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A winter weekend in Grand Lake, Colorado
We arrived in Grand Lake on a snowy Friday. The area had received three straight days of heavy snow, which made our first stop at On the Trail Rentals, even more fantastic. I previously wrote about this experience.
We were booked at the Western Riviera Lakeside Lodging & Events, soon to be world famous because on March 25, they will be featured on the Travel Channel's Hotel Impossible. Much to my delight, because I'm a fan, the crew of Hotel Impossible was filming on the weekend we were in town.
We were staying in The Tree House, a huge two bedroom apartment overlooking the lake and just a short distance from the Western Riveria's lake front property. It has a full kitchen, a living room and dining area with a fireplace. The master suite features a walk in closet the size of my first apartment, and the bathroom has Jack & Jill sinks.
We thoroughly enjoy the purple and pink sunrises from our big front window each morning, and we had a front row seat to the fireworks show on Saturday night. I was in town covering Grand Lake Winter Carnival, one of the best little winter festivals I've attended in Colorado.
Scheduled for a guided snowshoe trek in Rocky Mountain National Park, we got up early on Saturday. The Fat Cat had been recommended to us by the bartender at The Lariat. They serve a $12 breakfast buffet on the weekend and the selection included Scotch eggs, a personal favorite. A table heavy laden with desserts caught our eye. It looked like something right off the pages of Bon Appetit Magazine.
My husband, Ryan, is a big fan of desserts. Big. He looked at me, eyes full of excitement and asked, "Do you think that's part of the buffet?"
It was. The Fat Cat will now always hold a special place in my Ryan's heart, and mine too. We ended up there twice during our stay. We met and chatted with Sally, the owner, and felt like we'd discovered a home away from home. That's how I feel about Grand Lake in general – it is genuinely comfortable.
At 9 a.m. we arrived at the Kawuneeche Visitors Center for our ranger led snow shoe outing with Ranger Barb. This was one of the best mornings ever, in part because Barb's husband cut the trail for us. The scenery was breathtaking and it felt like we were snowshoers in a postcard. Barb is a gifted guide and amusing storyteller. Ranger led hikes at RMNP are free, but you must make reservations by calling 970-586-1223.
After our hike we sped back to Grand Lake to enjoy Winter Carnival activities. During winter carnival the businesses along Grand Avenue sponsor snow sculptures and this year's theme was Alice in Winterland, resulting in especially whimsical creations.
The bed races are the Carnival's most famous event. Eager festival goers were already lining the streets when we arrived and I noticed right way that Grand Lake is probably one of the most dog friendly towns in a state full of dog friendly towns.
The bed races lived up to the hype. It was a seriously entertaining event and since Hotel Impossible was in town they entered a team complete with their own camera crew caught here in our video of the races.
After the events were complete we made a quick trip out to Grand Lake Brewing which had moved out of town since our first visit. Their taster trays are huge and the staff is really nice. Back in town, we walked from The Tree House to Pancho & Lefty's, another one of our favorite places in Grand Lake. It's a super fun place that I mentioned in previous posts here on HeidiTown.
After a rather impressive fireworks show that we watched from our porch, we dined on delicious handmade pizza at the Grand Lake Bowling Alley.
The next morning, after filling up at The Fat Cat once more, we headed out to the picturesque Grand Lake Nordic Center for a morning of cross country skiing. The club house is beautiful and with 35 kilometers of groomed trails, you could easily spend a day here.
Sadly, we couldn't. We had to head back home, but we'll miss Grand Lake, and can't wait to go back.
Author's Note: Don't miss the historic Daven Haven Lodge while you're in town. It's been open since the 1930s, and has had just three owners over the years. For a delicious steak, or all you can eat shrimp on Friday nights, make a reservation at the Daven Haven.
See more photos from this trip on Instagram at @TheMayorofHeidiTown.
HOTEL IMPOSSIBLE featuring the Western Riviera airs Monday, March 25 on the Travel Channel.
Sponsored by the Grand County Colorado Tourism Board. Find them on Facebook here. and on Twitter at @GrandCountyCo. #VisitGrandCounty
A Colorado Valentine's Day Guide
Want to experience an authentic Colorado Valentine's Day? Here are my tips for infusing a little Rocky Mountain high into your relationship this February.
Colorado offers everything from well-appointed historic hotels like the Brown Palace and Hotel Colorado to the shiny and still-fairly-new Ritz-Carlton in downtown Denver to Movie Manor, a motel in Monte Vista offering a drive-in movie with your stay.
When it comes to lodging options, this state has it all, but it's Colorado's bed and breakfasts that offer the perfect romantic ambiance for a Valentine's Day escape. Many bed and breakfasts are located in the heart of Colorado's unique towns, making everything within easy walking distance including restaurants, bars and shopping.
If you have reservations about staying at a bed and breakfast, I encourage you to read my article, "Dispelling the myths about staying at a bed & breakfast."
Pick a bed and breakfast with a romance package, or make your own romance package. Pack a bottle of bubbly, some of your partner's favorite chocolates or other fancy foods and stay in for the evening. Wink, wink, nudge, nudge!
We recently stumbled across the Carr Manor in Cripple Creek, and toured this 14-room bed and breakfast in a converted high school. There are two spa suites that will blow your mind. One has a vaulted Jacuzzi tub that should come with a couple servants waving palm frawns.
For a romantic little get away in the charming town of Berthoud, Colorado, check out the Berthoud Inn. The owner is an accomplished chef who just might cook up something special for your dinner if prearranged.
When it comes to the culinary scene, Colorado has chefs who are pushing the boundaries and testing the palates of residents from the Front Range to Aspen. While not always known as a "foodie town," today, inspired cuisine can be found throughout Denver at places like Euclid Hall and Fruition Restaurant.
But let's face it, Colorado's known for meat, red meat like steak, lamb and wild game.
If you want to impress your date this Valentine's Day with an authentic Colorado meal, try the Frontier Platter at The Fort in Morrison. This sample plate includes a buffalo sirloin medallion, wild boar chops and grilled teriyaki quail. Start your meal with some Rocky Mountain oysters as a daring aphrodisiac.
Looking for Rocky Mountain oysters at a less fancy establishment and price? Try Bruce's in Severance, Colorado. A basket of oysters, fries and dip at Bruce's will only set you back $8.99.
Here at HeidiTown, I'm all about doing stuff, and Valentine's Day is your opportunity to get out and do something romantic with that special person in your life.
If you read HeidiTown then you're already an expert when it comes to doing fun Colorado stuff, but if not, here are a few suggestions for this Valentine's Day.
Dinner theater – This option kills two birds with one stone; dinner and the evening's entertainment. And if you want to keep the Colorado theme going, The Unsinkable Molly Brown is now onstage at the Candlelight Dinner Playhouse in Johnstown.
A free gondola ride in Breckenridge – The 143-cabin Breckenridge Gondola runs year-round for skiers and non-skiers. This ride could be part of a romantic getaway in the beautiful town of Breckenridge, Colorado. Current hours of gondola operation are 8 a.m. to 4:30 p.m.
A moonlight snowshoe outing – Check out Colorado's Best Snowshoeing Trails and do a little investigating to find out what resorts/ranches have this unique experience available.
HeidiTown Gives Back Campaign: Café 180 (Englewood, Colorado)
It's the first Monday in December and that means it's time to unveil the recipient of the HeidiTown Gives Back Campaign. This month's organization came to my attention through a citizen of my little town, and I'm so glad that she shared the story with me.
Café 180 is the brainchild of Julie Mihevc and Cathy Matthews. Their original idea was to create transitional housing in Englewood, but fate had other plans. Instead, following the "pay it forward" concept pioneered by SAME Café in Denver, they decided to open their own café with a "pay what you can" mentality. Continue reading
A 40th birthday party at Dave & Buster's
Birthdays are a big deal in our house and the milestone birthdays are an even bigger deal. A birthday usually involves a weekend away, but since my husband, Ryan, was turning 40 this month I asked whether he preferred a party. He informed me that he wanted a party, but "didn't want to just meet up for beers with friends like we do all the time."
So I had to put my thinking cap on. I came up with a hundred different ideas, but most involved meeting up with friends for beer like we do all the time. Then, Dave & Busters popped into my mind. We hadn't been in a while, but Ryan loves video games and I figured it would give our friends a fun evening out.
So I booked Dave & Busters in Westminster, Colorado and nearly 30 people showed up to help Ryan ring in this new decade of life and to rib him about turning the big 4-0. Due to Friday evening rush hour traffic we were running a few minutes late to our own party, but I got an excellent text from a partier who beat us there.
It read: Set up looks great! 🙂
And it did. We had booked a party room that included a pool table, shuffle board, tables and lots of seating. The space was large, but not too big, and it was staffed with three attentive wait staff. As party goers continued to arrive and people began to tip back beers, the atmosphere couldn't have been better.
I like to feed people. I got this trait from my mother. I'm always afraid people won't get enough to eat or won't like the offerings. I had ordered the fajita buffet and as they uncovered the food I was pleasantly surprised to see the party goers digging in.
When ordering a buffet for a party, I'm always a little nervous about the quality, but this buffet was a big hit. I was overjoyed to see people going back for seconds and thirds. The buffet included chips and queso, fajita stuff (tortillas, beef and chicken) and lettuce, cheese, fresh guacamole, pico de gallo, and an addictive green sauce.
Everything was fresh, as was noted by several guests, but despite the compliments on the food, the best praise is that people ate the heck out of it!
After a fine meal and conversation, party goers began to filter out onto the game floor. Our party package came with $10 power cards for each attendee. Our group took over air hockey, then basketball and then moved on to trivia. Smiles were wide and laughs were many.
My 7-year-old niece, the only child in attendance, made out like a bandit because everyone kept giving her their winning tickets. She was giddy with excitement as she "bought" nearly every jelly bracelet in the Dave & Busters store.
With the games and crowds, Dave & Busters is a noisy place, so it was good to have our room available as an escape from the hubbub of the game floor. Some in our party became addicted to shuffle board and never left our room. Our wait staff also remained on hand, making sure all the gifts, purses and coats were looked after and drinks were full.
Almost everyone stayed late and I deemed this birthday bash a complete success. Having a party at Dave & Busters makes it easy for everyone – the host and the guests. With food and fun at your fingertips, not to mention drinks, there's no reason to leave early.
Dave & Busters may not be the first place you think of when planning an adult birthday party, but maybe it should be. They also specialize in team building parties and office holiday events.
Dave & Buster's Westminster location (303) 217-7107
"Oklahoma!" comes to Colorado
It's time for another dinner theater review and this time I went back to Candlelight Dinner Playhouse in Johnstown, Colorado for "Oklahoma!" I've been going to theater since I was just a little girl, but I had never seen a production of this famous play until last week.
Today, "Oklahoma!" is considered a wholesome family musical and is often performed at high schools and even middle schools, so it's difficult to think of it as ground breaking, but when "Oklahoma!" hit theaters in 1943, it broke the rules. First, the musical begins with just one person singing, and this was unusual. At the time, musicals always opened with a score performed by the ensemble cast. Secondly, a ballet takes place during the play, something that had never been done before.
For those of you who are unfamiliar with dinner theater… continue reading this review by clicking here.
HeidiTown readers get special perks at Wine Fest!
Didn't win a pair of tickets to Wine Fest in Fort Collins in last week's HeidiTown contest? Well, here is your chance to buy tickets and get treated like a star! Wine Fest has created a special incentive package for HeidiTown readers.
If you purchase tickets through the link provided below, you will receive the following:
1. Two free raffle tickets for the Grand Wine Fest Prize, a handmade wooden wine rack with a gift certificate to fill it with wine. Check-in at the Wine Fest Will Call Table and show your badge (printed or smart phone version) to receive your bonus raffle tickets.
2. A wine sample exclusive to blog partner fans. Visit the Synergy Fine Wines table and show this badge, either printed or on your smart phone, to Mark Eppel, the Synergy wine expert, to receive your sample of Scott Base Pinot Noir, 2009, Central Otago, New Zealand, from the Allan Scott Family Winery.
Click the logo below to buy tickets.
Your tickets must be purchased through this link
to receive these special perks at Wine Fest.
This is the 30th Anniversary of Wine Fest in Fort Collins, a wine tasting to benefit Disabled Resource Services. DRS provides support for those living with disabilities in Larimer and Jackson counties. As one of the ten centers for independents in the state of Colorado, DRS provides resources to empower individuals with disabilities to achieve their maximum level of independence through advocacy, awareness and access to the community.
Wine Fest features 500 different wines paired with food from Fort Collins' restaurants. The evening also includes prizes, music, entertainment and a silent auction that raised $12,000 last year. For those who prefer beer, that delicious beverage will be available as well.
This year's Wine Fest is being held at the Hilton located at 425 W. Prospect Road in Fort Collins, Colorado. The event is scheduled for Friday, March 23 with general admission at 7 p.m.
Colorado Christmas musicals in review – Scrooge at Candlelight & White Christmas at Midtown
Two very different Christmas shows are now playing in Northern Colorado, and I have had the chance to see them. Read my reviews and decide which one is right for your family this season. The two musicals are strikingly different, and each has strong points, so you may decide to see both.
"Scrooge! The Musical" While the entire cast is talented, Brian Burron, as Scrooge, is the heart of this show. We watch as Scrooge transforms from a terrifying character into a man who is filled with a love for life, and Burron vividly emotes the difficult internal struggle his character goes through in this play… continue reading this review here.
"White Christmas" From the moment go, this musical is just plain fun. The cast kicks off the production with a rousing rendition of "Happy Holidays" and "Let Yourself Go." The young, energetic and talented cast of "White Christmas," works hard to make sure the audience is immersed in this charming tale. I'm a sucker for a good song and dance number and this show has many… continue reading this review here.
Win 2 tickets to the Oscar Party at The Kress
The Kress is rolling out the red carpet for YOU!
The Kress Cinema & Lounge is located in downtown Greeley, Colorado and it's a HeidiTown favorite! This is a classy lounge with a well-appointed theater. They have live music in the bar area on the weekends and old and new movies nearly every night of the week. They also have a full lunch and dinner menu. It's worth the trip to Greeley to check this place out! You can read my previous review of The Kress HERE.
The Kress is throwing their annual Oscar Party on Sunday, February 27 at 5:30 p.m. The event includes a red carpet, appetizer buffet, Oscar ballot and cash prizes and the 83th Academy Awards on the big screen and the smaller televisions in the lounge. Tickets are $20 each.
You can win 2 tickets to the Oscar Party at The Kress by leaving me a comment here. Tell me which of the ten best picture nominees you think should win. If you haven't seen any of the best picture nominees (and shame on you!) tell me your favorite movie of the past year.
A winner will be picked randomly on Thursday at 9 a.m. Good luck!
"Smokey Joe's Cafe" at Carousel Dinner Theater
My husband and I finally had the opportunity to check out Carousel Dinner Theater thanks to a great deal they ran on Facebook. Carousel has been in Fort Collins, Colorado since 2002, and they recently moved to The Midtown Art Center at 3750 South Mason.
The new location is classy and houses a foyer, bar area, a theater and a ballroom for conferences, parties and weddings. I never saw the original location of Carousel, so I can't compare the two, but I was impressed with Midtown Art Center.
Colorado Rapids v. FC Dallas MLS Cup Watch Party
It's no secret, the Mayor of HeidiTown is a fanatical fan of soccer.
Seriously folks, I LOVE soccer. LOVE.
And it's been a great year for Colorado soccer fans… first the World Cup and now the Colorado Rapids have made it to the MLS final in Toronto.
There are several watch parties in Denver, but there wasn't an organized event in Northern Colorado, so I decided to put one together and YOU ARE INVITED!
Here are the details:
When? November 21, 2010 at 5:30 p.m. (match starts at 6:30 p.m.)
Where? C.B. & Potts Restaurant and Brewery at 1441 E. Horsetooth at Collindale Golf Course in Fort Collins (we were originally holding this party at the Elizabeth location, but have decided to join the Fort Collins Soccer Club)
If you are on Facebook, please RSVP for the event BY CLICKING HERE.
Please come out and cheer on your Colorado Rapids to victory and win prizes from Rocky Mountain Soccer, HeidiTown.com and more!!!
Face paint and flags are not required, but highly recommended.


