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|20 Reviews
|Property type
|Minimum Stay
|3 - 4 nights
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3 bedroom 2 full baths, and newly remodeled waterfront home, with great view and peaceful Puget Sound setting gives guest peaceful sense of serenity. Just steps to our beach. Enjoy the view and roast marsh mellows at our fire pit. Enjoy a family barbeque on our deck with the gas barbeque. Large private master suite with two sinks, jetted tub, open shower, private toilet room, vanity with a wake up view of water and Mount Rainier from a new Queensize bed . Two bedrooms on main level with 1 new Queen bed, and a sleeper with comfortable upgraded mattress. The living room is large, with an area for an additional Aero pillow top bed. There is a fireplace, and ajoining dining room and completely remodeled gourmet kitchen that is fully provisioned with spices, dishes, condiments, basics for baking and cooking. There is a large deck, facing the water and mountain view. Young children and adults will love beach combing for treasures from the sea. Just 25 min south of Gig Harbor.
My husband and I bought our beach home because we love being on the water. We are sailors, and have a sailboat that we bring to our property, and park on our bouy which is right in front of our home, when we are there, during the summer moths. We decided to share our home with VRBO renters after a very successful experience with our West Seattle VRBO RENTERS. We have 24 reviews, and all 5 star. Please refer to listing number 399739 to get reviews from our recent vacationers at West Seattle. Since we have just listed our Longbranch home, we hope these reviews will give you an idea of how we maintain our homes, as well as our character. We do strive for our vacationers to have a memorable private, and enjoyable family vacation.
Scott VandeZande or Jan Place purchased this house in 2010
The sense of peacefulness, quiet, and beauty of the northwest surrounds us while staying at our home. The incredible sunrises, as well as when the sunsets, bring a warm orange cast on the snow capped Mount Rainier throughout the year.
Our home has been completely remodeled, so all appliances are new, including remodeled baths, new carpet, and a beautifully decorated, comfortable home, that most would not consider a beach home, but instead, a beautiful home at the beach. The wall to wall windows provide an incredible view from nearly every room. For summertime, there is a large deck with gas grill, as well as a fire pit for roasting marsh mellows. Just steps away to the beach for kayaking, and swimming.
I am not sure how Scott and Jan engineered it but we saw Mount Rainier every day...
Jan and Scott were delightful to work with, personalities that carried into the atmosphere of the house.
The home was lovely and well supplied and beautifully decorated. The electronics all worked well, the views of the water, walks on the beach all lovely. The kitchen was full of great utensils and appliances and the beds were very comfortable. Our adult children had a great time out in the canoe, biking and enjoying the outdoor fireplace.
I could go on but suffice it to say this will be a memorable holiday that will surely become part of the family lore. We will be back.
Searching for a place on the water that would accommodate our grown children from Seattle to Eugene -- luck was with us! We booked for 5 nights, with family coming and going for the 4th of July weekend. It was perfect! Amazing views of the Sound, right on the water so easy to canoe, kayak; or beach walk, bike on the local roads, or just relax on the large deck! The home is in terrific shape, beautifully decorated yet fitting for a home away from home. Previous reviewers were "right on" with the well-stocked, awesome kitchen. Jan and Scott were lovely to work with and very accommodating without being intrusive. We will look forwarding to returning.
Arriving from Sweden we spent 4 nights at this wonderful house and spectacular setting. We had beautiful weather which offered us a full view of Mt. Rainier and sitting on the front deck in the sun with no coats on. The kitchen has everything you will need for easy cooking or gourmet cooking. Cozy fireplace for evening relaxing. A beautiful view from the master bedroom offers a perfect start to the day. We hiked on the beach, enjoyed quiet time with computer or book and basked in the sun with no wind. It was a extremely pleasant stay and we can highly
recommend this for a quiet, beautiful and serene escape.
Let me start with the magnificent view of Mt. Rainier and the water! It was absolutely splendid! The house is well decorated, clean, comfortable and very "homey". We made our traditional turkey dinner for Christmas in the nicely stocked kitchen (spices, flour, sugar, etc. were all there to use). We had a fire going in the fireplace almost the entire time. The beds were super comfortable and the sheets, linens, etc. were all clean. It is also worth noting that the owner, Jan, was so easy to work with and very friendly. We are already talking about coming back this summer so we can enjoy the beach when the weather is warmer. If I could give more than 5 stars I would.
The house is beautiful, well taken care of and extremely comfortable. The view is spectacular and every need was met. The house had no let downs and the owners are so kind and Extremly helpful. I will be staying there again. Thanks again!
We are in love with this house and location! It was the perfect site for a family getaway with our young adult children and their significant others. Just enough out of the way so that we could spend time together, but plenty to do. Right on the water and because it's on a high bank beach (as they say in Puget Sound) we had stunning views of Mt. Rainier, and of the harbor seals that hauled out every day onto the raft below. Lovely home with plenty of room, great outdoor space, BBQ and a couple of small boats to row around in. We had perfect late summer weather and made great family memories. Thank you!
Just steps to our beach, and minutes from our home is a boat launch. We have a bouy in front of our home, where you can tie your boat. Or if you prefer, there is a Marina in Fulicy bay, just a mile from our waterfront home. Penrose State park is minutes away. There is a local winery for those that enjoy wine tastings. Side trips to Olympic National Park, Hood Canal, Mount Rainier, Mount Saint Helens, Port Townsend, Olympia , Seattle, Bremerton, and Gig Harbor are all points of interest. During the summer there are many local festivals. During the month of August we have delicious blackberries, pears, and plums growing on our property.
|Rate Period
|Weekend Night
Summer rates
May 15 2016 - Sep 15 2016
4 night minimum stay
Standard rate
Sep 16 2016 - Nov 14 2016
3 night minimum stay
Holiday rate
Nov 16 2016 - Dec 31 2016
4 night minimum stay
|Property Damage Protection
|Cleaning fee
|$200 + Tax
|Refundable damage deposit
|Pet Fee
|Tax Rate
Beautiful beach home with view of water and Mount Rainier from nearly every room.


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Sun Title Agency, one of Michigan's leading title and real estate transaction coordination companies, announced today the opening of the company's new Grandville office in a converted, 3,000 sq. ft. space at 4600 Ivanrest. The transformed space replaces the company's current location at 4693 Wilson Ave SW.
The converted space, which was formally a residential home, will accommodate as many as six employees. The new office will focus on providing commercial and residential title insurance and closing services and allows the company to expand its presence in the Grandville community.
About two years ago, I received an e-mail asking if I was interested in getting a Trivia Question on a daily basis? I was very skeptical with all that can happen with a computer when replying to the wrong person or site. Anyway, I replied with a YES and since that time, I get one or two Trivia Questions each day. In fact, not only do I get the questions, I usually receive another e-mail that gives information about subjects that are very interesting.
I look forward to learning something new each day by answering these questions without cheating. No looking it up on GOOGLE before answering. The site keeps track of my answers and my score stays up in the upper corner where I can see it. Why not try this? firstname.lastname@example.com
Let me know what you think about this site and if you enjoy it?
The public is invited to check out sky objects above, 2 Saturdays a month through October at the James C. Veen Observatory.
Visitors have the opportunity to experience the night skies on Saturday, June 25 from 9:30pm to midnight with Veen Observatory telescopes and member's equipment. The night sky will feature Mars, Jupiter, Saturn & deep Sky Objects such as nebulas, galaxies & star clusters.This is also a chance to tour the facility & check out the audio visual presentation about the Observatory & the GRAAA.
