieces to use on them!
The flowers were just a perfect match for the kit colors!
Well thats it for today! Watch the Flying Unicorn for presales for the November Kit..they start tomorrow! Take care and happy scrapping J

subset: commoncrawl


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Google Display Network Advertising Packages in Singapore
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Advertising on the GDN involves placing an ad for your business on another relevant website. Generally, this website needs to be not only industry-specific, but should also generate plenty of website traffic – if more people are visiting the site, more people will be exposed to your ad! For example, if you were selling sports gear online, you might place your ad on a sports blog, or a website that is dedicated to a relevant sport.
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For more information, feel free to contact our team today.


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First vice-president of the European Union Parliament Irish lawmaker Mairead McGuinness has asked EU Commissioners for agriculture and trade for clarity on allegations that Brazil has sold meat that is unfit for consumption.
McGuinness, a member of the parliament's agriculture committee, has written to the EU Commissioner for Agriculture Phil Hogan and the EU Commissioner for Trade Cecilia Malmstrom in light of recent reports about allegations that some of Brazil's biggest meat processors have sold meat, both domestically and internationally, that is unfit for consumption.
"I have requested clarity on whether the European Commission has received the full details on the extent of this scandal," the MEP and vice-president said.
These disturbing reports add further weight to my concerns that beef from Brazil, and Mercosur countries, should not be on the trade talks agenda at all.
"With negotiations on an EU-Mercosur trade deal continuing this week in Argentina, it is imperative that this issue is addressed. I put that to the commissioners and stressed that before these revelations, we had concerns about beef imports from Brazil. Given what we now know, I am firmly of the view that beef and meat generally may be off the agenda," McGuinness said in Brussels.
McGuinness has also sought information on what action the Commission will take in addressing food safety concerns in response to the reports.
"These disturbing reports add further weight to my concerns that beef from Brazil, and Mercosur countries, should not be on the trade talks agenda at all. The risks for EU producers and consumers are too great," McGuinness wrote in her letter to the commissioners.
Both Irish and Brazilian beef is on sale in our local Makro, has been for years. The Brazilian stuff is very cheap. Presumably samples will have been inspected on import. Personally I either buy Scottish or English beef. As it's Makro that steak you buy at the hotel or pub may be a Brazilian! I don't understand what this is about because Brazilin beef is already being imported in large quantities. You never see Argentinian beef or American beef. I believe that's because both countries allow steroids.Mar 23rd, 2017 - 09:23 pm +1
Given what we now know, I am firmly of the view that beef and meat generally may be off the agenda," McGuinness said in Brussels.
Well, all I have to say is that what McGuiness thinks, or says she knows, is not the full story. It's clear that her motivation to try to block Brazilian beef from entering the EU has always been based on economic reasons, not sanitary ones....but no doubt she'll explore it to the bitter end, even after the temporary ban to other countries, is lifted.
Ireland should be more worried about its recent stance with the UK...no deals unless through the EU...
When you consider the 240,000 tonnes of beef that is exported annually to the UK, Ireland's largest export market, which accounts for 53 per cent of total beef exports..
Perhaps the UK will instead strike a deal with Mercosur...
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text: How i can get the newest wine without broking my Ubuntu install? + Steam I have installed Steam I have installed on steam - Counter Strike source + Day of Defeat Source. They both need new font (CSS fonts are criples, but DODS fonts - black boxes). When i have lunched game and join to server - i get into it but i cant push the first button on game and it quits automaticaly after 3 seconds or so. THANKS!!!


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Massereene Upper
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Massereene Upper
Location of Massereene Upper, County Antrim, Northern Ireland.
|Sovereign state
|United Kingdom
|Northern Ireland
Massereene Upper is a barony in County Antrim, Northern Ireland.[2] To its east lies Belfast Lough, and it is bordered by four other baronies: Massereene Lower to the north; Belfast Upper to the east; Castlereagh Upper to the south-east; and Iveagh Lower, Lower Half to the south-west.[2]
List of settlements[edit]
Below is a list of settlements in Massereene Upper:[1]
Population centres[edit]
List of civil parishes[edit]
Below is a list of civil parishes in Massereene Upper:[3][4]
- Aghagallon
- Aghalee
- Ballinderry
- Blaris (split with barony of Iveagh Lower, Lower Half)
- Camlin
- Derriaghy (also partly in barony of Belfast Upper)
- Glenavy
- Lambeg (split with barony of Castlereagh Upper)
- Magheragall
- Magheramesk
- Tullyrusk
- ^ a b "Massereene Upper". Placenames Database of Ireland. Department of Community, Rural and Gaeltacht Affairs. Retrieved 5 June 2011.
- ^ a b PRONI Baronies of Northern Ireland
- ^ PRONI Civil Parishes of County Antrim
- ^ Baronies and parishes of County Antrim
|This article related to the geography of County Antrim, Northern Ireland is a stub. You can help Wikipedia by expanding it.