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Body weight is one of the aspects of the body which most of the people care about. That is the reason why it has been one of the determining factors of the quality of their confidence. In most of the time, the skinnier a person is, the more confident he or she can be. There are actually several factors which can affect the body mass of someone other than the amount of the food intake. One of them is called as the internal factors. Internal factors responsible for body mass are factors that cannot be consciously controlled by people who do a diet program or plan no matter how effective it is such as the 3 week diet plan. Read the 3 week diet reviews here. Rather, they are more of the innate of the body. There are several internal factors that affect body mass and the genetic factor is one of them.
One of the human genes which can affect the body mass is the INSIG2. Research conducted by the Boston University Medical School found that a gene called INSIG2 responsible for obesity. INSIG2 gene is responsible for inhibiting the synthesis of fatty acids and cholesterol. Some of the protein product of the gene variant had a power INSIG2 inhibition so low that people with this gene variant will tend to accumulate more fat in their bodies. Approximately one out of ten people (10%) believed to be carrying this gene variant.
Other than the INSIG2 gene, there is another gene which is influential in determining the body mass of a person. One of the other genes responsible for obesity is FTO gene. FTO is the name of a gene located on chromosome 16 human. Based on the results of the study, people who have a particular variant of the FTO allele homozygote and also a couple of these variants in their genome, will have a weight of 3 kg heavier than an ordinary person and have a risk of developing obesity 5 times greater than ordinary people.