This Public Night takes place only if the sky is clear. Starting times vary with the sunset. It is a quarter mile walk from the parking area to the top of the hill. If need, ask the lot attendant about assistance to the Observatory.
James C. Veen Observatory is located at 3308 Kissing Rock Ave. SE in Lowell Twp., MI 49331.
An anticipated 2,000 participants will bike through scenic West Michigan on June 25 for the fourth annual MSU Gran Fondo. The non-competitive cycling event benefits MSU College of Human Medicine's skin cancer awareness, prevention and research.
Italian for "big ride," cyclists of all skill levels will ride 12, 25, 40 or 80 miles. Some participants are avid cyclists with a passion for these types of "mass-start" events, while others are riding to honor a loved one – or their own – battle with skin cancer.
Cyclists can participate as individuals or teams and raise money to fight skin cancer. All fundraisers have a chance to win prizes including a commemorative jersey, a spot on cycling legend Bob Roll's honorary cycling team, road and cruiser bikes, racing wheels, a Herman Miller Aeron Chair and more.
The State Park in Grand Haven comes to life with incredible kite performances this weekend. Don't miss the fun!
For the 28th year, the world's best kite flyers - individuals, pairs & teams will be performing continuously on Saturday, May 21 from 10am – 5pm & Sunday, May 22 from 11am – 5pm.
Kite enthusiast and their families are invited to experience different size kites with amazing capabilities controlled by professional kite handlers. There will also be an Open Flying Field to fly your own kite.
Grand Haven State Park is located at 1001 Harbor Avenue in Grand Haven, Michigan 49417.
Call: 1-866-428-2335 or visit www.mackite.com for more information.
If you enjoy the planets & the stars above, this is a Saturday evening event that you don't want to miss.
The community is invited from 9:30pm – midnight on Saturday, April 30 to observe the night sky at the astronomical education & research center. The James C. Veen Observatory houses several equipped telescopes to explore the featured objects, such as Jupiter, nebulas, galaxies, & star clusters.
This Public Night takes place only if the sky is clear. Starting times vary with the sunset. It is a quarter mile walk from the parking area to the top of the hill of the Observatory, ask the lot attendant for assistance, if needed.
Admission: Adults - $3, Kids 17 & under - $2, under 5 & GRPM Members – Free. There will be more Public Nights, every month through October 2016.
The GRAAA is offering beginning amateur astronomer's the opportunity to learn resources that can assist with stargazing in their backyard, along with what to look for in the sky. Specialist of the Grand Rapids Amateur Astronomy Association will give important observing aids to help find your way around the sky during this afternoon class.
This session takes place on Saturday, February 13 at 1:00-3:00pm at Schuler Books and Music located 2660 28th Street SE in Grand Rapids. The hour long session will be followed by a Telescope Clinic at 2:15pm.
Visit www.graaa.org or call – (616)942-2561 for more information.
Ferris Coffee & Nut Company
Peter M. Wege Health & Learning
WZZM 13, 645 Three Mile, Grand Rapids, MI 49544
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Copyright 2016

subset: commoncrawl


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Courts in Barnstable County will be closed on Monday, February 11, as the region continues to recover from Nemo.Read More »
Lobbyists prevail in suit vs. Galvin
Asserting that part of Secretary of State William F. Galvin's arguments made "absolutely no sense," a Superior Court judge has ruled in favor of lobbyists who challenged Galvin's interpretation of a requirement that they disclose their "direct business associations" ...Read More »
Refusal to foreclose on abandoned home OK
A mortgage lender did not violate the Bankruptcy Code's discharge injunction by refusing to foreclose or otherwise take title to a residence belonging to debtors who sought to surrender the home, the 1st U.S. Circuit Court of Appeals has determined.Read More »
Courts to close at noon
Trial courts throughout the state are slated to close at noon today due to the the winter storm.Read More »
Bulger Timeline
The passage of time is just one of the many challenges facing the lawyers in the James "Whitey" Bulger prosecution, as the facts of the case will require jurors to sift through more than three decades worth of evidence. Key ...Read More »
Pedal mettle
Jason & Fischer served as lead ride sponsor at the Bike4Life Boston ride. Andrew M. Fischer participated in the fundraiser for LivableStreets, an organization that advocates for a better balance of walking, biking and public transit to create safer streets ...Read More »
Man's brain cyst goes undiagnosed for two years
The plaintiff, a male in his early 40s, went to his primary care physician with complaints of memory loss. A CT was ordered and interpreted by a radiologist.Read More »
2012 Lawyers of the Year
Introducing our 2012 Lawyers of the Year Frances S. Cohen, Bingham McCutchen John H. Cunha Jr., Cunha & Hilcomb Thomas J. Frain, Thomas J. Frain Law Office William F. Lee, WilmerHale Ashlee N. Logan, Essex ...Read More »
Criminal – Probation – Revocation – Hearsay
Where a Juvenile Court judge revoked a youthful offender's probation and committed him to the custody of the Department of Youth Services until his 21st birthday, the revocation order was justified by evidence that the offender committed armed robbery and assault by means of a dangerous weapon.Read More »
Motor vehicles – RMV – Mootness – Immunity
Where the registry of motor vehicles revoked and later restored the plaintiff's driver's license, a Superior Court judge correctly concluded that the plaintiff's subsequent claim for injunctive relief was moot, his claim for damages was barred by sovereign immunity, and his action was otherwise infirm as a matter of law.
Tagged with: RMVRead More »


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(Since this is a christian movie, it tackles a lot of different issues on sin)A woman wears tight fitting outfits throughout the film.In one of the skits where "satan" a character on a TV show sings, 2 women wear short outfits.In a dream sequence the main character (who is amish) see's the girl he loves getting dressed (behind a tree with no view of her) in amish clothing. This is very funny and no nudity is shown.Roy (when he was drunk the night before) left his wallet at Tiffany's house. She brings it to him and says "You left this at my place big boy." Everyone thinks that he slept with her, we later find out that he passed out by the time they arrived at her house and that nothing happened.Tiffany is released from prison for "good behaviour" and one man says "I think it had something to do with the warden's bachelor party." Showing that Tiffany is a temptress.
(Most of the violence in the film is comical)Two women have a catfight, this is comical, and one of the women has a black eye, and the other has a small cut under her eye.When Roy get's a job at a Hot Dog place and goes "a little crazy" he is tackled by the police, this is also very comical.Roy's uncle runs over 2 men with his car. Then the men get up and they aren't hurt, they only have a few cuts in their clothing. The 2 men were trying to steal Roy's money. This scene is also comical.
A little mild name calling, but no swearing or blasphemy.
Roy's uncle (who has lost his way as a christian) smokes throughout.Roy's uncle is a drug addict and he buy's cocaine and Roy thinks it's flower.Roy see's his uncle taking drugs and asks what it is but his uncle says it's medicine. Roy then wants to take some but his uncle stops him.Roy get's "bullied" into trying a rhode island ice tea, and he gets drunk. This scene though is very comical.
Nothing Frightening or Intense. This is a christian film with a good lesson, but I'd say it's safe for anyone 12 and up.
Find showtimes, watch trailers, browse photos, track your Watchlist and rate your favorite movies and TV shows on your phone or tablet!