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Having been a parent for 20 years I wanted to share some of my more humorous parenting fails. We have all had them, things that just didn't go to plan or worked out how we imagined and are the stories you look back on and howl with laughter, although at the time they are often not so funny.
The first nappy
When I had my first baby I had zero experience with babies. I hadn't even held one. So, it's no surprise that my first time putting a nappy on my daughter I seriously fluffed it up. Although it wouldn't become quite apparent until a midwife did some new baby checks. My partner at the time had done the first nappy under midwife supervision, whilst I had a much-needed bath. As it was very early in the morning when I arrived on the ward, my partner was ushered home, and I was left alone with my baby.
No one told be about the black poop, so i was deeply unprepared when she did the biggest rip-roaring fart and noise that signalled nappy Armageddon! I remember wondering how do I know if the nappy is too tight, so gave baby room to breathe. Which apparently wasn't wise as when the midwife lifted baby up to check her a short while later the nappy slipped right off, the midwife was very amused at my first attempt, although at the time i was mortified.
Car seats and nappies
By the time baby number 2 came along 4 years later I had nappies down to perfection. Although I was still to be caught out when my second daughter was 13 months old. We had just visited my parents as they holidayed in their static caravan. Travelling down the dual carriageway about 15 minutes from where we left my elder daughter announced that her sister was 'covered in poop'. My daughter was in a rear Facing car seat so I couldn't see what had happened. I said she probably just had it on her hands, but my daughter was very sure it was 'everywhere'.
Pulling over expecting it to be nothing I was hit by the horror of just how covered she was. And my goodness was she covered. She had emptied the contents of her nappy out all over her face, arms, legs, hair… everywhere! And then had promptly fallen asleep! I couldn't help but howl with laughter at the sight. In fact, I couldn't breathe for laughing so hard. I learned the hard way, kids and dresses in car seats do not mix as they can access the legs cuff and thus the contents of the nappy! We still laugh about this today. Although my daughter doesn't see the funny side.
Never leave a toddler in the front room to go get some more sleep in the morning!
My first daughter was an early riser. I was not. Once she reached 3 I used to put her in front of the TV put on a movie and go back to bed. It was a tried and tested method. She would watch TV happily munching on a piece of fruit and I got a good hour or so extra rest. This particular morning, I had been up during the night, so when I went back to bed exhausted I fell back into a lengthy deep sleep. I was woken up by the ringing of my flats intercom doorbell.
Nothing unusual, until my friend announced 'you have been a while are you coming down'? To which I replied, 'no, I was in bed'. 'Ah right, you do know your daughter is down here'? Many expletives later I ran downstairs not able to believe that my 3-year-old had unlocked the door and gone for a little walk! Lucky my neighbour had seen her and brought her back. Major parenting fail and ever since I have been super careful with toddlers and locked doors!
The toddler and the reed diffuser
After 2 children, you would think I was clever enough to know kids put everything into their mouths. Apparently not! My first son and baby number 3 was a real fire cracker and because of this there was a gate on everything. He was pretty much confined to the front room which was baby proofed to within an inch of its life. Yet I still made the rooky mistake of leaving a reed diffuser on the side. I walked into the front room to be hit by a wall of scent.
I knew immediately what had happened. My son had got hold of the reeds and started sucking them. In a blind panic, I washed him the best I could and called the emergency number for the hospital. I took him to A & E to get him checked as we had no idea if the diffuser liquid was poisonous. Luckily, he was fine apart from a rash on his face and a few yucky nappies. I learned the hard way to keep everything out of my sons reach.
The first day of a new school year
After I had baby number 5 I think I lost a considerable amount of my sane brain. Either that or the sheer amount of planning and time it took to coordinate everyone was getting too much. It was the first day of the new school year and I took my kids into school as I always did. I had no clue where their classrooms were, and owing to being late I hastily dropped the first one off and then asked my 5-year-old which classroom was his. He was unsure and I recognised some of the mum's coming out of a classroom, so without checking,
I ushered him in and went off home. On my return that afternoon and to my horror I was greeted in the playground by my son's teacher who told me I had dropped my son off at the wrong classroom. He had sat in the class on the mat with the other children who he didn't know and worse still, were in the year above him. It was not until after register that they realised that my son didn't belong there, only then was he taken to the correct classroom. They were impressed though as he sat really well and didn't seem out of place with the older children! To say I was mortified was an understatement. Needless to say, I was the butt of the joke for a few weeks after when the teacher's would check daily 'if i had the right classroom'.
Saturday school
Like most mum's I get mixed up with what day of the week it is, days often merge into each other and this particular day was no exception. The alarm went off as it always did and we got up, dressed and ate breakfast as normal. There was the normal fight to get shoes on in time, but we were out of the door a surprising 5 minutes ahead of time. As we walked up the road something was wrong, and I couldn't quite put my finger on what it was, but something was 'off'. The shops were beginning to open and there was people walking about like normal, I must be imagining it. As we got closer to the school I noticed a lack of parent and kids, that's strange I thought, I must be earlier than I thought. It only became obvious as I neared the school that all the gates were closed.
Was it a day off and I didn't get the letter? Or perhaps there was a school flood or some other equally as random disaster and it was closed? I had no idea. So, we turned around with me scratching my head wondering what was going on and headed off home. We got back indoors and I rang my friend, enquiring what had happened as the school was closed. I was met with shrieks of laughter, unbidden howls down the phone. What? I thought, what was so funny? After what seemed to be 5 minutes of constant laughter my friend replied. "It's Saturday"! Yup, I had gotten up forgetting what day of the week it was and went to school on a Saturday. I don't think I will ever live that one down!
Just eat it!
I love to experiment when cooking, it's so much fun. On this particular day, I had found a recipe for goulash and I wanted to try it out in my slow cooker. What could possibly go wrong?
I prepared the meat and vegetables and placed them in the slow cooker with the relevant amount of liquid and set it on low. The goulash bubbled away for most of the day and I thought it smelt great. My husband wasn't so sure. At teatime, I made some mashed potatoes to accompany the goulash. When I looked though the goulash was still very watery. I had forgot to coat the meat in flour. So, trying to avoid a tragedy I added some flour to thicken up the sauce. It kind of went a bit stodgy but I was confident it would taste fine. When I dished it up EVERYONE turned their noses up at it.
To be fair it did look akin to dog food on the plate. I was cheesed off at everyone's lack of faith and began ranting about how I had spent all day cooking and preparing it, and how ungrateful it was to be so rude about the food I had lovingly made. By the time we sat down to eat everyone had decided it would be better to shut up than say anything else to me. Everyone's faces were a picture as they tried to politely but very slowly eat the Goulash which really wasn't very nice. Trying to prove a point I forged ahead eating my dinner, but even I was struggling.
The whole table, even me started to push their food around the plate, waiting on who would be the first to say they were done. No one would relent. Eventually I crossly announced that I knew no one liked it and to go ahead and put it in the bin. Looking back the speed they jumped up from the table, is funny. They wanted to get rid of it before I changed my mind I think! 8 years on and no one will let me forget about just how disgusting the Goulash I made was. I still remain adamant though, it wasn't THAT bad, when in reality it really was!
Our budding Di Vinci
Kids are like little whirlwinds of mess. They have the ability to trash a room in 2.4 seconds. The amount of time it takes a skilled mum to do a pee! One particular day I had been tidying around the house. I had made the decision earlier that day to take away our fire guard, leaving the fake coals still in the fire. Up until this point my son had paid little notice to the fire, so I thought I had no worries. I had gone to the kitchen to grab something and whilst I was there I had a quick drink. I was gone for perhaps 3 minutes at most, and all was quiet so I didn't feel too worried anything was afoot.
WRONG! I walked into my son coal in hand creating his own da Vinci masterpiece on our wall, along with several other masterpieces and a scrawl joining each one on the 3 other walls as well as a nice drawing on my front room rug! I could muster nothing more than 'oh my goodness, what have you done'. He was smiling from ear to ear, proud of his achievements. In cases like these you can't do much else than laugh. I had pretty much invited him to do it, by leaving such fantastic charcoals within easy reach. I still have a faint reminder of his work on our exposed brickwork as nothing would remove it completely, it always makes me smile.
What goes up
When you are a first-time parent you are always told to never leave your baby in the bed unattended as they can roll off. However, as a bed-sharing mum it's easier said than done. My daughter crawled at 5 months, she was amazingly mobile very early and it made leaving her unattended in bed a challenge. I had a great set up of a cot attached to the side of my bed for her to sleep in and as she became mobile i lowered it down so there was a lip of about 10 inches enough to stop her rolling on me during the night. Although she was crawling she hadn't yet figured how to climb up on anything. as a result I didn't worry about her crawling off my bed. At night when I came downstairs after she fell asleep I would pop the camera on to watch her sleep, occasionally the camera would freeze but we usually realised quickly and could restart the camera.
On this particular night, we were engrossed in a movie and hadn't noticed the screen freeze. There was a faint thud, and I think my husband and I went white, we could see out daughter still in bed, phew, but then we noticed that the screen had frozen. On restarting the camera, the picture revealed my daughter no longer in bed. We jumped from the sofa and as soon as we started up the stairs we could here crying. My heart was thumping, wondering if I was going to find my daughter injured. Luckily picking her up and soothing her we found she was not injured just a little shaken from the fall. I was berating myself for allowing it to happen and not being in bed with her. As she had now figured how to climb up ledges we decided to move the cot from against our bed and put on the rail the following morning.
Happy Birthday
On my daughters 3rd birthday we got up stupidly early as she always did and unwrapped all her presents, had a 'birthday breakfast' and generally had loads of fun indoors waiting until my friends came round to celebrate with us. It got to quite late in the day and no-one including family had come over. I was a bit cheesed off as no one had seemed to be acknowledging my beautiful babies birthday. It wasn't until i called one of my friends asking if she was still coming over that i discovered i had got the dates mixed up and it was in fact my daughters birthday the following day! So, yeah, once i forgot my own child's birthday!
Other people's Parenting Fails
Teaching my then 1-year-old to walk, she fell and I grabbed her arm to stop her unfortunately, I tried to stop her too hard and dislocated her elbow. Worst moment!
Trying to combine potty training and a beach trip! Epic parenting fail !
Driving along my daughter managed to get her arms out the car seat, i couldn't pull over due to the road being super busy. She then leant over and undid the car door…child locks were not on for some reason, i only knew as she started shouting door mummy! I kept seeing the door starting to open so was yelling at her to hold the door closed. She's just over 2, she had no clue. Epic parenting fail!! We were 2 mins away from nursery thank god!
When my son was 18 months, we went to a friend's house. There was several of us there and the kids were playing in the lounge and hall. He always was a monster so I always had an eye or ear on him where ever I went. A moments lapse in concentration as a friend asked me something and half way through a sentence I stopped cos I couldn't hear him. He was in the hall, which was in sight of everyone but when I looked he wasn't there! I turned around and her front door was wide open and I found him a few doors down the road. Not only that he was rattling an old iron fence, which then fell and hit his bare foot as I ran towards him. I had never had to think about locking doors as ours is so high up, he can only just reach it now and he's a tall 4-year-old. Worst moment ever!
This morning I was trying to teach my 17-month-old to come down the stairs on her bum, this resulted in a carpet burn on her back and a new found fear of the stairs.
Forgetting to put the potty-training seat on the toilet then laughing too hard when he fell in that I couldn't pull him out for about five minutes.
Having him all dressed and proudly ready for his 1st day at nursery – photos taken, nanny's phoned and wished him luck, daddy had the day off work and everything… we were a day early *slaps head* so we went to McDonalds for breakfast instead! That was 15 years ago and it's still one of my biggest parenting fails!!
Got a funny story to tell? It would be great to hear them, so leave a comment and give us all a good giggle.
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Say Rappahannock heidi-ho and here we go!
The Brunch is now four years in a row!
We'll crank up the music and set out the food
To make sure folks are in a good mood.
At Washington Fire, in their meeting hall,
Ten in the morning, doors open to all.
This is on Saturday, 29th of March
Come on over, no admission charge.
Park there for free, wear casual duds
We serve plenty drinks, alas tho' no suds.
What's special this year? Two candidates live
Guys running for Congress in our District Five.
There's sturdy Ben Hudson, ex-Army is he
Versus Larry Gaughan to be our nominee.
(Ben and Larry? So near to Ben & Jerry
Such a word game woulda been so merry!)
Solid Virginians, good men and true
Helping to turn Commonwealth blue.
Come join in the fun with Smiggy's fine tunes
We'll keep on goin til the clock says tis noon.
This Brunch of the Dems is hardly exclusive;
We offer to all to be quite inclusive.
From Left o'er to Right, politically ecumenical
The java and chow are pure commonsensical.
The victuals will tempt you, as will the joe,
So chat with your neighbors and say heidi-ho!
Jed Duvall
Vice-chair, Rappahannock Democratic Committee