subset: commoncrawl


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Known until the early 1960′s as a quiet fishing village, Búzios skyrocketed to fame when actress Brigitte Bardot spent time in the town. Today, fishing still exists but the town has evolved into a world-class holiday and tourist destination while still preserving its rustic charm. Búzios is ideal for every type of traveller – whether you are looking for a laid back and relaxing vacation or 24/7 action, you can find it here! Located only 100 miles from the city of Rio de Janeiro, Buzios served as the Beck & Score getaway city during the 2014 FIFA World Cup™.
Considered by many as the "St. Tropez" of Brazil, you will never run out of exciting activities to pursue. Rua das Pedras and the Bardot Beachfront, are the main spots for bars and restaurants: This is where it all happens. The street is a little over 400 meters long, but it is charm laden from end to end. If it weren't for this street, Búzios just wouldn't be the same. Restaurants serve all kinds of food covering all gastronomic cultures. Friendly bars with live shows – some facing the ocean – invite you to sit and appreciate the ever-popular sport of people-watching. Art galleries, stylish stores featuring national and foreign brands, and beautiful sun tanned Brazilians make up the scene here all year long.
Visitors in Búzios love to stay active and enjoy the natural beauty of this place with outdoor activities. Paddle boarding, kayaking, and of course surfing are common activities. Another great way to explore Búzios is by boat. With more than 20 beaches to see, this is a boat ride to remember!
Beck & Score also curated unique experiences for our guests while in Búzios: big wave surfer Garrett "GMAC" McNamara took clients out to surf and paddle board and Beck & Score hosted exclusive weekly dinners for our clients at Rocka Beach Lounge.
Throughout his two weeks in Buzios, GMAC gave free surf lessons exclusively to our members, presenting the rare opportunity to venture past the break with the legend himself. On land, GMAC join our members as they soaked up the local nightlife and culinary scene
Beck & Score was also excited to share one of our favorite dining experiences with our clients: weekly dinners hosted at Rocka Beach Lounge. Voted to Condé Nast Traveller's Gold Standard list in 2013, this beach-side restaurant is home to one of the area's finest chefs, Gustavo Rinkevich. Chef Gustavo leads a kitchen that only uses the best of the best and the finest regional ingredients. The fish is always caught fresh by the local fishermen and a large part of the vegetables and spices are grown in their very own garden. Every meal here is one to remember!


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Stone Spray Sandwich Trailer (2005)
01 December 2005
Join TransWorld MX as they take you behind the scenes with the best racers and freestyle motocross riders on the planet. Features a variety of locations, from the legendary Castillo Ranch to the Mulisha Compound and everywhere in between. You ll see the pros in the business testing at top-secret locations, freeriders going huge in the natural terrain of Beaumont, Utah, Reche Canyon and more, and even score a pass into Ricky Carmichael s own backyard. Filmed entirely on 16mm film, Stone Spray Sandwich promises to be the ultimate moto film of 2005 and is packed with tons of bonus features.
Watch the official Stone Spray Sandwich 2005 trailer in HD below or find more Stone Spray Sandwich videos on Vidimovie.