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Single Black Sneaker
Credit: Jonathan Kantor
Creative #: 93338754
License type: Royalty-freeRoyalty-free pricing is based solely on the size of the product you need, not the specific use. You don't have to pay any additional royalties on a use-by-use basis. Once you license a royalty-free product, you may use it multiple times for multiple projects without paying additional fees. (Pornographic, defamatory, libelous or otherwise unlawful use of any image is, of course, prohibited.)
Collection: Photodisc
Release info: No release required
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Single Black Sneaker Stock PhotoBlack Color,Close-up,Comfortable,Fashion,No People,Photography,Protection,Reflection,Single Object,Sports Shoe,Studio Shot,Vertical,White BackgroundPhotographer Jonathan KantorCollection: Photodisc Jonathan Kantor Studio


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The NF-κB transcription factor plays a central role in diverse processes, including inflammation, proliferation and cell survival, and its activity is dysregulated in diseases such as auto-immunity and cancer. We recently identified the TRE17/ubiquitin-specific protease 6 (USP6) oncogene as the first de-ubiquitinating enzyme to activate NF-κB. TRE17/USP6 is translocated and overexpressed in aneurysmal bone cyst (ABC), a pediatric tumor characterized by extensive bone degradation and inflammatory recruitment. In the current study, we explore the mechanism by which TRE17 induces activation of NF-κB, and find that it activates the classical NF-κB pathway through an atypical mechanism that does not involve IκB degradation. TRE17 co-precipitates with IκB kinase (IKK), and IKK activity is augmented in stable cell lines overexpressing TRE17, in a USP-dependent manner. Optimal activation of NF-κB by TRE17 requires both catalytic subunits of IKK, distinguishing its mechanism from the classical and non-canonical pathways, which require either IKKβ or IKKα, respectively. TRE17 stimulates phosphorylation of p65 at serine 536, a modification that has been associated with enhanced transcriptional activity and nuclear retention. Induction of S536 phosphorylation by TRE17 requires both IKKα and IKKβ, as well as the IKKγ/NEMO regulatory subunit of IKK. We further demonstrate that TRE17(long) is highly tumorigenic when overexpressed in NIH3T3 fibroblasts, and that inhibition of NF-κB significantly attenuates tumor formation. In summary, these studies uncover an unexpected signaling mechanism for activation of classical NF-κB by TRE17. They further reveal a critical role for NF-κB in TRE17-mediated tumorigenesis, and suggest that NF-κB inhibitors may function as effective therapeutic agents in the treatment of ABC.
NF-κB is a ubiquitous transcription factor that plays a central role in innate immunity, inflammation, cellular proliferation, survival, and differentiation (Gilmore, 2006; Karin, 2006). This transcription factor comprises five family members, Rel (c-Rel), RelA (p65), RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52), which form various homo- and heterodimeric complexes to regulate the transcription of numerous target genes, including cytokines, components of the cell cycle machinery, and genes that control proliferation (Hacker and Karin, 2006; Rollins, 2006). In addition to its role in these physiological processes, dysregulation of NF-κB has been documented under pathological conditions, such as autoimmune disease and cancer.
NF-κB is typically divided into classical and non-canonical pathways, which are distinguished by the NF-κB subunits activated, the upstream regulatory proteins involved, and their biological functions. The classical pathway is associated with innate immunity and the inflammatory response, and is activated by agents such as tumor necrosis factor α (TNFα) and interleukin-1 (IL-1). Classical NF-κB consists of p65/p50 heterodimers or p65 homodimers, which are kept inactive in the cytoplasm through their association with Inhibitor of NF-κB (IκB). Agonists induce phosphorylation of IκB by IκB kinase (IKK), triggering its ubiquitination and proteasomal degradation, with subsequent translocation of p65/p50 to the nucleus. IKK consists of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ/NEMO. Both IKKβ and NEMO, but not IKKα, are required for phosphorylation and degradation of IκB.
The non-canonical pathway is associated with B cell differentiation and a distinct array of agonists, such as B-cell activating factor (BAFF) and lymphotoxin β. The NF-κB subunits in this pathway are RelB and p100/p52. Agonists of the non-canonical pathway induce phosphorylation of p100 by IKK, leading to its processing to the p52 form, and nuclear translocation with RelB. Notably, IKKα, but not IKKβ or NEMO, is essential for activation of this pathway.
While classical vs. non-canonical NF-κB are commonly depicted as described above, increasing evidence suggests a higher degree of complexity and crosstalk between these pathways. For example, the p100 gene is a direct target of classical NF-κB. In addition, recent work indicates that in mouse embryo fibroblasts with targeted deletion of IKKβ, IKKα can mediate IL-1-induced activation of classical NF-κB in a NEMO-dependent manner (Solt et al., 2007). Another example of atypical IKK subunit involvement is exemplified by a study showing that reovirus requires IKKα and NEMO, but not IKKβ, for activation of classical NF-κB (Hansberger et al., 2007).
Numerous components of the NF-κB pathway are regulated by poly-ubiquitination (Harhaj and Dixit, 2011). While some modifications occur through lysine 48 (K48) ubiquitin linkages, others arise through lysine 63 (K63)-linked chains, which promote the formation of signaling complexes. Multiple de-ubiquitinating enzymes (DUBs), including A20, CYLD, Cezanne, USP21, and USP15, have been shown to function as negative regulators of NF-κB (Brummelkamp et al., 2003; Kovalenko et al., 2003; Trompouki et al., 2003; Wertz et al., 2004; Harhaj and Dixit, 2011). However, only one DUB, TRE17/ubiquitin-specific protease 6 (USP6), induces NF-κB activation (Ye et al., 2010).
The TRE17 locus is translocated in aneurysmal bone cyst (ABC), an aggressive bone tumor characterized by inflammation and destruction of the surrounding bone (Mankin et al., 2005; Mendenhall et al., 2006; Saccomanni, 2008). The majority of ABC cases harbor rearrangement of TRE17, resulting in high level expression of wild type TRE17 (Oliveira et al., 2004a; Oliveira et al., 2004b; Oliveira et al., 2005). We recently undertook studies to determine the mechanism by which TRE17 contributes to ABC pathogenesis. The cells harboring TRE17 translocation/overexpression are believed to be pre-osteoblasts or fibroblasts (Oliveira et al., 2004a; Oliveira et al., 2004b; Oliveira et al., 2005), leading us to examine the effects of TRE17 overexpression in this cell type (Lau and Oliveira, 2010; Ye et al., 2010). We recently reported that TRE17 induces the expression of matrix metalloproteinase 9 (MMP9) through activation of NF-κB (Ye et al., 2010). In the current study we examine the mechanism by which it functions, demonstrating that TRE17 activates classical NF-κB through an atypical mechanism that is independent of IκB degradation. We further demonstrate that NF-κB is a critical effector of TRE17 in tumorigenesis.
We recently reported that TRE17 activates NF-κB, in a manner dependent on its USP activity (Ye et al., 2010). However, the mechanism by which TRE17 activates NF-κB and the selective involvement of the classical vs. non-canonical pathways were not examined. To address these issues, we initiated studies in two distinct cell culture models, HeLa cells and stable MC3T3-E1 cell lines. HeLa are human cervical carcinoma cells that endogenously express low levels of TRE17 (Martinu et al., 2004). MC3T3-E1 (referred to hereafter as MC3T3) are murine pre-osteoblasts, the cell type in which TRE17 is translocated/overexpressed in at least a subset of ABCs. MC3T3 cell lines stably expressing TRE17 in a doxycyclin (dox)-inducible manner were previously generated, and shown to recapitulate multiple features of ABC when xenografted into nude mice (Ye et al., 2010), validating it as a useful model for dissecting TRE17's functions in ABC pathogenesis.