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Seahawks Insider Blog
Ruskell on departure: 'I'm proud of what we've done'
An emotional and teary Tim Ruskell announced this morning that he had reached an agreement with Seahawks CECO Tod Leiweke to step down as the team's general manager with five games left in the season.
Leiweke cited the exacting standards of owner Paul Allen as one of the primary reasons for the team making the decision at this time, along with Ruskell pushing to seek clarity on what his role would be with the team in the future. Ruskell was in the final year of a five-year contract with the team, and the Seahawks had not offered to re-sign him to a new contract.
"Quite simply we didn't win enough games," Leiweke said. "And I think that when you're 4-12, there's obviously expectations that follow that in the next season, and we simply didn't win enough games."
Leiweke said the Seahawks vice president of player personnel, Ruston Webster, will serve as the interim general manager, and will be considered as a candidate. And the search will begin immediately for Ruskell's replacement.
"I'm proud of what we've done, and what we did," Ruskell said. "And I wish the new phase had gone quicker, but it didn't and that's the way the NFL works."
Leiweke credited Ruskell for helping to get the organization on the right track, creating a culture of accountability that allowed the Seahawks to make a Super Bowl run in his first season as the team's general manager, and also helping to facilitate the team building one of the best headquarters in the league here in Renton.
"Certainly there were challenges," Leiweke said. "But there were an amazing amount of things achieved."
Along with the Super Bowl appearance, the Seahawks made the playoffs three times during Ruskell's tenure, and finished with a 40-38 record. However, since the 2008 season the Seahawks are 8-19.
Leiweke would not address the potential of Mike Holmgren as a candidate, stating that they will go through an in-house evaluation process of the organization before interviewing potential candidates.


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Overview of Sciatica
Sciatica (pronounced sī-at-ĭ-kă) is a term often used to describe low back pain that spreads (radiates) through the hip, to the back of the thigh, and down the inside of the leg. There are many causes for low back pain; however, true sciatica is a symptom of inflammation or compression of the sciatic nerve.
The sciatic nerve is the largest nerve in the body. It originates in a bundle of nerves in the lower back (called the sacral plexus), and passes through the pelvis and down the back of each thigh. In the back of the thigh, the sciatic nerve branches into two smaller nerves called the tibial nerve and the peroneal nerve. The sciatic nerve carries impulses between nerve roots in the lower back and the muscles and nerves of the buttocks, thighs, and lower legs.
Nerve roots are extensions of the spinal nerves. They emerge from the spinal cord through spaces on both sides of the bony segments (vertebrae) in the backbone (spine). There are 31 pairs of nerve roots.
- eight pair originate in the neck (called the cervical spine)
- 12 pair originate in the upper back (called the thoracic spine)
- 5 pair originate in the lower back (called the lumbar spine)
- 5 pair originate in the lowest area of the back (called the sacral spine)
- 1 pair originate in the tailbone (coccyx)
Pressure or irritation in one of the nerve roots in the lumbar spine (which lead to the sciatic nerve) or compression of the sciatic nerve itself can cause sciatica.
Sciatica is a type of radiculopathy. Radiculopathies are disorders that affect the nerve roots or nerves. Compression of a nerve root often occurs as a result of damage to one of the discs between the vertebrae (called intervertebral discs). In some cases, sciatic pain radiates from other nerves in the body. This is called referred pain.
Pain associated with sciatica often is severe, sharp, and shooting. It may be accompanied by other symptoms, such as numbness, tingling, weakness, and sensitivity to touch. Sciatic pain usually occurs on one side of the body and may extend into the lower leg.
Incidence and Prevalence of Sciatica
Although low back pain is a common condition that affects as many as 80–90 percent of people during their lifetime, true sciatica occurs in about 5 percent of cases. Sciatica is more common between 30 and 50 years of age. Although the condition occurs in men and women about equally, some studies have shown that sciatica often is more severe in women.


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Chief Executive Leung Chun-ying has unveiled new measures to boost Hong Kong's tourism industry through diversified and value-added offerings, the Hong Kong Economic Journal reported.
In the last policy address for his term, Leung said the government will pursue a balanced, healthy and long-term development of the sector.
He cited the "one trip per week" individual visit scheme for Shenzhen residents, which has proved effective in minimizing the pressures of tourism on society.
The chief executive said an animation park will be opened in Wan Chai to promote the city's innovative culture to tourists.
It will showcase 30 animation characters from movies and cartoon strips such as My Neighbor Totoro, McDull and Old Master Q.
The park is expected to open in May with free entrance to the public, sources said.
Meanwhile, Lantau Island will be developed with 14 tourist facilities and attractions, along with the expansion of the Hong Kong Disneyland, Leung said.
The government also plans to develop Kai Tak into a tourism hub and scrap entrance fees to certain permanent exhibition halls of local museums.
Tourism is a major industry that accounted for 5 percent of the city's GDP in 2013 and provides nearly 270,000 jobs, he added.
[Chinese version中文版]
– Contact us at [email protected]