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The signaling mechanisms underlying cell differentiation have been extensively studied with the use of rat PC12 cells as a model system. Nerve growth factor (NGF) is a trophic factor inducing PC12 cell differentiation through the activation of the p35/cyclin-dependent kinase 5 (Cdk5) complex. It has been reported that adenylyl cyclase activation and cAMP production may be involved in NGF-dependent actions. Our previous results indicate that cAMP activates the p35/Cdk5 complex in reproductive cells. Therefore, the role of cAMP in NGF-triggered p35/Cdk5 activation and PC12 differentiation was interesting to explore. Our results indicate that roscovitine, a molecular inhibitor of Cdk5, blocks cAMP-triggered PC12 differentiation, which was evaluated by neurite initiation, a decrease in proliferation, and cell cycle G1 arrest. The following data show that cAMP treatment increased Cdk5 activity through p35 upregulation. cAMP downstream components, protein kinase A (PKA) and phosphorylated cAMP response element binding protein (CREB), are involved in this regulation. The immunocytochemical results indicate that PKA inhibition disrupted cAMP-triggered p35/Cdk5 localization in PC12 cells. In addition, adenylyl cyclase inhibition was found to diminish NGF-induced intracellular cAMP production, CREB phosphorylation, and p35 expression. The cAMP antagonist and the PKA inhibitors reduced NGF-induced p35 expression. Finally, NGF-triggered PC12 differentiation was partially decreased by adenylyl cyclase or PKA inhibitors. In conclusion, these results demonstrate that cAMP may play a role in NGF-p35/Cdk5-dependent PC12 differentiation.
- adenosine 3′,5′-cyclic monophosphate
- nerve growth factor
- cyclin-dependent kinase 5
the pc12 cell line was cloned from rat adrenal pheochromocytoma and is characterized based on its capacity to cease proliferation and promote differentiation when exposed to nerve growth factor (NGF) (8, 11, 18). In recent decades, the PC12 cell line became a useful model system for investigating the genes and proteins involved in the effects of NGF on cell differentiation (31, 42). NGF signals through two different types of receptors to promote differentiation: TrkA, a receptor tyrosine kinase, and p75NGFR, a low-affinity neurotrophin receptor (4). The finding that Src and Ras enhance NGF-induced differentiation led to the identification of Ras and Src signaling pathways that are a part of the total differentiation response to NGF (6). NGF was also reported to upregulate cyclin D1 expression and promote neurite outgrowth through Ras/ERK pathway (25). In addition to NGF, cAMP is also thought to be an important stimulator of PC12 differentiation (36). A recent report suggests that NGF and cAMP have a differential and synergistic effect on PC12 differentiation through transcriptional regulation (27). Furthermore, several lines of evidence indicate that NGF-triggered responses are correlated to cAMP-related signals (9, 13, 32, 39). NGF induces cAMP formation via a p75NGFR/G protein-mediated mechanism (13). Also, cAMP response element binding protein (CREB) appears to mediate NGF-induced Bcl-2 expression and promotes sympathetic neuron survival (32). A recent study revealed that NGF-like neurotrophins elevate cAMP and overcome inhibition by myelin-associated glycoprotein (MAG) (9). Stessin et al. (39) identified soluble adenylyl cyclase as a mediator of NGF signaling and revealed the existence of distinct pathways leading to cAMP-dependent signal transduction.
Cyclin-dependent kinase 5 (Cdk5) plays important roles in the development of the central nervous system and neurodegenerative diseases (7). Association with its regulatory partner, p35, is required for Cdk5 activation, which maintains the physiological functions of Cdk5 (7, 44). The expression of the p35/Cdk5 complex in extraneuronal cells has been linked to various biological functions (19, 26, 37, 41). Our previous results (14, 20, 22, 23) indicate that Cdk5 affects the fate of neuronal cells as well as tumor cells. In addition, we also identified that cAMP modulates Cdk5 kinase activity in mouse Leydig cells (21). Alternatively, NGF induces p35 expression through ERK/Egr-1 activation and causes Cdk5 activity-dependent differentiation of PC12 cells (12). In light of these findings, the involvement of cAMP in NGF-induced p35 expression and PC12 differentiation becomes interesting to investigate. Our present data indicate that NGF treatment stimulates intracellular cAMP production and causes p35 expression and Cdk5 activation, which induces growth arrest and neurite initiation of PC12 cells.
Cell culture.
The PC12 cell line (ATCC no. CRL-1721) was purchased from the Culture Collection and Research Center, Taiwan. PC12 cells were cultured in Ham's F-12K medium (Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum (Invitrogen, Carlsbad, CA), 10% horse serum (Invitrogen), 1 mM sodium pyruvate (Sigma), and penicillin-streptomycin (Sigma) at 37°C in a humidified atmosphere with 5% CO2.
Measurement of neurite outgrowth.
Cells were seeded on 24-well culture plates and treated for 48 h with cAMP or NGF with or without specific inhibitors. Neurite outgrowth was measured with an optical microscope, and five random fields were observed in each culture dish. A neurite is defined as an extension longer than the diameter of two cell bodies (3, 38, 40). The mean number of neurites per 100 cells for each well was calculated in 3 independent experiments. The values are expressed as means ± SE.
Western blotting.
Cell lysates were extracted with lysis buffer as previously described (20, 21), and the proteins were analyzed by Western blotting (20–40 μg/lane). Antibodies included anti-Cdk5 antibody (DC-17, Upstate, Lake Placid, NY), anti-p35 antibody (sc-820, Santa Cruz Biotechnology, Santa Cruz, CA), anti-actin antibody (Chemicon, Temecula, CA), and peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Jackson ImmunoResearch Laboratory, West Grove, PA). Enhanced chemiluminescence (ECL) detection reagent (PerkinElmer, Shelton, CT) was used to detect the immunoreactive proteins.
In vitro Cdk5 kinase assay.
Cdk5 protein was immunoprecipitated from PC12 cell lysate with a specific antibody (DC-17, Upstate). The kinase assay was performed by washing immunoprecipitates three times with kinase reaction buffer [in mM: 50 HEPES (pH 7.0), 10 MgCl2, and 1 DTT]. ExactaCruz matrix beads (sc-45039, Santa Cruz Biotechnology) bound to target proteins were incubated in kinase reaction buffer containing 10 μg of substrate (histone H1, 14-155, Upstate) and 20 μCi of [γ-32P]ATP (NEG502Z001MC, PerkinElmer) in a final volume of 40 ml at 30°C for 30 min.
Measurement of cAMP.
PC12 cells were primed for 30 min with 1 mM 3-isobutyl-1-methylxanthine (IBMX, phosphodiesterase inhibitor) to prevent the degradation of cAMP before other treatments. PC12 cells were then scraped and lysed with lysis buffer (no. 250006, Cell Biolabs, San Diego, CA). The intracellular content of cAMP in cell lysates was measured with a cAMP ELISA kit (Cell Biolabs).
Reverse transcription-polymerase chain reaction.
Total RNA was isolated from PC12 cells with the Spin Column RNA Miniprep Kit according to the manufacturer's instructions (Genemark). Reverse transcription-polymerase chain reaction (RT-PCR) was performed with the One-Step RT-PCR Kit (Genemark). Primer sets were designed and synthesized according to database sequences (p35: 5′-taggatccgccatcacctctgcagggac-3′ and 5′-tagtcgactcaccgatccagccccag-3′; GAPDH: 5′-tgatgacatcaagaaggtggtgaag-3′ and 5′-tccttggaggccatgtgggccat-3′; β-actin: 5′-ttgccgacaggatgcagaa-3′ and 5′-gccgatccacacggagtact-3′). The RT-PCR reactions were performed with 10 pmol of each primer and the following program: 30 min at 50°C and 2 min at 94°C, followed by 18 cycles of denaturing at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, ending with a final extension at 72°C for 7 min. The amplified PCR products were separated by electrophoresis on a 0.8% agarose gel and visualized by ethidium bromide staining. A 1-kb DNA ladder (Genemark) was used as a size marker.
MTT assay.
A modified colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to quantify the proliferation of PC12 cells (14). Yellow MTT compound (M5655, Sigma) is converted by living cells to blue formazan, which is soluble in dimethyl sulfoxide (DMSO, D8418, Sigma). The intensity of blue staining in the culture medium proportionally represented the number of living cells and was measured by an optical density reader (Anthos-2001) at 570 nm (20).
Analysis of cell cycle distribution.
Propidium iodide staining was used to measure the DNA content of PC12 cells as previously described (14). DNA content was quantified by flow cytometry (FACSCalibur, BD). The percentage of cells in the G1 phase of the cell cycle was determined with Cell Quest software (BD) (14).
After two phosphate-buffered saline (PBS) washes, PC12 cells cultured on coverslips were fixed for 10 min in 4% paraformaldehyde and 2% sucrose in PBS at room temperature (RT). After fixation, the cells were permeabilized in PBS buffer containing 0.3% Triton X-100 and 3% bovine serum albumin (USB, Cleveland, OH) for 2 min at RT. A PBS wash and a subsequent block step in 3% BSA-PBS were performed for an additional 15 min at RT. Antibodies used for immunostaining included anti-Cdk5 (DC-17, Upstate), anti-p35 (SC-820, Santa Cruz Biotechnology), and anti-StAR (FL-285, Santa Cruz Biotechnology). Images were scanned and recorded with Leica confocal microscopy (TCS NT).
8-Bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP, B7880), 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-BrcAMPS, B2432), 2′,5′-dideoxyadenosine (ddAdo, D7408), H-89 (B1427), IBMX (I7018), KG-501 (70845), KT-5720 (K3761), roscovitine (RV, R7772), and SQ-22536 (SQ, S153) were all purchased from Sigma. Recombinant NGF (no. 450-1) was purchased from PeproTech (Rocky Hill, NJ).
Data analysis.
All values are denoted as means ± SE. In all cases, the Student's t-test was used. A difference between two means was considered statistically significant with P < 0.05.
Cdk5 inhibition blocks cAMP-induced differentiation of PC12 cells.
RV is a Cdk5 molecular inhibitor and prevents NGF-triggered differentiation (12). On the basis of analysis of differentiation indicators such as neurite initiation (defined as extensions longer than 2 cell body diameters), proliferation, and the cell cycle G1 distribution of PC12 cells, our results confirmed the RV inhibitory effects on NGF-triggered PC12 differentiation (Fig. 1A). In addition to the inhibition of NGF-triggered responses, RV blocked the morphological changes induced by 48-h treatment with 8-BrcAMP (a permeable analog of cAMP) in PC12 cells (Fig. 1B, top left). The quantitative results indicate that RV treatment significantly prevented 8-BrcAMP-triggered neurite initiation (Fig. 1B, top right). Alternatively, a decrease in cell proliferation and cell cycle arrest are phenomena of cell differentiation progress (24, 35). Therefore, MTT assay and flow cytometric analysis were performed, and the results showed that 8-BrcAMP treatment decreased PC12 cell proliferation (Fig. 1B, bottom left), while cell death was unaffected (data not shown). In addition, 8-BrcAMP-triggered G1 phase arrest of PC12 cells was also observed (Fig. 1B, bottom right). In regard to the influence of Cdk5 inhibition, RV treatment significantly prevented 8-BrcAMP-induced morphology changes, neurite initiation, decrease in cell proliferation, and cell cycle arrest. These findings suggest that Cdk5 activation may be involved in cAMP-induced changes of PC12 cells. Subsequently, the effects of cAMP on the regulation of Cdk5 activity were explored. p35 is the major regulator of Cdk5 and is associated with PC12 differentiation (12); therefore, the involvement of 8-BrcAMP in p35-dependent Cdk5 activation was investigated. Our results indicate that a 48-h treatment with 0.1 mM 8-BrcAMP significantly increased p35 protein expression and Cdk5 activity in PC12 cells (Fig. 2, top). NGF treatment (100 ng/ml) was used as a positive control for the induction of p35/Cdk5 activation in PC12 cells. The quantitative results of cAMP-dependent regulation on p35 expression and Cdk5 activation are also shown (n = 3; Fig. 2, bottom). These results indicate that cAMP may regulate p35/Cdk5 activation and also suggest the possibility that p35/Cdk5 activation involves cAMP-triggered PC12 differentiation.
PKA inhibition partially blocks 8-BrcAMP-induced PC12 differentiation.
Protein kinase A (PKA) is a common downstream component of cAMP signaling in neurons (43). Here, we used two molecular inhibitors (H-89 and KT-5720) to inhibit 8-BrcAMP-dependent activation of PKA. The serine-133 phosphorylation of CREB (PKA downstream target) and p35 protein expression were then monitored. The data indicate that both PKA inhibitors suppressed 8-BrcAMP-induced CREB phosphorylation and p35 protein expression in PC12 cells (Fig. 3A, top). The quantitative results of the effects of PKA inhibitors are shown in Fig. 3A, bottom (n = 3). In addition to CREB phosphorylation and p35 expression, the effects of PKA inhibitors on 8-BrcAMP-triggered differentiation indicators are also reported in Fig. 3, B (for KT-5720) and C (for H-89). We discovered that PKA inhibition was able to reverse the differentiation-related actions (including neurite initiation, cell proliferation, and cell cycle distribution) induced by 8-BrcAMP. Alternatively, immunocytochemical analysis was utilized to investigate the subcellular localization of the p35/Cdk5 protein in PC12 cells. Corresponding to the results of the Western blot, the immunocytochemical images indicate that p35 protein levels in the 8-BrcAMP group were apparently higher than the levels in the control group; however, Cdk5 levels in both groups were comparable (Fig. 4, left and center). Although few processes of PC12 cells were observed in the control group, p35 protein was absent in these regions, which suggests that the original processes in the control group are p35/Cdk5 independent (arrows in control group, Fig. 4). After 8-BrcAMP treatment, the morphology of neurite initiation was shown, and the colocalization of p35 and Cdk5 proteins was observed in the periphery of PC12 cells, especially in the termini of the neurites (arrows in 8-BrcAMP group, Fig. 4). After PKA inhibitor (KT-5720) treatment, 8-BrcAMP-induced neurite initiation and p35 protein expression were apparently diminished, and the colocalization of Cdk5 and p35 was also absent (8-BrcAMP+KT group, Fig. 4). These data suggest that cAMP may modulate the function of p35/Cdk5 in the periphery (or termini) of PC12 cells and cause neurite initiation partially through PKA activation.
CREB blockade downregulates cAMP-induced p35 expression.
As shown by the results in Fig. 3, cAMP may stimulate p35 expression through CREB activation; therefore, the importance of CREB activation in cAMP-dependent p35 expression was subsequently examined. A blocker of CREB activation, KG-501, was recently reported (1, 28) and was utilized in our experiments. KG-501 treatment for 48 h in culture medium of PC12 cells was found to dose-dependently inhibit both p35 mRNA and protein expression induced by 8-BrcAMP (Fig. 5, top). The quantitative results of KG-501 effects on p35 mRNA and protein expression are shown in Fig. 5, bottom (n = 3).
Adenylyl cyclase inhibition blocks NGF-stimulated cAMP production in PC12 cells.
NGF induces cAMP elevation in neuronal cells (9, 39); therefore, NGF itself is a possible upstream regulator that controls cAMP production and the subsequent differentiation of PC12 cells. We found that 48-h treatment with NGF elevated the accumulation of intracellular cAMP, while the addition of IBMX (a phosphodiesterase inhibitor; 1 mM) to NGF treatment inhibited the drastic digestion of cAMP by phosphodiesterase (Fig. 6). In addition, the NGF-stimulated increase in cAMP was significantly blocked by adenylyl cyclase inhibitors [SQ (10 μM) and ddAdo (50 μM)] (Fig. 6). These data suggest that adenylyl cyclase is involved in NGF-increased cAMP production in PC12 cells.
Inhibition of cAMP pathway interferes with actions of NGF in PC12 cells.
Adenylyl cyclase inhibitors (ddAdo and SQ), cAMP antagonist [Rp-8-BrcAMPS (21)], and PKA inhibitors (H-89 and KT-5720) were used to investigate the involvement of adenylyl cyclase, cAMP, and PKA in NGF-induced responses in PC12 cells. When adenylyl cyclase was inhibited, we found that NGF-induced CREB phosphorylation and p35 mRNA/protein expression were significantly decreased (Fig. 7, A–C). Furthermore, cAMP antagonism also significantly diminished NGF-induced p35 mRNA/protein expression levels (Fig. 7, D and E). Finally, when PKA was inhibited, NGF-induced p35 protein expression was reduced (Fig. 7F). The quantitative results of CREB phosphorylation, p35 expression, and Cdk5 activation are shown in their respective panels (n = 3) in Fig. 7. In addition to the above findings, the effects of adenylyl cyclase inhibition on PC12 differentiation were further identified. The differentiation indicators of PC12 cells described in Fig. 1 were observed after NGF treatment for 48 h. Inhibition of adenylyl cyclase (by SQ, 10 μM) was shown to inhibit NGF-triggered morphology changes (Fig. 8A, top left) and effectively prevent NGF-induced neurite initiation (Fig. 8A, top right). The results also show that 48-h treatment with NGF reduced PC12 proliferation, and SQ reversed the reduction without cell death (Fig. 8A, bottom left). G1 phase distribution of PC12 cells increased after 48-h treatment with NGF, and the distribution was reversed by cotreatments with SQ (Fig. 8A, bottom right). In addition to SQ inhibition, PKA inhibition (by KT-5720 and H-89) was also tested, and the PKA inhibitors prevented the actions of NGF (Fig. 8, B and C). These results suggest that the one of the numerous pathways triggered by NGF that promote PC12 differentiation may involve cAMP production, PKA activation, CREB phosphorylation, p35 expression, and Cdk5 activation. A schematic representation of our findings is provided in Fig. 9.
The PC12 cell line is a popular tool for investigating the mechanisms underlying neuronal differentiation. Upon treatment with NGF, PC12 cells differentiate into sympathetic-like neurons (11, 18, 35). p35/Cdk5 activation was previously believed to be a main cause of NGF-induced PC12 differentiation, in which NGF mediates p35/Cdk5 activation through the ERK pathway (12). Some reports suggest that cAMP is also involved in NGF-induced responses of PC12 cells (9, 13, 39). However, cAMP was found to both induce the transactivation of the NGF receptor as well as modulate ERK1/2 activation and neurite outgrowth (30). Therefore, a complicated correlation exists among NGF, cAMP, and p35/Cdk5 in promoting the differentiation of PC12 cells; however, the link between cAMP and p35/Cdk5 in neuronal differentiation remains unclear. Our previous findings indicate that cAMP regulates p35/Cdk5 activity in mouse Leydig cells. Here we determined that cAMP also acts as a mediator in the NGF pathway and promotes p35/Cdk5-dependent differentiation of PC12 cells.
RV inhibits NGF-dependent differentiation through Cdk5 inhibition (12). To understand whether our evaluation of PC12 differentiation is appropriate, the effects of RV on the actions of NGF were monitored. Our data indicate that RV prevented NGF-induced morphology changes, neurite initiation, a decrease in proliferation, and G1 phase arrest (Fig. 1A). In regard to cAMP, our data show that RV blocked cAMP-induced PC12 differentiation (Fig. 1B). Although RV partially rescues NGF/cAMP-differentiated PC12 cells from cell death induced by withdrawal of trophic factors (15), our data demonstrate the involvement of p35/Cdk5 in cAMP-dependent PC12 differentiation. Several lines of evidence indicate that cAMP may cause apoptosis of various tumor cell lines (2, 5, 17); however, we did not detect any cell death by Trypan blue staining and sub-G1 phase distribution (data not shown). Although the neurites initiated by cAMP or NGF are reportedly different (10, 27), our data showed that the increasing levels of p35 protein and Cdk5 activity induced by cAMP and NGF are comparable (Figs. 1, 2, and 8). These findings imply that even though different signaling pathways play a role in differentiation, NGF and cAMP do share partial downstream p35/Cdk5 signaling components. A recent report indicates the synergistic effect of NGF and dibutyryl cAMP (DBcAMP) on neurite outgrowth; it also indicates that >100 genes appeared to be similarly regulated by both NGF and DBcAMP. These findings suggest that these genes may contribute to the induction of neuronal differentiation by NGF through the downstream activation of the cAMP pathway (27). In addition, it has been reported that the GC box-binding protein Sp1 contributes to the regulation of p35 expression in neurons (34), and Sp1 was also found to be modulated by cAMP-dependent protein kinase (PKA) (33). Together with our findings, these findings imply that cAMP may be an upstream regulator of p35 expression in PC12 cells. Alternatively, cAMP regulates a PKA-independent signaling pathway that mediates neuritogenesis through Egr1 in PC12 cells (31). Therefore, the detailed mechanisms of cAMP-dependent p35/Cdk5 regulation in cell differentiation are still unclear and are worthy of further investigation.
To unravel the above enigma, the involvement of PKA and CREB in cAMP-p35/Cdk5 regulation was investigated by utilizing their molecular inhibitors. In addition to the common PKA inhibitors (H-89 and KT-5720) utilized, KG-501 is a new and original blocker of CREB activation that blocks cAMP induction of CREB-dependent target gene transcription through interference of the binding between the kinase-inducible domain (KID) domain of CREB and the KIX domain of the CREB-binding protein (1). Our results suggest that cAMP may regulate p35 expression, at least through PKA and CREB activation (Fig. 3). Supporting our findings, although the authors do not mention this point, Ravni and colleagues (31) showed that PKA inhibitor (H-89) significantly decreases forskolin (adenylyl cyclase activator)-induced total length of neurites, and there is a tendency that the number of neurites induced by forskolin per cell is affected by H-89. Alternatively, several lines of evidence indicate that p35/Cdk5 colocalization may be important for neuronal differentiation (12, 45). Our previous results (14, 20, 23) also show that the localization of the p35/Cdk5 protein complex can be an indicator of cell fate in both neuronal cells and tumor cells. Therefore, it is worthwhile to understand whether the p35/Cdk5 localization in PC12 cells changed after cAMP treatment. The immunocytochemical results indicate that cAMP increased p35 protein levels in PC12 cells and p35/Cdk5 colocalization in the periphery (arrows in 8-BrcAMP group, Fig. 4). In the termini of neurites, 8-BrcAMP-induced colocalization of p35/Cdk5 may promote neuritogenesis. Moreover, inhibition of PKA both prevented the 8-BrcAMP-induced increase of the p35 protein level and eliminated p35/Cdk5 colocalization (8-BrcAMP+KT group, Fig. 4). Interestingly, although inhibition of PKA decreased p35 expression in the termini, a few processes still existed (arrows in 8-BrcAMP+KT group, Fig. 4), which supports the previous findings indicating that the cAMP-dependent, PKA-independent pathway mediates neuritogenesis in PC12 cells (31). In addition to PKA inhibition, a CREB activation blocker (KG-501) significantly prevented 8-BrcAMP-induced p35 expression (Fig. 5). These results suggest that PKA and CREB activation were involved in cAMP-induced p35 expression and PC12 differentiation.
After understanding that cAMP acts via p35/Cdk5 to promote PC12 differentiation, the correlation of NGF and cAMP production in PC12 cells becomes the next question that needs to be further examined. Several lines of evidence indicate that NGF induces cAMP formation in neuronal cells including PC12 cells (9, 13, 39). Here we provided evidence indicating that NGF stimulates intracellular cAMP production through activating adenylyl cyclase (Fig. 6). However, we observed that NGF-induced stimulation of cAMP production was not obviously detected in the absence of IBMX treatment (data not shown). This observation may explain why the relationship between cAMP and NGF was previously questionable. Subsequently, the involvement of adenylyl cyclase activity, downstream cAMP, and PKA activation on NGF-induced p35 expression was confirmed (Fig. 7). The blockade of NGF-induced morphological changes by inhibition of adenylyl cyclase or PKA was also identified (Fig. 8). Therefore, we suggest that cAMP plays a role in NGF-induced p35/Cdk5 activation and PC12 differentiation. Alternatively, it has been demonstrated that p35 and Cdk5 expression in human neuroblastoma cells is regulated by activating Erk/Egr-1 and PKA/CREB, respectively (16). However, our present data provide evidence showing that PKA/CREB is important for cAMP-regulated p35 expression, while Cdk5 protein expression only slightly increased after cAMP treatment (Fig. 2). Correspondingly, our previous results in Leydig cells (21) also support the idea that Cdk5 is upregulated by cAMP. Importantly, H-89 did not fully reverse NGF-induced actions in Fig. 7F and Fig. 8C, which suggests that there are still additional PKA-independent regulations under NGF stimulation. ERK-related inhibitors were used to determine whether ERK-dependent steps are involved in NGF-induced Cdk5 activation. The results revealed that ERK inhibition partially blocked NGF-induced p35 expression and subsequent Cdk5 activation (Supplemental Fig. S1), which corresponds to the study of Harada et al. (12).1 Our unpublished data also indicated that some kinases might be involved in NGF-induced Cdk5 activation through direct phosphorylation. In addition, another Cdk5 activator, p39, is also a possible regulator of NGF-induced Cdk5 activation (44). On the other hand, PKA phosphorylation of Src is essential for cAMP and NGF signaling in PC12 cells, and PKA was identified as an important downstream target of NGF (29). Here we used molecular inhibitors to identify that cAMP plays a role in activating p35/Cdk5, and this regulation is one pathway that NGF acts through to promote the differentiation of PC12 cells. These observations imply that differentiation is a process that occurs through the simultaneous activation of multiple parallel signaling events rather than a single master pathway (31). This study sheds light on the involvement of cAMP in NGF-p35/Cdk5-related development of the central nervous system and provides a new target for future research in neurobiology.
This work was funded by the National Science Council (NSC 97-2320-B-010-017-MY3 to P.S.Wang, National Yang-Ming University and NSC 96-2628-B-005-013-MY3, to H. Lin, National Chung Hsing University), Taipei City Government (No. 99001-62-020). and the Ministry of Education, Taiwan, R.O.C. under the ATU plan (to H. Lin, National Chung Hsing University).
No conflicts of interest, financial or otherwise, are declared by the author(s).
The authors thank Dr. Shih-Lan Hsu and Mei-Chun Liu (Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan, R.O.C) and Dr. Chuan-Mu Chen, Dr. Hong-Chen Chen, and Dr. Chi-Mei Hsueh (Department of Life Sciences, National Chung Hsing University) for their full support.
↵1 Supplemental Material for this article is available online at the Journal website.
- Copyright © 2010 the American Physiological Society