To determine whether TRE17 activates classical or non-canonical NF-κB, electrophoretic mobility shift assays (EMSAs) were performed. As shown in Figure 1A (left panel), expression of the full length TRE17 isoform, TRE17(long), in HeLa cells induced activation of NF-κB. This NF-κB complex was supershifted by antibodies against classical NF-κB subunits p65 and p50, but not non-canonical subunits RelB and p52. To ensure that antibody against non-canonical NF-κB was functional, we confirmed its ability to perturb NF-κB activation induced by overexpression of NIK, an activator of the non-canonical pathway (Figure 1A, right panel).
We next examined the composition of NF-κB complexes in the MC3T3 stable cell lines. Addition of dox induced expression of HA-TRE17(long) (Figure 1B, left panel), with concomitant activation of NF-κB (Figure 1B). Interestingly, only antibody against p65 but not p50 caused a supershift, indicating activation of a p65 homodimer. We also tested a point mutant of TRE17 that ablates its de-ubiquitinating activity (TRE17(long)/USP-) (Figure 1B). This mutant failed to activate NF-κB above levels seen in control samples (Figure 1B). Furthermore, as with HeLa cells, we confirmed that TNFα activated classical NF-κB in control vector-expressing MC3T3 cells (Figure 1B, right panel). The major TNFα-induced complex in these cells was composed of p65 homodimers, rather than the p65/p50 heterodimers commonly seen. This led us to consider whether the p65 homodimers induced by TRE17(long) in MC3T3 might reflect an atypical classical NF-κB response in this cell line.
To explore this issue, we examined NF-κB complexes in NIH3T3 cells, where TNFα has been shown to activate p65/p50 heterodimers. Stable NIH3T3 cell lines expressing TRE17(long) or TRE17(long)/USP- in a dox-inducible manner were generated (Figure 1). EMSA confirmed USP-dependent activation of NFκB by TRE17 (Figure 1C). Supershift analysis revealed that the TRE17-induced complexes were composed of p65 and p50 (Figure 1C).
As an independent means of confirming selective activation of classical NF-κB, we examined the subcellular localization of NF-κB by immunofluorescence confocal microscopy. In both transiently transfected HeLa (Figure 1D) and stable MC3T3 cell lines (Supplementary Figure 1), TRE17(long) induced nuclear accumulation of p65, but not of non-canonical NF-κB, as monitored by p100/p52 staining. In contrast, a constitutively active mutant of IKKα stimulated translocation of p100/p52 to the nucleus (Figure 1D). In sum, these data indicate that TRE17(long) selectively activates classical NF-κB, consisting of either homo- or heterodimeric p65 complexes, in three different cell types.
In resting cells, classical NF-κB is sequestered in the cytoplasm by Inhibitor of NF-κB (IκB). Upon agonist stimulation IκB is phosphorylated by IKK triggering its proteasomal degradation. Since TRE17(long) induced activation of classical NF-κB, we anticipated that it would induce IκB degradation. However, immunoblotting revealed that steady state levels of IκBα and IκBβ, the two major IκB isoforms, were comparable in TRE17(long) and control cells (Figure 2A). Given this discrepancy, we analyzed IκB processing in greater detail. Following initial agonist-induced degradation of IκB, activation of NF-κB leads to re-synthesis of IκB within several hours. Thus, transient downregulation of IκB might have been missed due to the chronic nature of TRE17(long) expression in our system (i.e. over a 24–48 hour time period). To address this issue we exploited our dox-inducible expression system to monitor IκB phosphorylation and degradation upon acute TRE17(long) expression. As seen in Figure 2B, a time-dependent increase in TRE17(long) expression was observed from 8 to 24 hours of dox treatment. However, no consistent phosphorylation or reduction in total levels of IκBα or IκBβ was observed in TRE17(long)/MC3T3 (Figure 2B). In contrast, TNFα induced phosphorylation and degradation of IκBβ, but not IκBα, in MC3T3 cells (Figure 2B). Probing of TNFα-treated HeLa extracts confirmed that the phospho-IκBα antibody was functional (Figure 2B).
As an alternative approach to unmasking TRE17's potential effects on IκB degradation we tested the effects of cycloheximide (CHX) to monitor the stability of the pre-existing pool of IκB in the absence of de novo synthesis. Both IκB isoforms were highly stable in control and TRE17(long)/MC3T3, with comparable levels present throughout the 2 hour time course of CHX treatment (Figure 2C). As a positive control, we confirmed that TNFα caused a significant reduction in the half-life of IκBβ but not IκBα (Figure 2C). In aggregate these data indicate that TRE17(long) activates classical NF-κB through a mechanism that does not appear to involve phosphorylation and degradation of IκB.
This uncoupling of p65 activation from IκB degradation prompted us to further characterize how p65/IκB complexes are regulated by TRE17. Immunoprecipitation of p65 followed by IκB immunoblotting revealed that TRE17 did not induce dissociation of p65 from either IκB isoform (Figure 2A, right panels). Nevertheless, TRE17 induced the accumulation of nuclear p65 which was free of IκB (Figure 2D). We speculated that the only means by which this could occur is if there was an IκB-free pool of p65 in resting cells. Supporting this notion, we detected a population of p65 that was resistant to immunodepletion using anti-IκB antibodies (Figure 2E). Notably, TRE17 did not increase levels of IκB-free p65, suggesting that it does not function by stimulating dissociation of p65 from IκB.
The findings above suggest that TRE17(long) may activate NF-κB by regulating nuclear translocation of an IκB-free population of p65. To explore how this might occur we examined whether TRE17(long) associates with the IKK complex, which consists of the catalytic subunits IKKα and IKKβ, and the regulatory subunit NEMO. TRE17(long) co-immunoprecipitated with endogenous IKKα but not control IgG in MC3T3 cells (Figure 3A). Association did not require USP activity, since TRE17(long)/USP- bound at levels comparable to the WT protein (Figure 3A). Similarly, TRE17(long) tagged with either HA or GST co-immunoprecipitated with endogenous IKKα in transiently transfected HeLa cells (Figure 3B). To confirm this association, the reciprocal immunoprecipitation/blot was performed. As seen in Figure 3C, IKKα and IKKβ were present in anti-HA immunoprecipitates from TRE17(long) but not vector-expressing control cells.
We next sought to determine which IKK subunits were required for activation of NF-κB by TRE17. In the classical pathway, IKKβ and NEMO, but not IKKα, typically mediate activation of p65 complexes by inducing IκB phosphorylation and degradation. However, since IκB degradation was not elicited by TRE17, it was unclear which IKK subunit(s) might be required. To address this question, siRNA-mediated depletion of IKK was performed in HeLa cells. Specific knockdown of IKKα, IKKβ, and NEMO by their respective siRNAs was confirmed (Figure 4A). Depletion of each subunit significantly inhibited TRE17(long)'s ability to induce NF-κB activation and p65 nuclear translocation (Figure 4B).