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Girls Get I.T. scouts, recruits and encourages talented girls to focus on science, technology and math in school, college and as a career.
Nearly 75% of future jobs in the United States will require the use of technology. Fewer than 33% of students in computer courses are female, and women comprise only 20% of IT professionals and 13% of engineers. We all pay the price when half our population can't compete on a global scale, so encouraging our girls to enter in these important fields is crucial.
The Florida Distance Learning Consortium and Cisco Systems, Inc. forged a partnership with Florida's college and university system to develop a network of statewide and community partners, including policy makers, industry leaders, middle and high school teachers and their students.
Girls Get IT scours the state, recruiting talented young girls who show an aptitude for math and science, encouraging them to pursue science, technology, engineering, and math (STEM) career paths in school, college, and as a career.
Girls Get IT kindles and sharpens the recruits' interest and aptitude in STEM through learning and mentoring. These girls participate in "girlified" activities using high-tech equipment and lesson plans that build from their existing non-academic interests. A typical GGIT recruit might participate in a "Surfing Science Camp"; star gaze through a hot-pink telescope; or develop a line of make-up, complete with a business plan.
Our facilitators are math and science teachers. These teachers guide and mentor our girls to help them avoid the pitfalls that often cause girls to give up on technology careers.
The results have been remarkable:
89% of GGIT girls reported that their parents and friends support their interest in science more than they did before.
After one week, 1.3 reported an increase in their belief that they could "make it into a good college"
After two weeks, 50% show an increase in their attitudes towards getting a job in science or technology.
Without access to Girls Get IT, statistics state:
80% would not go on to a career in science or math
Only 10% would major in a technology field
One Comment
I provide curriculum support to the CTE programs in our K-12 schools. Do you have resources for teachers to incorporate into their instruction? Thanks for any information you can share.


text: Hello guys, my tests: - http://source.winehq.org/patches/data/78080 - http://winetestbot.winehq.org/JobDetails.pl?Key=13790 - http://winetestbot.winehq.org/JobDetails.pl?Key=13791 - http://winetestbot.winehq.org/JobDetails.pl?Key=13792 - http://winetestbot.winehq.org/JobDetails.pl?Key=13793 show that native dsound has only one IDirectSoundBuffer implementation. That shouldn't matter as per the COM rules that is an implementation detail but there are broken apps out there (http://bugs.winehq.org/show_bug.cgi?id=28207) that rely on that fact. Not sure how many of the crashes due to dsound http://bugs.winehq.org/buglist.cgi?cmdtype=runnamed&namedcmd=dsound can be traced to that as PrimaryBufferImpl and IDirectSoundBufferImpl had (before my dsound changes) only the first three fields in common. Well PrimaryBufferImpl has only three fields as it stores its info in its parent object aka the device (this is a very common "design" in our dsound). Keeping the two objects/implementations in sync is brittle and will litter the code. Thus my goal is to merge PrimaryBufferImpl into IDirectSoundBufferImpl. My plan of battle is (to keep the patches small and limit the scope of the regressions I'll introduce during this): - Make the primary buffer use struct IDirectSoundBufferImpl. - Move fields that belong to the primary buffer out of struct DirectSoundDevice. The structs DirectSoundDevice and IDirectSoundBufferImpl share quite a few fields. - For methods that have the same implementation I'll use the IDirectSoundBufferImpl_<Method> - Methods that differ considerably between primary and secondary buffer I'll make IDirectSoundBufferImpl_<Method> just a wrapper that calls out into the respective implementation, something along the lines of: if(is_primary) return primarybuffer_<method>(...); else return secondarybuffer_<method>(...); - Once all methods are converted I'll switch the primary buffer to use the same vtbl. - Profit! Comments? bye michael