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It's with a heavy heart that I'm writing to you not from a cosy little room by the sea but from my (admittedly cozy) little couch situated in cold and rainy Bristol. We had the most lovely weekend in Bournemouth, thanks to Travelodge and I think I've felt a bit glum by comparison since we got back!
As you can tell from my archives, Ryan and I love to get out and explore the bits of England that we can get to by train or bus! When Travelodge offered us a two night stay, we pounced on the opportunity and decided on a place that we'd never been together and, despite the weather, is close to the water.
I was really blown away by how roomy and comfortable everything was upon arrival! We've had a few lackluster hotel stays in the last few months, so it was nice to be met with a warm, huge room with a view of the ocean! I'm a sucker for blackout curtains but I was very tempted to leave the windows bare in the hopes that we'd get woken up to a gorgeous view of the sun rising over the water!
After we unpacked and tucked into warm clothes, we went out to explore a bit to catch the sunset and map out our game plan for the second day! I (per usual) have a stack of photos that I can't wait to show you!
I'd had a really busy week leading up to our trip and man was I rewarded with a good night's sleep! We were really pleased with the cleanliness and comfort level (and hey, the prices aren't bad, either!) and will absolutely be making it a point to head back. Considering that we were just steps from the water, we really couldn't have asked for a better location!
Before we left, we enjoyed a yummy breakfast (and bottomless coffees! joy!) downstairs in the restaurant and it really ticked all of the boxes. Food? Good! Service? Amazing! (In fact, the staff might been one of the best parts of our stay. I can't shower them with enough praise!) We left feeling full and happy and so relaxed.
Mission: accomplished!
(Ryan would just like to add that he really liked the bay windows, which I, too, enjoyed!)
Happy Wednesday!