The contribution of IKKα in particular was surprising, since it is largely viewed as dispensable for activation of classical NFκB. We therefore confirmed that this result was not due to non-specific effects of IKKα depletion on IKKβ or NEMO function. Indeed, in the absence of IKKα, TNFα was able to elicit normal degradation of IκB and phosphorylation of p65 on serine 536 (see below for further discussion of this regulatory site) (Figure 4C). These results indicate that IKKβ, NEMO and, unexpectedly, IKKα all contribute to activation of NF-κB by TRE17(long). Thus, TRE17(long) functions through a novel mechanism distinct from classical and non-canonical NFκB activators, which typically require IKKβ or IKKα, respectively.
The requirement for all IKK subunits raised the possibility that TRE17 induces activation of the complex. To test this, in vitro kinase assays were performed. MC3T3 extracts were immunoprecipitated with anti-IKKα, then subjected to in vitro kinase assays using recombinant I B or a fragment of p65 as substrates. As seen in Figure 5A, IKK activity was elevated in TRE17(long)/MC3T3 relative to vector control cells. This required TRE17's de-ubiquitinating activity, since TRE17(long)/USP- failed to induce IKK activation (Figure 5A).
While IκB served as a convenient in vitro substrate for the kinase assays, our results above indicated that it is not targeted by TRE17 in vivo, leading us to explore p65 as a potential substrate. Serine 536 (S536) of p65 is a major regulatory site within the carboxy-terminal transactivation domain (Viatour et al., 2005). Phosphorylation of S536 has been associated with enhanced nuclear localization of p65, decreased association of p65 with co-repressors, enhanced association with co-activators, and elevated transcriptional activity (Yang et al., 2003; Hu et al., 2004; Mattioli et al., 2004; Viatour et al., 2005; Hoberg et al., 2006). TRE17(long) induced phosphorylation of S536-p65 in both the MC3T3 stable cell lines and transiently transfected HeLa cells (Figure 5B,C).
We next assessed the contributions of the IKK subunits to S536 phosphorylation induced by TRE17. Previous studies have indicated that a number of kinases, including IKKα and IKKβ, can mediate phosphorylation of this site in vivo (Sakurai et al., 1999; Sizemore et al., 2002; Sakurai et al., 2003; Buss et al., 2004; Viatour et al., 2005). As seen in Figure 5C, TRE17-induced phosphorylation of p65 was significantly attenuated in cells depleted of each of the IKK subunits. To confirm the functional specificity of the siRNAs, we determined that NEMO but not IKKα was required for p65 S536 phosphorylation in response to TNFα (Figures 4C and and5C).5C). Together, these data suggest that TRE17 activates NF-κB by promoting S536 phosphorylation of p65, in manner that optimally requires IKKα, IKKβ, and NEMO.
Our results showing that TRE17 overexpression induces NF-κB activation and p65 phosphorylation is relevant for understanding its pathological functions in ABC, which is a disease arising from TRE17 overexpression. However, we also sought to determine whether TRE17 modulates NF-κB activation in response to physiological agonists. Knockdown of TRE17 in HeLa cells using SmartPool siRNA partially attenuated p65 phosphorylation in response to TNFα and LIGHT, an agonist for the lymphotoxin β receptor (Figure 5D). Inhibition of NF-κB was further confirmed by EMSA and blotting for nuclear translocation of p65 (Figure 5E, F). Thus, TRE17 also participates in agonist-mediated NF-κB activation when expressed at native levels.
We recently demonstrated that overexpression of TRE17(long) in MC3T3 osteoblasts induces tumor formation in nude mice, in a USP-dependent manner (Ye et al., 2010). Interestingly, our results contradicted an earlier report suggesting that when overexpressed in NIH3T3 fibroblasts, the truncated, catalytically inactive TRE17(short) isoform is tumorigenic, but TRE17(long) is not (Nakamura et al., 1992). To determine whether this difference arises from the use of different cell types, we examined the tumorigenic potential of our NIH3T3 cell lines. As seen in Figure 6A, TRE17(long)/NIH3T3 were highly tumorigenic, with all injected animals exhibiting palpable tumors visible as early as 5 days post-injection. In contrast, no tumors formed in mice injected with NIH3T3 cells expressing TRE17(long)/USP-, vector control NIH3T3, or TRE17(long)/NIH3T3 in the absence of dox (Figure 6A and data not shown). These results thus refute initial reports suggesting that USP activity inhibits TRE17's tumorigenic potential in NIH3T3 (Nakamura et al., 1992), and instead confirm our recent work in MC3T3 cells demonstrating that USP activity is essential for TRE17's oncogenicity (Ye et al., 2010).
We next examined whether activation of NF-κB by TRE17 was required for tumorigenesis. We performed these studies in NIH3T3 rather than MC3T3 cells, since the former yielded much larger tumors, providing a wider dynamic range to assess the effects of NF-κB inhibition. Our studies above indicated that depletion of any of the IKK subunits might block activation of NF-κB. However, we encountered difficulties with sustained and efficient knockdown in NIH3T3 cells (unpublished observations). Although IκB degradation is not normally elicited by TRE17, we speculated that overexpression of the IκB Super Repressor (IκB-SR) might nevertheless effectively sequester p65 and block NF-κB activation by TRE17(long). IκB-SR is an amino-terminal truncation mutant lacking the IKK phosphorylation sites, and has been used as a potent, agonist-resistant inhibitor of classical NFκB (Brockman et al., 1995). IκB-SR was introduced under the control of a dox-inducible promoter into the TRE17(long) cell line (Figure 6B). IκB-SR completely abrogated activation of NF-κB by TRE17 as determined by EMSA (Figure 6C).
Xenografting of the TRE17(long)/IκB-SR cells in parallel with the parental TRE17(long) cells revealed that inhibition of NF-κB significantly attenuated tumor formation (Figure 6D). The average mass of TRE17(long)/IκB-SR tumors was approximately 10% that of TRE17(long) tumors (0.83 g for TRE17(long); 0.07 for TRE17(long)/IκB-SR). However, equally striking to the reduction in tumor mass was the virtually complete inhibition of tumor vascularization (Figure 6E). These results demonstrate that NF-κB is a critical effector of TRE17 in tumorigenesis.
Here we demonstrate that TRE17 activates canonical p65-containing NF-κB through an atypical mechanism that is independent of IκB phosphorylation/degradation. TRE17 associates with IKK and promotes its activation, leading to phosphorylation of p65 on S536. Our data suggest that TRE17 targets a pool of p65 that is free of IκB. Optimal phosphorylation of S536 requires both IKKα and IKKβ, distinguishing TRE17's mechanism from the proto-typical classical and non-canonical pathways. We further demonstrate that NF-κB is a critical effector of TRE17 in tumor formation. Inhibition of NF-κB dramatically attenuates tumor growth and vascularization, indicating that this may be a highly effective avenue for treatment of ABC patients.
Our finding that TRE17 activates classical NF-κB in the absence of IκB degradation, and in a manner requiring both IKKα and IKKβ, adds to the growing list of stimuli that activate NF-κB through mechanisms distinct from simplified depiction of the classical pathway (Hansberger et al., 2007; Solt LA, 2007). Interestingly, it has been shown that agonists such as glutamate, angiotensin II, lysophosphatidic acid, and thrombin activate p65 NF-κB by inducing S536 phosphorylation, independently of IκB downregulation (Douillette et al., 2006; Choudhary et al., 2007; Sitcheran et al., 2008; Sun et al., 2009). Additional studies have shown that IKKα and IKKβ can phosphorylate a pool of p65 that is not bound by IκB (Sasaki et al., 2005), and we suggest TRE17 functions by such a mechanism. S536 phosphorylation of p65 has been reported to promote its nuclear retention, enhance its association with the transcriptional co-factors, inhibit its association with co-repressors, and potentiate activation of NF-κB target genes.