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- Research
- Open Access
Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach
- Jairam Vanamala1, 2Email author,
- Sridhar Radhakrishnan†1,
- Lavanya Reddivari†1,
- Vadiraja B Bhat3, 5 and
- Andrey Ptitsyn4
© Vanamala et al; licensee BioMed Central Ltd. 2011
- Received: 11 January 2011
- Accepted: 17 August 2011
- Published: 17 August 2011
We and others have previously reported that resveratrol (RSV) suppresses colon cancer cell proliferation and elevates apoptosis in vitro and/or in vivo, however molecular mechanisms are not fully elucidated. Particularly, little information is available on RSV's effects on metabolic pathways and the cell-extra cellular matrix (ECM) communication that are critical for cancer cell growth. To identify important targets of RSV, we analyzed whole protein fractions from HT-29 advanced human colon cancer cell line treated with solvent control, IGF-1 (10 nM) and RSV (150 μM) using LC/MS/MS-Mud PIT (Multidimensional Protein Identification Technology).
Pentose phosphate pathway (PPP), a vital metabolic pathway for cell cycle progression, was elevated and suppressed by IGF-1 and RSV, respectively in the HT-29 cell line. Enzymatic assays confirmed RSV suppression of glucose-6 phosphate dehydrogenase (rate limiting) and transketolase, key enzymes of the PPP. RSV (150 μM) suppressed, whereas IGF-1 (10 nM) elevated focal adhesion complex (FAC) proteins, talin and pFAK, critical for the cell-ECM communication. Western blotting analyses confirmed the suppression or elevation of these proteins in HT-29 cancer cells treated with RSV or IGF-1, respectively.
Proteomic analysis enabled us to establish PPP and the talin-pFAK as targets of RSV which suppress cancer cell proliferation and induce apoptosis in the colon cancer cell line HT-29. RSV (150 μM) suppressed these pathways in the presence and absence of IGF-1, suggesting its role as a chemo-preventive agent even in obese condition.
- Resveratrol
- Proteomics
- Talin
- Focal Adhesion Kinase (FAK)
- Pentose Phosphate Pathway
- Insulin-like Growth Factor-1 (IGF-1)
Cancer is a multifactorial disease whereas cancer cells can upregulate multiple defense mechanisms to evade drug treatments and therapies. It is therefore important to study the different mechanisms of compounds showing promise in chemopreventive efficacy to further enhance targeted therapy development. Proteomic analysis, a powerful method for discovery of new biomarkers and pathways, has recently been used in studies of obesity, diabetes, and especially cancer [1–3]. Proteomic profiling not only offers a method to study cancer but has also indeed broadened our understanding of multiple cancers. Nowadays, profiling and discovery of novel biomarkers are required for not only diagnosis but also for accurate understanding of mechanisms and causes of metabolic disorders [2].
Resveratrol (RSV, 3,5,4'-trihydroxy-trans-stilbene), a stilbenoid and a potent chemopreventive bioactive compound, is found in the skin of red grapes, and peanuts. RSV exerts anti-cancer properties by inhibiting three major stages of carcinogenesis, namely tumor initiation, promotion and progression [4]. RSV has been studied extensively as a chemopreventive/anti-proliferative agent in multiple cancer types including colon and prostate cancers [5, 6]. We reported previously that RSV suppressed colon cancer cell proliferation and induced apoptosis even in the presence of IGF-1, a well-known growth factor elevated during obesity which has shown to enrich colon cancer stem cell populations [6, 7]. RSV targets p53 and IGF-1R/Wnt signaling pathways to suppress colon cancer cell proliferation and induce apoptosis. RSV interactions with p53, Akt and other effector proteins that regulate proliferation and apoptosis are well documented [6, 8–13]. However, the effects of RSV on metabolic pathways like the pentose phosphate pathway (PPP) and the cell-extracellular matrix (ECM) protein interaction, important in cancer cell growth and proliferation are not clearly understood.
High concentrations of RSV used in this study and other studies on colon cancer cell lines are quite achievable in the colon (luminal- as epithelial cells are exposed to RSV directly) with novel pectin based formulations, as most of the trans-resveratrol reaches the colon unaltered. The pectin formulation protects RSV from upper GI tract enzymes and allows for targeted release in the colon (i.e., colon-specific drug delivery system). Pectin (biopolymer) used in such formulations is safe for oral intake and almost completely degraded by colonic bacteria [14–16]. Moreover, studies with human subjects revealed that resveratrol is well tolerated even at high oral doses (daily intake of up to 5 g) without adverse effects [17–19]. Juan et al [20] suggested that wines with 14.3 mg/L RSV could provide a luminal concentration of around 80 μM of trans resveratrol. Thus, novel formulations that protect RSV from first-pass metabolism or presystemic metabolism during GI transit and with RSV being safely tolerated in high doses, it is quite possible to achieve 100-150 μM RSV in the colon. Thus, use of dietary bioactive compounds at pharmacological doses, is emerging as a therapeutic approach to target colon cancer in humans.
Over-activation of the IGF system is frequently observed in obese conditions and plays a key role in obesity-promoted colon cancer [21]. Activation of the IGF system due to elevated circulating levels of IGF-1 stimulates colonocyte proliferation [21–24]. IGF-1 bound to IGF-1R activates downstream signaling pathways to promote proliferation and cell cycle progression [25, 26]. We have previously shown that RSV suppressed IGF-1 signaling via suppression of IGF-1R resulting in G1 cell cycle arrest and suppression of proliferation in HT-29 and SW480 colon cancer cells [6].
The metabolic pentose phosphate pathway (PPP) produces ribose-5-phosphate required for the synthesis of nucleotides. Cell proliferation requires metabolic sources for the duplication of DNA and cell size [27]. Therefore, PPP is frequently up-regulated in cancer cells; especially in HT-29 cells where, it has been shown that the PPP is very important for cell cycle progression [28]. This pathway is divided into two branches. The oxidative branch uses glucose-6-phosphate as a substrate to generate NADPH, NADPH acts as a reducing agent for the maintenance of reduced glutathione levels and fatty acid synthesis. On the other hand, the non-oxidative branch recycles pentose phosphates to glycolytic intermediates and generates de novo ribose-5-phosphate for nucleotide biosynthesis. Key regulatory enzymes of PPP are glucose-6-phosphate dehydrogenase (G6PDH) rate limiting enzyme in the oxidative branch and transketolase (TKT) in the non-oxidative branch [28, 29].
Cell-cell and cell-ECM interactions are important for viability and cell growth. Cell adhesion and migration contribute to normal processes such as differentiation, embryonic development, and wound healing. However, in cancer cells such processes are also significant for invasion and metastasis [30]. Key mechanistic steps in these processes involve the extracellular protein interaction with cell specific adhesive receptors such as integrins [31]. In cancer cells, these interactions serve as a link between extracellular and intracellular signals and regulate cell adhesion leading to invasion, proliferation, anoikis (anchorage independent apoptosis), survival and tumor progression [32]. Even though interruption of cell-ECM adhesion is a potential strategy for cancer prevention and treatment, and in spite of the cell-ECM components frequently modulated in chemoprevention studies, not much is known about the potential effects of the bioactive compounds like RSV on cell-ECM and integrin dynamics [33]. Talin is an integrin regulatory protein that translates the external message into regulatory intracellular signal transduction cascades. Cell-ECM interactions mediated via integrin/talin convey vital information to the cell interior to regulate cell proliferation and differentiation [34]. Focal adhesion kinase (FAK) is another member in the family of molecules that regulates cell adhesion dynamics, stimulates multiple cellular signal transduction events leading to cell motility, proliferation and survival [35, 36]. Talin has also been suggested to mediate FAK activation upon integrin stimulation [34]. FAK and IGF-1R have been shown to activate common pathways, leading to increased cell proliferation and survival. These studies suggest that talin-FAK signaling plays an important role in signaling initiated by integrins that translates into downstream signaling pathways [34].
In the present study, we used the power of functional proteomics to unravel critical components of the cancer chemoprevention ability of RSV in HT-29 advanced human colon cancer cells. The whole protein fraction of RSV (150 μM) or IGF-1 (10 nM) treated HT-29 cells was analyzed using LC/MS/MS. IGF-1 elevated and RSV suppressed G6PDH and TKT, the 2 key enzymes of oxidative and non-oxidative branches of the PPP, respectively, indicating that RSV suppressed cell cycle progression of HT-29 cells by down-regulating the PPP. Proteins in the focal adhesion complex were also found to be differentially regulated by RSV and IGF-1. RSV (150 μM) suppressed the talin and phosphorylated Fak protein levels even in the presence of IGF-1, a potent mitogen, indicating that RSV anti-cancer effects against human colon cancer cell might be partly due to disruption of cell-ECM interaction. On the basis of these results, we found the PPP and the talin-FAK signaling as critical targets of RSV.
Proteomic Analysis of HT-29 colon cancer cells treated with RSV and IGF-1
IGF-1 and RSV modulate the PPP to target cell cycle progression in human colon cancer cell line HT29
RSV suppresses while IGF-1 elevates the talin-Fak signaling pathway
The modern-day approach to cancer management is multidisciplinary, consisting of surgery, radiation therapy and chemotherapy with potential side effects. Several investigations are underway to improve the efficacy of these treatment modalities or to find new ways to treat or prevent cancer. Proteomics technology plays an important role in finding and validating biomarkers for cancer. Bioactive compounds like RSV have multiple mechanisms of action. It is vital to discover novel targets/biomarkers of chemopreventive agents like RSV that has multiple mechanisms of action so that those targets could be harnessed to develop targeted therapies. The goal of the study was to identify RSV target proteins and mechanisms implicated in its anti-neoplastic activities. The use of the proteomics technology enabled us to identify an array of proteins modulated by RSV. Although our present analysis accessed a small window in the cellular proteome, it was possible to detect RSV modulation of 1231 proteins in the HT-29 cell line. To determine if the expression of many of these proteomic markers was modified at both low and high concentrations of RSV, our confirmatory analysis included western blots and enzyme kinetic assays using RSV at concentrations 50-150 μM alone or after pre incubation with IGF-1, a mitogen elevated during obesity.
We have previously reported that at concentrations > 100 μM RSV suppressed colon cancer proliferation and up-regulated apoptosis even in the presence of IGF-1, elevated during obesity, and that has shown to enrich colon cancer stem cell populations [6, 7]. Apoptotic levels correlated with pp53 levels and suppression of proliferation transpired via attenuation of IGF-1R/Akt/Wnt signaling pathways. The present study focuses on novel targets of RSV that govern the proliferation, apoptosis and growth of a cancer cell. Using the pathway analysis software the important targets from the pool of proteins differentially altered by IGF-1 or RSV treatment were selected to devise pathways that have not been studied.
Genego Metacore software provided a basis to evaluate pathways based on differentially expressed proteins. As expected major canonical pathways affected were from the actin cytoskeleton signaling, oxidative stress response pathways, glucose metabolism, apoptosis and cell cycle etc. Among the significantly expressed proteins, G6PDH and TKT from the pentose phosphate pathway family and talin and FAK from the cytoskeleton family were found to be differentially expressed in RSV or IGF-1 treatments (Figure 1, 2 and 4). This along with the data showing that the glycolytic pathways [45, 46] and the cell-ECM interaction [1] to be frequently deregulated in cancer cells, fueled interest in RSV, the PPP and the talin-pFAK interaction.
Cell division is an energy-demanding process and its correct progression depends on sufficient metabolic resources to support a doubling of cell mass. Though nutrient availability is a key factor for cell proliferation, nucleotide synthesis is a rate limiting step in cancer cell replication [29]. Ribose-5-phosphate, which is a key nucleotide component, is synthesized from glycolytic intermediates in the PPP. PPP is considered important in tumor proliferation processes because of its role in supplying tumor cells with reduced NADP and carbons for intracellular anabolic processes [27]. In particular, the two key enzymes G6PDH and TKT have been shown to play a critical role in cancer cell cycle progression in the HT-29 cell line [28, 29].
In this study, we have demonstrated, using two different experimental approaches, that the PPP which is specifically elevated during cell cycle progression in the highly proliferating advanced human adenocarcinoma cell line HT-29 [28, 29] is further elevated by IGF-1 but suppressed by the bioactive compound RSV. Thus, a specific decrease in the activity of 2 key enzymes G6PDH and TKT, may lead to suppression of PPP that provides precursors of nucleotides for cancer cell cycle progression. Resveratrol at high concentration (150 μM) showed pronounced suppression of G6PDH and TKT in the presence of IGF-1. IGF-1 has been shown to elevate cancer cell proliferation via elevation of ROS (reactive oxygen species) [39]. At high concentrations where resveratrol has been shown to be pro-oxidant, the pronounced effect of resveratrol on pentose phosphate pathway in the presence of IGF-1 may be due to elevated ROS levels that promotes apoptosis [20, 44]. Detailed mechanistic reason for this potentiated effect is still need to be delineated, however, based on our earlier studies we propose that this might be due to elevated suppression of pAKT, Cyclin D1, nuclear β-catenin and SP1, proteins critical for cancer cell proliferation and cell cycle progression, in the presence of IGF-1 by resveratrol [6].
Suppression of PPP by RSV may be via inhibition of mTORC1. Recent evidence has shown clearly that the mTORC1 controls downstream metabolic pathways like the PPP via S6K1/SREBP-1/2 [47]. Our earlier experiments with mTOR signaling revealed that RSV activated tuberous sclerosis protein 2 (TSC-2, an mTORC1 inhibitor) and suppressed two best characterized downstream targets of mTORC1 (mammalian target of rapamycin complex 1), p70S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) [48].