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Playa Santana, Nicaragua offers the right climate for learning
The LeaderSurf program is located at the Buena Onda Beach Resort in Playa Santana, Nicaragua. This remote location on the Pacific coast of Nicaragua is the ideal spot to match perfect surf for learning with a modern hotel featuring the creature comforts of home. LeaderSurf has chosen this location for the leadership development program after an exhaustive search for a site that meets its high standards for delivering world class leadership development.
"I loved this place. It was perfect for the course. It was simple yet authentic and had a great ambience. I miss the hammock every single day."
-LeaderSurf executive participant
Buena Onda beach resort
Buena Onda Beach Resort is a boutique surf retreat located in Playa Santana, Nicaragua. The hotel is situated on 5 acres on the Pacific shores of Nicaragua about 2.5 hours drive from Managua International Airport. The resort offers private accommodations for each program participants. All rooms include the following amenities:
· Queen size beds
· Air Conditioning
· Safe
· Refrigerator
· Satellite TV
· Coffee Maker
· Private porch
· Wifi
· Private bathroom with hot water shower
The Buena Onda Beach Resort is located on 5 acres of beachfront property. The resort features plenty of shaded typical rancho areas for lounging with beautiful tropical landscaping. There is very fast wifi service located throughout the resort. Other amenities include a pool with swim up bar, private beach access, a pool table and covered lounge area. Learning sessions will utilize several of the resorts unique venues including indoor air conditioned meeting space, covered breezy patios, outdoor meeting space and of course the beach.


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By BGEA Staff • June 1, 2004 • Topics: Faith, Salvation
Threatening world events should not be a source of anxiety to true believers who are trusting in Christ as their personal Savior. We who know the Lord are looking forward to His soon return, and we are assured that our future is in His hands.
Perhaps you do not feel this assurance. The key is to realize that God made us so we could have a personal relationship with Him. This relationship gives us peace. The Bible says, "He will keep in perfect peace all those who trust in Him, whose thoughts turn often to the Lord" (Isaiah 26:3, TLB). No matter the troubles in the world, you can have peace with God.
The Scriptures give us many promises to keep us in God's perfect peace when the world is shaking with fear. We are to "watch and pray," but we are not to have an unwholesome fear, which is equivalent to doubting God's Word. Jude 24 describes God as "Him who is able to keep you from falling and to present you before his glorious presence without fault and with great joy." In Ephesians 6:13 we read, "Therefore put on the full armor of God, so that when the day of evil comes, you may be able to stand your ground, and after you have done everything, to stand."
It is important that we make our relationship with Christ a priority, that we faithfully serve Him in consistent Christian living, and that we eagerly look forward to His coming again. Some relevant Bible passages we encourage you to read are Matthew 24:42; Luke 21:36; Ephesians 6:10-18; 1 Thessalonians 5:1-11; 2 Timothy 4:8; Titus 2:11-15; 1 Peter 4:7; and 2 Peter 3:9-18. May God bless you as you trust in Him.
I haven't felt the anxiety and stress more than I feel the last few years. It is a tangible feeling that I express as "having a bad feeling" but not being able to give a definitive reasoning to this emotion. The bills, the jobs, the economy, the politics it's all madness. I strive for a balance in my life but continually struggle with feeling secure and seeing positivity.
When you open your eyes every day when you awake, rebuke the devil…..before you get out of bed…do it…that feeling will be gone!!!


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My mother and I have been suffering from financial difficulties and we are living with my aunt. We need to move out and own our own home so we can be away from our family because the family threatens to take me away from my mother just because I am homeschooled. I am a helpless 13 year old girl and I pray for Jesus to protect me from this terrible family. Please pray for me and my mother that our lives will get better and that we will have enough money to buy a nice home for us to dwell in.
Join in and write your own page! It's easy to do. How? Simply click here to return to Daily Prayers.
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|23 Sep 2013
Win 7 64 bit
Issues connecting to \\servername
When I try to connect to \\servername to see the available shares.....I get permission error 53 the network path not found.
If I do \\servername\folder1 - I can successfully view the folder.
Not sure why? Anyone can shed some light?
|My System Specs
|25 Sep 2013
windows 7
try to connect from a different computer to \\servername. If you get the same error, it may be a security setting on the \\servername computer. If you DON'T get an error from the other pc/laptop...check your pc/laptop for viruses or even changes viruses COULD have made to your permissions..
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The Old Village Association of Port Royal has announced the date and line-up for the upcoming 14th Annual Soft Shell Crab Festival. Perhaps one of the most popular and always-anticipated Lowcountry events, the big day this year is on Saturday, April 15th from 11am to 5pm.
Port Royal goes nuts over soft shell crabs and the Annual Soft Shell Crab Festival will be full of them (yes, crabs and nuts). The opportunity for soft shell crab is quick. It only comes once a year and the fun community festival offers a day of enjoyment and a fantastic opportunity to savor one of our Lowcountry delicacies.
And, boy…do they come. If you'll be one of the usual crowd of 8,000+, expect a day full of fun.
Live music will be performed by The Bull Grapes, Broke Locals and Trey's Aliens and there will be plenty of artisan craft vendors and an expanded kids' fun zone.
The Beaufort's Classic Car & Truck Club will be on hand showing off their spectacular collection of vehicles and the Rotary Club of Beaufort will host their annual Charity Crab Race along Battery Creek, with an available top prize of $40,000 to one possible winner and 10 chances to win $5,000! To enter the race, visit www.portroyalcrabrace.com.
It's always a good time to indulge in the sites of the beautiful wetlands and historical charm that Port Royal has to offer, while experiencing food, drink, and fun for the majority of the afternoon.