We recently reported that activation of NF-κB by TRE17 is mediated in part by RhoA and ROCK (Ye et al., 2010). Previous work from others has shown that RhoA and ROCK can promote p65 S536 phosphorylation (Khandaker et al., 2004; Shimada and Rajagopalan, 2010). However, we found that ROCK inhibitors did not significantly affect TRE17's ability to induce S536 phosphorylation (unpublished observations). Thus, our current results suggest that TRE17 functions through multiple parallel pathways to activate NF-κB, one of which involves interaction with IKK, a core regulatory component of the pathway. It remains to be determined if this interaction is direct, and how it contributes to IKK activation. However, TRE17 does not appear to directly activate IKK activity (Supplemental Figure 2). TRE17's USP activity is not required for association with IKK, but is required for NF-κB activation. Thus, one possibility is that TRE17 promotes interaction of IKK with an upstream positive regulator, perhaps by de-ubiquitinating it and rescuing it from proteasomal degradation. It is most likely that TRE17 targets a protein modified by K48-linked rather K63-linked poly-ubiquitin, since all K63-mediated ubiquitination events described thus far in the pathway promote NF-κB activation. Nevertheless, it remains possible that TRE17 acts on an as-yet identified K63-modified protein which functions as a negative regulator of NF-κB. Given this complexity, identifying the substrate of TRE17's USP domain and determining how it induces the activation of IKK remain goals for the future.
Our xenograft studies establish that USP activity is critical for TRE17's tumorigenic potential. Two prior studies yielded contradictory results: one, performed using MC3T3 cells, demonstrated TRE17(long), but not the catalytically inactive point mutant, induced tumor formation (Ye et al., 2010). The other study utilized NIH3T3, the cell line used in our current work, yet arrived at the opposite conclusion regarding the role of TRE17's USP activity in tumorigenesis. The reason for this discrepancy is unclear. However, it is noted that the earlier studies did not confirm expression of the different TRE17 isoforms (Nakamura et al., 1992). Furthermore, their tumors arose with much greater latency than we observed (4–6 weeks vs. 5–14 days).
Our study establishes NFκB as an essential effector of TRE17 in tumorigenesis. Although TRE17 does not normally elicit IκB degradation to activate NF-κB, overexpression of IκB-SR served as an effective tool to extinguish NF-κB activation in TRE17(long)/NIH3T3 cells, and revealed its requirement in tumor formation. TRE17(long)/IκBSR tumors were not only significantly smaller but also much less vascularized. It remains to be determined how inhibition of NF-κB contributes to these responses. It does not appear to be required for cell survival in vitro (Supplemental Figure 3), but may function cell autonomously to promote proliferation of the xenografted cells. Another possibility, which is not mutually exclusive, is that NF-κB is required non-cell autonomously. NF-κB has been shown to regulate production of vascular endothelial growth factor (VEGF), a key physiological regulator of angiogenesis. Whether NF-κB plays a direct role in angiogenesis, or functions indirectly by regulating proliferation of the TRE17(long) cells remains to be determined.
ABC is a tumor for which there are few treatment options, in part because little is known about its pathogenesis (Mendenhall et al., 2006; Cottalorda and Bourelle, 2007). They are typically treated by surgical curettage, however recurrence is relatively common. Furthermore, ABCs can arise in inoperable locations. Thus, alternative modes of treatment would be of great value. Our work strongly suggests that inhibitors of NF-κB might serve as effective therapeutic agents for the treatment of ABC. A variety of NF-κB inhibitors are being avidly explored for the treatment of cancer, autoimmune and inflammatory diseases (Pahl, 1999; Orlowski and Baldwin, 2002; Dobrovolskaia and Kozlov, 2005; Hacker and Karin, 2006; Rollins, 2006; Calzado et al., 2007; Lee and Hung, 2008). Our studies reveal that all three IKK subunits are required for TRE17-mediated activation of NF-κB, suggesting that inhibitors for any of them might be efficacious for management of ABC.
HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin, streptomycin, and fungizone. Transfections were performed with Lipofectamine 2000 (Life Technologies, Inc.) or Fugene (Roche Biochemicals) according to the manufacturer's instructions. MC3T3-E1 (hereafter referred to as MC3T3) pre-osteoblastic stable cell lines that express TRE17 alleles in a doxycyclin (dox)-inducible manner have been described previously (Ye et al., 2010). MC3T3-derived lines were grown in α-Modified Eagle Media with tetracycline-free FBS, penicillin/streptomycin, sodium pyruvate, and GlutaMax. All cell lines were maintained in 5% CO2. NIH3T3 cell lines expressing TRE17 in a dox-inducible manner were generated, using the methods previously described (Ye et al., 2010). NIH3T3-derived cell lines were grown in DMEM containing 10% calf serum and the supplements described above.
TRE17 constructs have been described (Masuda-Robens et al., 2003; Martinu et al., 2004; Shen et al., 2005). c-myc-NIK was provided by Dr. Jing Hu (University of Pittsburgh Medical Center). GST-p65 (354–551) was provided by Hiroaki Sakurai (Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd.). Purified recombinant GST-IκB was provided by Dr. Sergei Fuchs (University of Pennsylvania School of Veterinary Medicine). IκB-SR cDNA (provided by Dean NF-KB. Ballard, Vanderbuilt University) was subcloned into pRevTRE (Clontech) prior to introduction into the TRE17(long)/NIH3T3 cell line.
Antibodies against p65 (sc-372 and sc-8008), p100/52 (sc-7386), IKKα (sc-7218), IKKγ/NEMO (sc-8330), IκBα (sc-847 and sc-371), IκBβ (sc-945), HDAC2 (sc-7899), and actin (sc-8432) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against IKKα (#2682), IKKβ (#2678), IKKγ/NEMO (#2695), phospho-serine 536 p65 (#3031), phospho- IκBα (#9246) and phospho-IκBβ (#9245) were purchased from Cell Signaling Technology. Affinity purified anti-TRE17 antibody was generated against the N-terminus of TRE17. TNFα (210-TA) and LIGHT was purchased from R&D Systems.
Cells were harvested in ice cold PBS, then resuspended in hypotonic lysis buffer (10 mM HEPES, 0.1 M EDTA, 10 mM KCl, pH 7.9), and supplemented with 1 mM DTT, 5 mM β-glycerophosphate, and protease inhibitors) and incubated for 30 min on ice. Nonidet P-40 was added to a final concentration of 0.1%, and extracts were incubated for 5 min at room temperature. Samples were centrifuged at 375 × g for 10 min at 4°C. The supernatant (cytoplasmic fraction) was collected, and the cell pellet (containing nuclei) was resuspended in ice cold nuclear extraction buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 5 mM β-glycerophosphate, and protease inhibitors), incubated for 30 min on ice, then centrifuged at 18,000 × g for 20 min at 4°C. The supernatant containing nuclear proteins was used for EMSA or western.