Talin-FAK interaction has been well established and is implicated in various cancers [49, 50]. Talin plays an essential role in integrin activation and acts as a link between cell and ECM to regulate cancer cell kinetics [49]. Talin has been shown to engage in focal adhesion interactions with Akt signaling as the intracellular survival mechanism to confer anoikis (anchorage independent apoptosis) [51, 52] resistance and promote cancer cell invasion, especially in prostate cancer [49]. IGF-1 has been consistently linked to increased cell proliferation and cell migration, elevating cell invasion and metastatic properties of cancer cells [53]. Our earlier work with systems biology to identify biomarkers in metastatic progression of cancer featured talin as one of the differentially expressed genes in metastatic tumors in the context of cytoskeleton remodeling pathway [1]. Even the current proteomics data had talin as one of the differentially expressed proteins in IGF-1 and RSV treatments (Figure 4). RSV elevated talin levels at low concentration (50 μM) and suppressed talin, and concurrently elevated apoptosis at high concentration (150 μM). This may be due to the action of RSV as an antioxidant at low concentrations and pro-oxidant at high concentrations [38, 44, 54]. Anti-oxidant action at lower doses could protect DNA damage via scavenging of ROS, whereas at high concentration RSV acts as pro-oxidant leading to oxidative breakage of cellular DNA in the presence of transition metal ions such as copper causing apoptosis [44, 54]. This could possibly explain differences in talin activity at low vs high concentrations of RSV (RSV at 50 μM is not sufficient to induce apoptosis [20]). However, RSV was effective in suppressing IGF-1 stimulated talin expression, irrespective of concentration used. "FAK carries out protein-protein interaction adaptor functions at sites of cell attachment to the extracellular matrix (ECM), thereby contributing to focal-adhesion 'scaffolding'. FAK also transmits adhesion-dependent and growth-factor-dependent signals into the cell interior [50, 55]." The synergistic signaling between growth-factor receptors like IGF-1R and FAK might be particularly relevant as both are often up-regulated in tumor cells [36, 55]. FAK has also been shown to work similar to the IGF-1R to activate common pathways, leading to increased proliferation and cell survival. At least in pancreatic cancer, it has been shown that dual inhibition of FAK and IGF-1R led to a synergistic decrease in cell proliferation and increase in cell detachment and apoptosis compared with inhibition of either pathway alone [56, 57]. We have shown previously that RSV inhibits IGF-1R in HT-29 cells [6]. RSV suppressed FAK activation in the presence and absence of IGF-1. These results indicate that RSV suppression of cell proliferation and elevation of apoptosis involves modulation of FAK signaling, considering that the integrin-mediated FAK signaling regulates both proliferative and apoptotic signaling pathways [58, 59].
Proteomic profiling enabled us to identify novel targets of RSV. Our results establish PPP and the talin-pFAK as targets of RSV to suppress cancer cell proliferation and induce apoptosis in colon cancer cell line HT-29. These studies may prove germane to the envisaged use of RSV as a colon cancer chemopreventive agent as well as provide novel biomarkers to target and halt colon cancer cell kinetics.
Materials and methods
RSV and other cell culture materials were obtained from Sigma Chemical Co. (St. Louis, MO). IGF-1 was purchased from R&D Systems (Minneapolis, MN). Fetal bovine serum (FBS) was obtained from HyClone (Thermo Fisher Scientific, Inc., Pittsburgh, PA).
Cell line
Colon cancer cell line HT-29, was obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2 and grown in Dulbecco's Modified Eagle's Medium (DMEM) F-12 supplemented with 10% fetal bovine serum (FBS), 2.2 g/L sodium bicarbonate, 0.2 g/L bovine serum albumin and 10 mL/L streptomycin-penicillin mix.
Sample preparation
HT-29 cells were seeded at a density of 1.5 × 105 cells/mL in DMEM F-12 media with 5% charcoal-stripped FBS. Next day, cells were treated with DMSO (solvent control), IGF-1 (10 nM) or RSV (150 μM) for 24 h. We found from dose response studies with IGF-1 (5-20 nM) that 10 and 20 nM IGF-1 treatments did not differ (p < 0.05) in elevating cell proliferation (data not shown). Therefore, we used 10 nM concentration of IGF-1 for our experiments, which is close to normal circulating levels. Protein was extracted into a high-salt buffer containing 1% protease and phosphatase inhibitor cocktail, and protein concentrations were determined by a BCA Protein Assay kit (Pierce, Rockford, IL). The lysate samples (200 μg) were reduced, alkylated and double digested with trypsin to generate peptides. The digested peptides were completely dried in a SpeedVac and suspended in 100 μL of 5% acetonitrile acidified with 0.1% formic acid (mobile phase A). 200 μg of peptides (40 μl) were directly loaded onto a 1 × 150 mm Poly-SEA strong cation exchange column (Michrom Bioresources, Auburn, CA) using Agilent 1200 auto sampler. Peptides were eluted to 10 fractions using 0-100 mM ammonium formate for 40 min (mobile phase B: 1 M ammonium formate, 10% Formic acid in 5% acetonitrile) and 5 fractions in 100-1000 mM ammonium formate for 10 min on Agilent 1200 Capillary LC and Analytical-fraction collector at a flow rate of 50 μL/min. Peptides were dried and reconstituted in 10 μl of 0.1% TFA for LC-MS/MS analysis.
HPLC-Chip/MS analysis
A 3 μl volume of peptides (30% of SCX fraction) were injected into an LC/MS system consisting of an 1100 Series liquid chromatograph, HPLC-Chip Cube MS interface, and 1100 Series LC/MSD Trap XCT Ultra ion trap mass spectrometer (all Agilent Technologies, Santa Clara, CA). The system was equipped with an HPLC-Chip (Agilent Technologies) that incorporated a 160 nL enrichment column and a 150 mm × 75 μm analytical column packed with Zorbax 300SB-C18 5 μm particles. Peptides were loaded onto the enrichment column with 97% solvent A (water with 0.1% formic acid). They were then eluted with a gradient from 3% to 45% solvent B (acetonitrile with 0.1% formic acid) in 25 min, followed by a steep gradient to 90% solvent B in 5 min at a flow rate of 0.3 μl/min. The total runtime, including column reconditioning, was 35 min. The column effluent was directly coupled to an LC/MSD Trap XCT Ultra ion trap mass spectrometer from Agilent Technologies via a HPLC-Chip Cube nanospray source operated at ~1900 volts in ultra-ultra mode. The gain control (ICC) was set to 500,000 with a maximum accumulation time of 150 milliseconds. Collision induced dissociation (CID) was triggered on the six most abundant, not singly charged peptide ions in the m/z range of 450- 1500. Precursors were set in an exclusion list for 1 min after two MS/MS spectra. Results were expressed as mean for three replicate experiments for each treatment group.
Data analysis
CID data was searched against the NCBInr human database, using the Agilent Spectrum Mill Server software (Rev A.03.03.) installed on a HP Intel® Xeon (TM) dual processor server. Peak lists were created with the Spectrum Mill Data Extractor program with the following attributes: scans with the same precursor ± 1.4 m/z were merged within a time frame of ± 15 s. Precursor ions needed to have a minimum signal to noise value of 25. Charges up to a maximum of 7 were assigned to the precursor ion, and the 12C peak was determined by the Data Extractor. The NCBInr database was searched for tryptic peptides with a mass tolerance of ± 2.5 Da for the precursor ions and a tolerance of ± 0.7 Da for the fragment ions. Two missed cleavages were allowed. A Spectrum Mill auto validation was performed first in the protein details followed by peptide mode using default values [Minimum scores, minimum scored peak intensity (SPI), forward minus reversed score threshold, and rank 1 minus rank 2 score threshold]. All protein hits found in a distinct database search by Spectrum Mill are non-redundant.
Data scaling and normalization
Quantitative estimation of differences between physiological states by analysis of proteomics data has a number of challenges. Only a fraction of proteins actually present in each sample is identified and a still smaller fraction is quantified. Technical variation overlapped with these fractions results in substantial differences in the range of variation for identified fraction even though the overall range of variation between samples is similar (see Additional file 1, Figure S1). The resulting distribution of quantified protein abundance units has long tails of low-abundance proteins and proteins identified in only one of the samples. It is commonly advised that for MS proteomics (unlike microarrays) more technical replicates should be done in order to control variation and achieve more reliable quantitative estimation of change between samples. However, this expensive and labor-intensive "brute force" approach is not always feasible. We have taken an alternative data analysis approach which allows quantitative estimation of changes between samples with limited number of estimates. Our method is based on a reasonable assumption of little or no change in abundance for the majority of proteins in all samples. None of the experimental conditions we create in this project can be associated with lethality or high stress. There is also no indication of experiment-induced stress response in the pattern of expressed proteins. Assuming that relatively small fraction of genes have large fold changes we can apply quantile normalization algorithm, similar to that described by Bolstad et al. [60] for microarray normalization: x norm = F -1 (G(x)), where F is the distribution function of the actual sample, and G is the reference distribution function. We estimate G by the empirical distribution of each sample and F by using the empirical distribution of the averaged quantitative estimations of peptide abundance in samples across all experimental conditions, not just replicate groups. This approach has proven to be effective in low-replicate microarray studies [1, 61]. In our implementation G is more smoothly estimated by application of additional Savitski-Golay polynomial smoothing [62]. In addition after scaling, we zero down scaled values for proteins not identified in particular sample. Quantile normalization scales all protein abundances in all samples to the same distribution and allows realistic estimation of fold change between experimental conditions.
Biological pathway analysis
All lists of identified proteins and respective normalized expression values were imported to GeneGo Metacore (GeneGo INC, St. Josef, MI). Analysis of overrepresentation of canonical pathways, co-regulation/protein interaction pathways and comparative network analyses are performed using intrinsic tools and pre-formed analysis pipelines of Genego Metacore.
Western blot analysis
HT-29 cells were seeded at a density of 1.5 × 105 cells/mL in Dulbecco's Modified Eagle's Medium F-12 with 5% charcoal-stripped fetal bovine serum for 24 h. Cells were treated with solvent control (DMSO), IGF-1 (10 nM), different concentrations of RSV (50, 100 and 150 μM) with and without IGF-1 for 24 h. Protein was extracted into a high-salt buffer containing 1% protease inhibitor cocktail from Sigma-Aldrich (St. Louis, MO), and protein concentrations were determined by a BCA protein assay kit from Pierce (Rockford, IL). Cell lysates (45 μg) were incubated at 98°C for 5 min and separated on 4-12% Criterion XT bis-tris gel at 120 V for 2 h in 1X XT MOPS Running Buffer (Bio-Rad Laboratories, Hercules, CA) and electrophoretically transferred to Immuno-Blot PVDF membranes (Bio-Rad) at 95 V for 35 min in tris-glycine transfer buffer with 0.025% SDS. PVDF membranes were blocked with Superblock buffer (Thermo Fisher Scientific Inc.) for 1 h at room temperature. The membranes were incubated with rabbit polyclonal anti-pFAK antibody (1:500; Cell Signaling, Danvers, MA) or mouse monoclonal anti-talin antibody (1:750; Millipore, Billerica, MA) or goat polyclonal anti-β-actin antibody (1:2500; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were also probed with respective IgG-HRP secondary antibodies from Santa Cruz Biotechnology (1:20,000; Santa Cruz, CA) and scanned using UVP imaging software (UVP BioDoc-It® Imaging System; Upland, CA). β-actin served as a loading control.
Enzymatic analysis
G6PDH activity was measured using Glucose-6-Phosphate Dehydrogenase assay kit (Biovision, Mountain View, CA). G6PDH activity was measured at absorbance 450 nm using the manufacturer's protocol. TKT activity was determined using the method of de la Haba et al [63]. Briefly, protein extracts were added to a 96-well plate containing 216 mM glycylglycine, 3.3 mM xylulose-5-phosphate, 1.7 mM ribose 5-phosphate, 0.002% (w/v) cocarboxylase, 0.14 mM ß-nicotinamide adenine dinucleotide (reduced form), 15 mM magnesium chloride, 20 units α-glycerophosphate dehydrogenase/triosephosphate isomerase (based on triosephosphate isomerase units). Decrease in absorbance was recorded over 15 minutes at 340 nm and activity was determined based on the method of de la Haba et al [63]. Protein concentration of cell extracts was determined using the BCA protein assay (Pierce) to calculate the specific activity of the enzymes.
Conflict of interests
The authors declare that they have no competing interests.
Grant Support
This work was supported by College of Applied Human Sciences Challenge Grant (2009-2010) and National Research Initiative Grant 2009-55200-05197 from the USDA National Institute for Food and Agriculture.
We would like to thank Scott and White Healthcare Proteomics Core Facility, Temple, TX for their help in proteomic analysis of our samples.
Authors' Affiliations
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Nowadays,the number of smartphone manufacturers producing smartwatches is increasing.As a captive product of smartphone,smartwatch has received more and more attention which becomes an indispensable part of smart life.We Bluboo, having released a smartwatch Uwatch previously,now launch a new one Xwatch,the strap of which is made of silicone.It's well-known that Apple Watch uses silicone as its strap as well.
Our Xwatch is a brand-new one equipped with the advanced MTK2601 chip with 1.3 inch IPS oncell screen and IP 65 waterproof rating.Why we choose silicone strap for Xwatch? Of course, strap of silicone does have lots of advantages.First of all,users will feel very comfortable when wearing it because of the special texture.Unlike metal strap ,silicone makes you not feel cold at all even wearing Xwatch in chilly days.Moreover, another feature of silicone is its durability which can stand the temperature ranging from minus 40 degrees to 250 degrees,not easily getting discolored or deformed due to sweatiness in tropical days.Besides,silicone also possess the characteristics of being flexible,indehiscent, Shrink-and-wrinkle-resistant and resilient. If wearing properly,you could keep it intact for a rather long time.Last but not least,silicone is environmentally friendly which is oderless and non-toxic and easy-to-clean.Customers could wear it at ease.
Apart from silicone,we Bluboo also provide straps made of leather and metal for users.Different straps match for different Xwatch series. Xwatch's strap is replaceable,customers could substitute other straps for the original ones at will.
Another highlight of Xwatch is that users could customize the function of the three buttons according to their needs,making the watch suits you best instead of one getting used to the fixed settings. Xwatch will be available for sales in May with affordable price nearly 100 dollars.
Source: BLUBOO