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"My work considers social ordering, or how a society determines what is tasteful, while engaging domestic materials to challenge traditional notions of art. I alter familiar household items like vinyl flooring and carpets and use these low materials as a source of inspiration.
I choose domestic materials for both practical and formal purposes and alter them in the attempt to derive meaning. I make works to consider taste and time. In these new pieces, I have added some drawing elements, reminiscent of coloring book pages, as maybe a nod to having a plan, but not really. I continue to search for ways to escape and exist without a clear narrative present, as this seems to be the only sane way for me to operate."
Originally from Nova Scotia, Leigh Anne earned her BFA from East Carolina University and MFA from Vermont College of Fine Arts.


text: Through the assistance of this forum I have definately decided a 48" deck will more practical for my application than the 44" I was considering. I hate to open up this next can of worms, but I want to be sure I have covered all the options. I have yet to see first hand the new Ferris 1000Z ZTR. The local dealer expects a new shipment in the next week or so. He has quoted me a price of $5,650 with the 48" 21Kaw engine. The Exmark with the 48" and 21 Kaw is about $300 more. If I knew for sure the Exmark was a much better mower I wouldn't hesitate spending the extra money and getting it. The Exmark dealer is about 20 miles closer to me. Has anyone had experience with the Ferris 1000Z and make any direct comparison between the two? I know that the Exmark is a quality machine. Thanks for any help. Wade


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AIOU Solved Assignment FA (Iqbaliat, 303-3rd) Autumn 2014 available now
Jan 022015
Allama Iqbal Open University (AIOU) solved assignment FA, Iqbaliat, Book code 303-3rd, Autumn-2014 is available now according to AIOU pattern, please visit following links for more AIOU assignments available on the website. Contact for assignment delivery on Helpline number. View more Solved assignments Autumn 2014
3 Responses to "AIOU Solved Assignment FA (Iqbaliat, 303-3rd) Autumn 2014 available now"
sir, pls send me code 303 assinment pls
iqbal ka majmoa hay kalam


text: NWIHA Regional Meeting | September 12-14 | Suquamish, WA 2017-09-13 14:22 - 2017-09-15 14:22 http://nwiha.org/events-2-2/ [{"latlong":["",""],"zoom":"9","infow":"<\/span><\/div>\n\t\t\t <\/label>\n\t\t\t 2017-09-13 14:222017-09-15 14:22 <\/span>\n\t\t <\/div>","google_map_url":""}] September 12-14, 2017 Clearwater Casino Resort Suquamish, WA September 2017 Annual Awards MORE INFORMATION In addition to our elections, at the upcoming quarterly meeting, we will be conducting our Annual Awards Ceremony at the banquet. Please nominate your colleagues and leaders for consideration by the NWIHA EC to receive one of our NWIHA Annual Awards. Download a nomination form by clicking here and then following the instructions to complete the form and submit it to NWIHA by the deadline indicated. Thank you. Related upcoming events NWIHA Regional Meetings | January 23-25 | Grey Wolf 2018-01-23 14:24 - 2018-01-25 14:25 Registration not open


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Cloud 9Cloud Nine Hair has been revolutionising the face of modern hair since 2009 when we launched our original Cloud Nine temperature control Iron, a new breed of hair straightener. Since then our new product launches have continued to stun the industry. The O in 2010 introduced a fantastic new volumising solution. More recently we launched The Wand, which gives unprecedented control and freedom when creating curls.
DelorenzoThe company, founded by brothers Vincent and Anton de Lorenzo remains a family owned and operated company. Staying true to its core values, it continues to develop and manufacture all of its products on Australian shores, proudly supporting Australian jobs and local economy when many of its competitors have since moved their manufacturing offshore.
Evoevo is an innovative, professional hair and beauty product manufacturer with individuality and integrity; a manufacturer that speaks the truth.
born from a desire to produce an exclusive salon range, evo steps outside the normal realm of truth-stretching invention in a 'wake up and smell the coffee' crusade of twisted honesty – designed to make people think. and so comes the catch phrase – saving ordinary humans from themselves™


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Y-Kids Are Fit
Moving Kids Towards a
Healthy Lifestyle
The YMCA of Central Ohio contracts with schools & organizations to provide the Y-Kids Are Fit curriculum or programs can opt to have their own staff implement the curriculum.
About the Program
Giving children the opportunity to participate in regular physical activity and to learn about healthy choices is what Y-Kids are Fit is all about. The focus of the program is on fitness, health and nutrition.
Created for use in after-school programs, the Y-Kids Are Fit user-friendly curriculum includes lessons for 30-45 minutes of physical activity per day (i.e. games, exercises, stretching, etc.) designed to be implemented 3-5 days per week. Youth learn about and experience hands-on activities relating to health and nutrition.
The Y believes that a healthy lifestyle is important for all ages and that the family's involvement is imperative to the program's success. Through the Y-Kids Are Fit program family component, families are encouraged to be active with their children. Leading a healthier lifestyle becomes a family-focused.Y-Kids Are Fit Curriculum
Y Kids Are Fit Physical Activity Cards
"I have benefited (from Y-Kids Are Fit) by being an exercise leader with my (after school) teachers. When I first started the program, I could only do 5 push-ups and now I can do 15 push-ups"
"One of my favorite things about YKAF are the cool games. Before we play games, we do stretches and exercises so we don't pull a muscle. YKAF has helped me do exercises and learn how to do them the right way."
"Y-Kids Are Fit program gives my daughters energy and endurance to carry on through their day. They come home and show me what they've learned in Y-Kids Are Fit. It's a learning experience for the whole family."
LinksFood & Fun - Harvard School of Public Health
Program Contact: Becky Ciminillo, 614 224 1142 Ext. 120 email@example.com


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In 2003, Pixar captivated audiences with a glimpse of the ocean that stuck with us for years. As Pixar started to make a couple sequels, people started clamoring to more of our favorite fish, and in June 2016, they gave us exactly what we wanted. Finding Dory is the movie that is great for kids who weren't around 13 years ago for the original, and a delight for the overgrown kids that were.
I remember watching Finding Nemo when I was little. I remember those spectacular seascapes that Pixar had crafted for the movie. I remember how authentic the characters felt, and I remember how connected I felt to them. I remember everyone talking about this movie, and I know for the last 13 years, we've all still been talking about it. Which is why it's so special that Pixar delivered such an incredible film. Finding Dory takes what was great from the first movie, and turns it up to 11.
One of the pitfalls of a sequel is the flanderization of characters and personalities. Flanderization is most certainly not an issue with Finding Dory, though. The returning cast of characters retain the personalities that made them so unforgettable, and added even more dimension to them. The new cast of characters manages to be just as charming, funny, and timeless as the originals. It's this fantastic troupe that Disney|Pixar use to guide through this new adventure. Characters like Hank the octopus and Bailey the beluga whale are standouts in a sea full of memorable creatures.
All of this is to say nothing of the breathtaking view that Pixar has been able to craft. In 2003, we knew that Pixar was capable of incredible CGI. They've proven that time and time again. What's remarkable is just how much they've improved on the original. The colors, textures, and just overall panoramic shots are absolutely stunning throughout the film. The way grains of sand flow and pieces of kelp move really do make it difficult to forget that this isn't real life. When these views are paired with the great score by Thomas Newman, you'll be hooked in the best way possible.
Best of all, the movie had heart. Pixar didn't forget how to make the kind of stories that made them absolute legends in the business; they gave us the story that will definitely go down as unforgettable. While, yes, it's a sequel, it's the best kind of sequel, the kind that truly adds value to the original and really deserves to have its story told. And boy am I glad I was there to see it!


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Marisol Bib Green Necklace
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Document's "first-party" takes ancestors into account.
I'd like to make a small change to the definition of "first-party" that we use for third-party cookie blocking: we currently look only at the top-level origin to determine the first-party origin for a request. I'd like to start walking the whole ancestor chain of a frame.
Status in Chromium
Enabled by default (tracking bug) in:
- Chrome for desktop release 45
- Chrome for Android release 45
- Android WebView release 45
- Opera release 33
- Opera for Android release 33
Consensus & Standardization
- Shipped
- No public signals
- No public signals
- Positive
Last updated on 2017-06-14


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Recently, one of the tools that I have been using to keep my life organized is Google Calendar. I have been tempted and tried many different apps for my calendar, to do list, menus, etc., and I keep coming back to the simplicity of Google Calendar. Google Calendar can be used across multiple devices, meaning that I can add, change, or delete something one place and it is changed on all my … [Read more...]
Using and Keeping a Calendar: The Foundation of a Peaceful Home
Using and keeping a calendar is one of the foundations of our everyday lives that will help to bring peace to our home. If we don't have a system in place to keep track of those events in our lives that are tied to a specific date, we will be overwhelmed trying to remember it all. Some families will have many more items that need to be placed on the calendar such as sporting events for the … [Read more...]


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Island of Blood Trailer (1982)
"The creep island slayings…" 08 January 1982 Horror 82 mins
A movie company is filming a murder mystery on an island. Soon afterwards cast and crew members start getting murdered, and the rest of the company must find out what is happening and who is behind it before they are all killed.
Watch the official Island of Blood 1982 trailer in HD below or find more Island of Blood videos on Vidimovie.