EMSA was performed as described (Ye et al., 2010). Antibodies for supershift assays were purchased from Santa Cruz Biotechnology: p65 (sc-372X), p50 (sc-7178X), RelB (sc-226X), and p52 (sc-7386X). For supershift assays, antibodies were incubated with nuclear extracts for 40 min prior to the addition of labeled oligonucleotide. Complexes were separated by electrophoresison 5% native polyacrylamide gels, and subjected to autoradiography.
Cells were seeded, transfected, and treated as previously described (Martinu et al., 2004). Samples were viewed on a Zeiss confocal microscope with LSM510 software, using excitation wavelengths of 488 nm (FITC), 546 nm (Cy3), or 358 nm (DAPI).
Cells were lysed in Lysis Buffer (50 mM HEPES (pH 7.2), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 5 mM β-glycerophosphate, 10% glycerol) supplemented with leupeptin, aprotinin, pepstatin, and 10 mM N-ethylmaleimide (NEM) and pelleted at 16,000 × g for 10 min at 4°C. The clarified supernatant was immunoprecipitated with the indicated antibody for 4 hours to overnight. Beads were washed three times in Lysis Buffer then separated by SDS-PAGE and blotted as indicated.
Cells were treated as indicated, then lysed in Lysis buffer. Extracts were immunoprecipitated with anti-IKKα (Santa Cruz Biotechnologies, Inc; sc-7218) overnight at 4°C, then washed three times in Lysis buffer, and twice in kinase buffer (25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 5 mM β-glycerophosphate, pH 7.4). Immunoprecipitates were incubated at 30°C for 30 minutes in kinase buffer containing 10 μCi [γ-32P]-ATP and substrate. GST fusions of IκB or a fragment of p65 encompassing amino acids 354–551 (Sakurai et al., 1999) were purified from E. coli; 0.5 μg of each was used as substrate. Reactions were terminated by boiling in sample buffer, and subjected to SDS-PAGE and autoradiography.
SMARTpool siRNAs targeting IKKα (#M-003473-02), IKKβ (#M-003503-03), NEMO (#M-003767-02), and TRE17 (M-006096-03) were purchased from Dharmacon RNA Technologies (Lafayette, CO). As a negative control, non-specific siRNA (#D-001210) was used. HeLa cells were subjected to two rounds of transfection with the indicated siRNAs using Oligofectamine (Invitrogen) according to manufacturer instructions. Cells were subsequently transfected and treated as indicated.
Mouse procedures were performed in accordance with the institutional animal care and use committee of the Children's Hospital of Philadelphia. Nude mice (Jackson Laboratories, Bar Harbor, ME, USA) were fed water with dox (1 mg/ml containing 5% sucrose) for 1–2 weeks before xenografting. NIH3T3 cells were pre-treated with dox for 1 day before subcutaneous injection (2.5E6 cells per site), using 4–7 mice per cell line, per experiment. Dox was maintained in the drinking water.
This work was supported by the National Institutes of Health Grant RO1 CA081415 (to M.M.C.) and Grant P30 AS050950 (Penn Center for Musculoskeletal Disorders). This work was also supported by the National Service Research Award Grant F31CA126488 (to L.M.P.).
The authors declare no conflicts of interest.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Sixty percent of Americans now take prescription drugs—more than ever before. This not only creates unprecedented problems for municipalities whose water filtration systems were developed before wide drug use—but for marine life. Continue reading
Sixty percent of Americans now take prescription drugs—more than ever before. This not only creates unprecedented problems for municipalities whose water filtration systems were developed before wide drug use—but for marine life. Continue reading
Did you ever wonder why new medications so often debut right after awareness of the condition they treat increases? It is no coincidence. The tactic is called unbranded advertising and "disease awareness," and drug companies spend more on it than they do for regular advertising. Continue reading
Padded bras for kindergarteners with growing breasts to make them more comfortable? Sixteen percent of U.S. girls experiencing breast development by the age of 7? Thirty percent by the age of 8? Clearly something is affecting the hormones of U.S. girls—a phenomenon also seen in other developed countries. Girls in poorer countries seem to be spared—until they move to developed countries. Continue reading
In April, Pennsylvania became the 24th state to legalize medical cannabis, a form of the plant popularly known as marijuana. That makes nearly half of US states. A major barrier to broader legalization has been the federal law under which all cannabis—even the very useful form known as industrial hemp—is classed as a Schedule I controlled substance that cannot legally be grown in the US. But that classification could change soon. In a letter sent to federal lawmakers in April, the US Drug Enforcement Administration said it plans to release a decision on rescheduling marijuana in the first half of 2016. Continue reading
Just as the public is digesting the fact that former chairman and CEO of drug giant Genentech, Art Levinson, is now the CEO of a new Google life sciences venture with Big Pharma and that he also serves as chairman of Apple Inc., there are more insidious "partnerships" between Pharma and top corporations. Walgreens has now announced a "partnership" with Mental Health America, an advocacy group so steeped in Pharma money, it was investigated by Congress. Continue reading
Despite limited advances provided by the Affordable Care Act, the U.S. healthcare system remains "uniquely wasteful" and profit-driven, leaving tens of millions without any insurance and even more underinsured. Continue reading
Forgetfulness. Falls. Adding a new prescription or over-the-counter drug to address problems that are side effects of a previous drug. It is an increasingly common problem says a new book from Hazelden because people are taking more drugs than ever before and not always aware of their side effects and interactions. This "polypharmacy" can produce everything from falls and accidents to behavior that is quickly termed "dementia" in the elderly even when it is clearly from drug effects. The problem is compounded by doctors not always aware of what other doctors are prescribing a patient and the very addictive nature of many popular drugs today. Continue reading
Big Pharma received $127 billion of our tax dollars in 2014 through the federal programs Medicare, Medicaid, VA, and TRICARE. But just because they live on our tax dollars, doesn't mean Pharma companies want to pay taxes. Increasingly, they seek tax inversions, reincorporating in countries like Britain, Ireland or the Netherlands, often merging with a European entity to duck U.S. taxes. Continue reading
From mercury in tuna and wood pulp in parmesan cheese to ground beef treated with ammonia to retard E. coli ("pink slime"), the press does a good job exposing the dangerous and deceptive practices of Big Food. The problem is, the public forgets about the food risk or contamination, assuming that reform is in the works and that is just fine with Big Food. Often nothing changes. Continue reading
Information on the important documentary to be released online April 12 states, "CANCER WILL KILL NEARLY 8 MILLION PEOPLE THIS YEAR." Continue reading
I recently (2016) underwent radiation for breast cancer so I titled this piece "Act II" because this is the second round of radiation for me. The first episode occurred in 2000 for treatment of inoperable throat cancer so I consider myself fortunate that this was not a recurrence of squamous cell cancer of the neck, but a different type of cancer to breast tissue. The breast cancer is a ductal cell type and according to the surgeon and radiologist at Seattle Cancer Care Alliance they are not related. Continue reading
Asthma is big business for Big Pharma. Advair was the third best selling drug in the world in 2013 and the asthma drugs Singulair and Symbicort were also blockbusters. So it is no surprise the prospect of a high-tech injectable drug that stops an allergic response by binding to immunoglobulin E (IgE) made Big Pharma sit up and take notice. Continue reading
After an 18-month investigation into the high cost of Gilead's hepatitis C drug Sovaldi—initially listed at $84,000 for a course of treatment or $1,000 per pill—the Senate Finance Committee said the prices did not reflect the cost of research and development and that Gilead cared about "revenue" not "affordability and accessibility." That sounds like an understatement. Sovaldi and the related pill Harvoni cost Medicare and Medicaid more than $5 billion in 2014, charged senators. Continue reading