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Come out and see Sea Lions Splash!
The Amazing California and South African Sea Lions will make a big splash for you. With there amazing and educational show for everyone. You will leave with a big smile on your face with their talent!
BMX Biking
Come out for an open practice or you can register to race the ultimate track with steep hills, huge jumps and lots of speed!! See below for the complete schedule.
Live Petting Zoo
Come out and let the kids be animals with animals at the Davis County Fair Petting Zoo!


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This was the scene on our back deck the other day. Em and Soph are aspiring artists - their handiwork covers their bedroom walls, and their little sisters love to be wherever they are. It is a pain to carry all the equipment down the steps to the deck from the house, but to see all of them together, busy in the sunshine, is great.
All this happened in the shade of our favourite tree - Tree of Heaven - which has grown each year to cover more and more of the deck. Aptly named, it reminds of us of the shade of our Father's hand.
Today was a great day. One of those days where all good things happen. Christina came to watch Rachel and Janneke, so Ralph and I could take Em and Soph for a hike in the Short Hills Provincial Park. Sometimes, when we are with the four of us, I feel as if we are caught between two families - with two daughters or with four daughters. I am so thankful that Em and Soph miss their sisters when we are apart. They love being able to do "normal" things - so they say, but they also reflect in passing with comments such as, "I don't think Rachel's chair would fit on this trail" or "Janneke would love to touch the moss and flowers."
Some time ago, I came across a number of documentaries that came from people just a bit older than me who discovered in their adulthood that they actually had a sibling they never knew. A disabled sibling who had been sent away - because that is what you did back then. Fascinating stories that evoke a range of emotions. Check out this website: The Teachings of Jon.
I am so thankful for the ability and means to care for all four girls in our home. Even if we take a few hours away from Rachel and Janneke, everyone is excited to go home and see them again. We are also blessed with loving respite workers and nurses who love them a lot.
Speaking of being loved and cared for, we continue to be humbled by the efforts with fundraising for our wheelchair-accessible van. This past week, Sarah V made cookies and sold them to people who stopped in to buy Norwex cleaning supplies from her mom Lisa. The other day, my neighbour Tracy told me her kids are collecting their loose change for our van. We are so loved.
Rachel is not impressed by something?
Soon, we will enjoy a pork roast in St. Anns and a wine/cheese evening at Club Roma. The excitement is building! Please check out the Appeals for Wheels website. I overheard Carolyn saying that tickets must be purchased in advance for the Club Roma event, so they know how many to plan for. Harv and Carol would also like to hear from those who are coming to the pork roast for the same reasons. Their RSVP is via Facebook.
If you can't reach either, you can always connect with Ralph or with me.
Hope your weekend goes well. Enjoy the long Canadian holiday weekend!
I often find Rachel sleeping in this relaxed pose.