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Shopping is one of the best things that I like to do during my leisure time. I love to go in the shopping mall to buy clothing and other stuff but I'll prefer to shop online when it comes to Lingerie and perfumes. I am somewhat frustrated when I went to the mall because I was looking for extra large size lingerie but most of their lingerie is for petite size. Before I got married I belong in the petite size but when I got married, I become bigger and fat. Now, it is hard for me to find a larger size.
I was in a hurry to find a nice lingerie for some reason, I will be attending a wedding. My dress is some kind a sexy look and I need a good lingerie. Good thing my friend told me about Boudiche dot com. This website is selling luxury lingerie. They have wide selection of design that you can choose from. The one that I like is the Freya Lingerie. The Designer Lingerie is really nice and very stylish, even though I am fat but the fitting is perfect for me. These lingerie is so modern, sexy and elegant looking. The colors of the design are so nice, the floral print, embroidery is fantastic. If you are interested, don't forget to check out the Fayreform Lingerie too.
Now, I am able to find the right size for me because at boudiche.com, they offer different sizes of bras. They are available of plus size like up to D+ cup sizes. So if you are looking for designer lingerie, bra, French knicker and other lingerie; just visit at boudiche.com or simply click the preceding links above. Check them out now and pamper yourself with these sexy and stylish lingerie.


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A moderate level of competition exists among the key players in the global personal mobility devices market. As per the findings of Transparency Market Research (TMR), the top three players in the personal mobility devices market, Invacare Corporation, Hill-Rom Holdings, Inc., and Sunrise Medical LLC, together accounted for approximately 35.6% of the overall market in 2015. Presently, the industry comprises a few major players operating across each product segment. In the forthcoming years, TMR expects price wars to persist between leading players operating in the different market segments.
Spurred by the aforementioned factors, competition prevailing in the personal mobility devices market will intensify through the course of the report's forecast period between 2015 and 2023. Furthermore, threat of substitution and considerable bargaining power of buyers will possibly escalate rivalry among the key market players.
Read Full Report: http://www.transparencymarketresearch.com/personal-mobility-devices-market.html
"The rising geriatric population around the world has been a key target market for personal mobility devices," observes the TMR report's author. In India and China, the population of aged people is particularly expected to surge at an unprecedented rate through the forecast period. Aging is often associated with lower bone density, and, consequently, mobility in numerous cases. The prevalent social structure has led to a decline in long-term caregiving support from families. This creates an environment conducive to the expansion of the personal mobility devices market.
"Besides the geriatric population, caregiving services also become necessary for people with physical disabilities creating a window of opportunity for personal mobility devices," added the TMR analyst. As per annual report on Disability Statistics published in 2014, by the University of New Hampshire, the rate of disability increases with age. Thus the increasing number of people suffering from disabilities will fuel requirement of advanced personal mobility devices. The demand for these devices is also expected to surge in the near future in response to the favorable government regulations and increasing grants from the United Nations and other international organizations. The advent of latest technologies will also allow the market to pace up considerably.
Download exclusive Sample of this report: http://www.transparencymarketresearch.com/sample/sample.php?flag=S&rep_id=6715
However, the high cost of many of these mobility devices is hampering the market's trajectory to an extent. Due to their high cost, the sales prospects of these devices are compromised with in developing or underdeveloped economies. Furthermore, TMR has observed low acceptance for highly digitized mobility devices. This could be because of the limited utilization of computers by disabled individuals. Moreover, digitization is pushing up the cost of products, which limits the market's growth.
In addition, the social stigma attached to using mobility devices such as crutches, wheelchairs, and cranes could inhibit the market's expansion as well.
TMR expects that policies delaying the age of retirement will positively influence the sales of personal mobility devices. Implementing such policies would require a flexible working environment and adequate facilities for the elderly working in the organization. This in turn will increase the demand for personal mobility devices in the forthcoming years. Furthermore, the market is also expected to gain significant impetus from the increase in productive employment of people with disabilities.
The WHO has been encouraging governments across various nations to develop policies for assistive and mobility devices as a part of the strategy for human development to leverage untapped potential of the disabled population. Such regulations will augur well for personal mobility devices sales.
In terms of device type, the global personal mobility devices market was led by the medical mobility aids and ambulatory devices segment in 2015. On the basis of revenue, the segment is expected to surge at a CAGR of 8.5% from 2015 to 2023. Regionally, North America led the overall market with a share of 41.4% in 2014.
As per TMR, the global personal mobility devices market, which stood at US$6.65 bn in 2014, is expected to reach US$12.7 bn by the end of 2023. The market is thus expected to exhibit a CAGR of 7.30% between 2015 and 2023.
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Fixing the System Trailer (2015)
"An Unprecedented Look Inside the U.S. Prison System with President Obama." 24 September 2015 Factual 67 mins
Along with the trip to an Oklahoma federal prison with Obama, Fixing the System takes an in-depth look at all the interlocking pieces of the complicated criminal justice system, from prisoners and their families to the judiciary and community reformers.
Watch the official Fixing the System 2015 trailer in HD below or find more Fixing the System videos on Vidimovie.


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Mukti - details and analysis
× This information might be outdated and the website will be soon turned off.
You can go to http://surname.world for newer statistics.
The word Mukti has a web popularity of 10700000 pages.
What means Mukti?
The meaning of Mukti is unknown.
Web synthesis about this name:
...Mukti is a registered voluntary organisation striving to free the rural masses from ignorance.
Mukti is not in fact the salvation offered at calvary.
Mukti is having a waiting list of nearly 600 amputees.
Mukti is an entering into the immortality of the spirit.
Mukti is not reflected by them because those three kinds of mukti are not permanent.
Mukti is the transcendental state of perfection obtained after a jivan mukta leaves his body.
Mukti is the cessation of identification with individual samskara.
Mukti is wrong as an aim with such attachment is damaging.
Mukti is achieved atma and parmatma become one them how was he still separate when he said as is there were two.
Mukti is considered to be the inherent state of our existence.
What is the origin of name Mukti? Probably Indonesia or UK.Mukti spelled backwards is Itkum
This name has 5 letters: 2 vowels (40.00%) and 3 consonants (60.00%).
Anagrams: Muitk Kitum Kuitm Tkium Mkitu Ikumt Tiumk Tuikm Ukitm Tikum Mitku
Misspells: Muktti Mukty Muktia Mkuti Mukit Mutki
Image search has found the following for name Mukti:
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|Mukti Dhawan
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|Mukti Bosco
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Welcome to the future of treasure hunts. If you love the thrill of hunting for treasure and need to get your bearings in a new city – this iPad treasure hunt is the ideal activity for you and your group. Each team will be given puzzles, games and tasks to complete during a treasure hunt through central Poznan – using iPads to document your challenges, as well as look back over and laugh.
You'll have an English-speaking guide to accompany you throughout, as well as all the instructions you could need, as you battle it out to be the first team to complete the tasks and reign victorious over your mates. This is a cracking way to see the city, without having to trudge around on walking tours or freeze on an open top bus.
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Queen Anne's Lace - Cross Stitch Pattern
Lavender and Lace
$14.00 $12.59 + $3.29 shipping within the U.S.
In Stock. Usually ships in 1-2 business days.
Item: 9397 - Type: Cross Stitch Patterns.
Stitched on either 32 count Barnwood Linen or 32 count Dirty Belfast Linen with DMC floss, Kreinik #4 Braid and Mill Hill Beads. The stitch count is 186W x 279H.
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text:
S.1370 - Common Sense Medical Malpractice Reform Act of 2001107th Congress (2001-2002)
|Sen. McConnell, Mitch [R-KY] (Introduced 08/03/2001)
|Senate - Judiciary
|Latest Action:
|Senate - 08/03/2001 Read twice and referred to the Committee on the Judiciary. (All Actions)
This bill has the status Introduced
Here are the steps for Status of Legislation:
- Introduced
Subject — Policy Area:
- Law
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Summary (1)
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Summary: S.1370 — 107th Congress (2001-2002)All Information (Except Text)
There is one summary for S.1370. Bill summaries are authored by CRS.
Shown Here:
Common Sense Medical Malpractice Reform Act of 2001 - Declares that, in a health care liability action that is subject to this Act: (1) the action may not be initiated unless a complaint is filed within two years, with exceptions; (2) the amount of non-economic damages shall not exceed $250,000; and (3) an award for punitive damages may only be made if proven by clear and convincing evidence that the defendant intended to injure the claimant for a reason unrelated to the provision of health care services; understood the claimant was substantially certain to suffer unnecessary injury and deliberately failed to avoid such injury; or acted with a conscious disregard of a substantial and unjustifiable risk of unnecessary injury which the defendant failed to avoid in a manner which constitutes a gross deviation from the normal standard of conduct.
Introduced in Senate (08/03/2001)
Establishes additional limitations on punitive damages, including specified requirements for the pleading of punitive damages, and a requirement (at the request of any defendant in a health care liability action) that the trier of fact consider the issue of punitive damages in a separate proceeding.
Sets forth provisions regarding periodic payments, the scope of liability (the liability of each defendant shall be several only and not joint), mandatory offsets for damages paid by a collateral source, and a cap on attorney's fees (limited to 25 percent of any judgement or settlement recovered).
Encourages each State to establish or maintain ADR mechanisms. Directs the Attorney General to develop guidelines regarding such mechanisms.