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You see, only the progressive socialist left gets to determine what group needs protection. So this is what I want my brothers and sisters in law enforcement to understand. The liberal left in America does not deem you worthy to be protected. They believe your commitment and honorable service to protect us does not allow you to be protected from specific attacks because of your chosen profession. These socialist leftists see you as their necessary agitators for Black Lives Matters. These are the same leftists who don't believe a young black mother like Bianca Horton, gunned down by gang members, deserves protection. Sadly, our law enforcement officers and black mothers like Bianca Horton or children, like her daughter who was shot and paralyzed, don't fit the "identity" ideological agenda of the progressive socialist leftists, and so they don't deserve protection.
h wait...It's only Alan West and we know how THAT guy thinks so we can just write this off as some "prejudice" article as usual.


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27th of June 2009
The videos are from the Ahyausca ceremony on the 2nd of July
Met with my Shaeman Torongo to do Ahyasca around 5pm and we set off for the temple that is 15 minutes from the village, so after sitting inside for a few hours and waiting for the dog (Oso, means Panda in Spanish) to stop barking, we took the first batch of the medicne (about a half a litre of San Pedro)
It was funny and maybe a bit strange that he called the bottle in front of him ´San Pedrito´, i was thinking ´But this supposed to be Ahyuasca´
As it turns out, when i asked him about San Pedro the previous day he thought i wanted San Pedro, not so, but, no matter, I´ll take anything, even abuse, i meant Ahyasca, he thought i meant San Pedro :)
Which is funny in itself, i think San Pedro found me, i didn´t go looking for it and it was better i too San Pedro, Ahyasca kicks you ass, you need to Detox more for it, if you´ve been partying more, i was partying for 2 months.
The San Pedro took ages to kick in and then i took i some more. The temple he has, has a toilet, very convenient indeed and it´s true, a sort of convenient truth, dont yea think?
After a while we went out to look at the stars, and it was back into the temple for more singing, there was a lot of singing one of the jams went on for about half an hour. But i didn´t know any songs to sing. I might have scared off (some) of the spirits with a bit of Garth Brooks, i left the singing to Torongo.
At one point i could hear the crystal in my hand resonate as the Shaeman was singing, that was incredible!!!
We didn´t need to talk, it felt like the more you tried to described anything the less it meant.
Torongo was telling me you didn´t need to read books, all you need to do is read him, i totally understood, there is no one (hopefully) like you in the world.
I told him my life is a series of moments, none of them ordinary and at the end, that´s my life, he understod this too.
He told me 2 days later he used 4 San Pedro Cactus and when he was chopping and boling them (with Charcoal) he was singing to them i believe hime too.
It felt like there was lots of people around me all night and i was having interactions/conversations with some girl next to me (or just behind me)
And then came the ´Bad thoughts´ of naked chicks, this would explain the strange look i got off of Torongo at one point.
When i went to lie down (sleep) lots of promotors were coming up to me asking me if they used the temple for a club/strip club and i was like ´Sorry man, this is a temple´ you cant.
When it was all over i couldn´t believe i was the only one in there, it felt like lots of people were there.
God, if you want to call it that is in everyone and there is only you and the pacha mamma and there is nothing else, well worth the $100, i got 3 dosages of the medicine.
Great Shaeman, good medicine, good location and very professional.
It was as good as Ahyasca, but San Pedro is more grounding you with Mother Nature (Pacha mamma) and holding that crystal felt so good, i remember telling
Torongo it was like holding the heart of of the world, it was an amazing spirutal experience.
I ended up buying the crystal off of Torongo, the world is MINE!!!
All in all, i had a great time, he left at 6am, 13hrs later, i left at 1.30pm in the afternoon, so surreal with everyone saying ´Good afternoon´ to me.
It would be interesting to find out who that girl i was talking to was, i´m sure it was some other side of me, very cool indeed!
One of the lessons from the first Ahyasca session was to connect with my female side, maybe something is clicking there?