They are so common no one thinks twice about them: drug ads that tell you about a disease you might have, a pill that could treat it, and tell you to "ask your doctor" if the pill is right for you. Continue reading
It is no secret that our bodies and our environment are swimming in estrogen. Puberty is occurring in children as young as eight and, in 2010, babies in China were reported to be developing breasts. In 2011, the United Kingdom's Daily Mail observed that women's bra cup sizes were growing even when the women themselves were not gaining weight and speculated it was estrogen exposure. And frogs and fish are becoming "intersex" and losing their male characteristics from endocrine disrupters in the environment and waterways. Continue reading
You would think the drug company that made the best selling drug in the world—Lipitor—and the blockbuster Viagra would appreciate the country and patients who have been so good to it. You'd be wrong. Continue reading
Consumers, safety activists, Big Food, biotech companies and many of the US's importing and exporting partners have been closely watching to see if the FDA would approve the genetically engineered AquAdvantage Salmon, which it did late in 2015. Of course unlabeled GE crops are eaten by millions and GE animals have been created to make human drugs largely under the public radar. Still the AquAdvantage Salmon is the first approved GE animal destined for the US dinner table. Continue reading
Most people couldn't name one animal drug used to produce their food. Yet conventionally produced US meat is grown with antibiotics, vaccines, anti-inflammatory drugs, hormones and other chemicals, most of which people would want to avoid if they were on the label. Continue reading
A House of Representatives Bill, short titled "Vaccinate All Children Act of 2015," has been referred to the Subcommittee on Health and is awaiting committee action. Continue reading
When the mainstream corporate media raise alarms over the rising costs of health insurance and major problems involving the misnamed Affordable Care Act (a.k.a. Obamacare), you can be sure corporate interests are threatened. Since the open enrollment period for 2016 ACA policies began on November 1, National Public Radio, the New York Times, CNN, etc., all supporters of Obamacare, have issued warnings couched in typical reassuring double-talk about developing problems. Continue reading
The advent of direct-to-consumer advertising made billion dollar blockbuster drugs possible for the first time. Often the drugs were rushed to market before their side effects were fully known or admitted like the cardiac side effects with COX-2 inhibitors like Vioxx and osteonecrosis of the jaw (ONJ) side effects with bone drugs like Fosamax and Boniva. Continue reading
For an example of how Big Money in politics is causing real harm to average Americans, look at the practice of dentistry in this country. Continue reading
Americans should be the healthiest people in the world with our advanced medical care. But thanks to direct-to-consumer advertising, they are some of the sickest in the world suffering from seasonal allergies, depression, mood disorders, dry eye, GERD, perimenopause, adult ADHD, sleep/wake disorders, restless legs, high cholesterol, thinning bones and spectrum disorders which may or may not even have symptoms. (Like people with "epilepsy spectrum disorder" who never had a seizure). Continue reading
If you'd like to meet someone who is truly "part of the solution," someone who understands the problems of American health care in a way that few politicians do—and someone who is putting her money where her mouth is to get us healthier—meet Esther Dyson. Continue reading
How much of what we believe about the "safety" of chemical and drug products is planted in journals by industry itself? A lot! Continue reading
If you've watched all of the presidential debates so far—both Democratic and Republican—and you were waiting for the candidates to tell you if and how they would change Medicare, you are still waiting. Continue reading
The years between the debut of direct-to-consumer drug (DTC) advertising in the 1990s and passage of the Physician Financial Transparency Reports (Sunshine Act) in 2010 were a kind of "Wild West" for the drug industry. Continue reading
Minnesotans who get health insurance through the individual market last week became the latest in the country to get unwelcome news: the cost of their coverage will likely go up significantly next year. According to the state's Commerce Department, which regulates insurance in the North Star state, some customers of Blue Cross and Blue Shield of Minnesota may see their premiums jump nearly 50 percent beginning January 1. Continue reading
Presidential candidates from both parties are full of sound and fury about various aspects of the U.S. health care system, but unless we as a nation get serious about big money in politics, all the noise will ultimately amount to nothing. Continue reading
Beneath a three-column headline in my local newspaper was a barely-edited press release. Continue reading
I periodically meet John at the rehabilitation center where I receive occupational therapy for my arthritic fingers, and where he receives treatment for the stroke he suffered several years ago. The stroke left his right arm and leg paralyzed. Continue reading
Health insurers have been telling us for years that their Medicare Advantage plans, which are federally funded but privately run alternatives to traditional fee-for-service Medicare, can provide better care—at lower cost—than the government. Continue reading


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Ronix 2016 One Cable Front Zip Comp Vest
Four Way Stretch Neoprene – A material that moves in any direction you do.
Synthetic PU Foil
2 Buckles
1 Zip Closure
Non Coast Guard Approved
Water Resistant Liner – Featuring a liner that greatly reduces water seepage.
Coast Guard Approved:NoMaterial:Neoprene, Polyurethane (PU)Warranty:1 Year Learn More
Ronix Bandwagon Camber ATR Wakeboard 2015
Regular Price: $479.99
Special Price: $299.99
Ronix 2016 Krush
A hot off the press new shape with a thinner profile for reduced swing weight in the air, and more contact/feel with the water. Along with a new rockerline that naturally puts you underneath the board in a more centered position. The innovative new Krush recognizes that your body is crossed up riding toeside, and more inline heelside, and every design aspect of this board takes this into consideration for the proper building block of wakeboarding.Learn More
Ronix 2016 Dempsy Wakesurf Bag
Twice as much padding as any of our board bags. We want to make sure your surf constructed Honeycomb Powertail can get there safely.
Super durable padded shell
Shoulder strap
Fin protector
External pocket
2 sizes: Pointed nose up to 5ft / Round nose up to 6.2ft Learn More
Ronix 2016 Hex Shell - The Blender Wakesurf (PRE ORDER)
Hex Shell Construction//
For 2016, we created a torsionally stiffer board using a new material we call Hex Shell. This layup removes any delay
that a rider might have on the next move, for the quickest response of any construction in our line. Created for a rider looking for a new molded construction reducing the reaction time between surfer and board in a couple of our latest shapes. Time is money – and this new core takes away any of the downtime in surfing. Learn More
Ronix 2016 Code 55 Boot
Generate superior board control rocking these customizable sex machines. Incredible response driven by our most supportive asymmetrical liners wrapped in our Brainframe chassis. Maximize full potential board control whether you are leaning on a nose press or charging hard into the liquid kicker. Great looking, unbelievably comfortable, and high performance boots. These will get the job done, wherever your riding takes you. Key features being the brainframe technology, intuition moldable foam, dual density double inected "rad" footbed foam with ronix's baseless tech, along with feetbelts, and a new lacelock system, Dean Smiths pro model boot will not dissapointLearn More
Ronix 2016 Koal with Technora Thruster Wakesurf
to ride without a rope - this is the fastest and hardest charging line of boards we have created in our many swells of producing high end surfers. Thought you knew what a wakesurfer was capable of? This electric Thruster is hands down the most responsive surfer we have ever tested. Need more bite in your bottom turn with a quicker release off the peak? This stealth creation comes with 10 adjustable fin slots and enough fins for over a hundred different customizable combinations.This speedy squash tail deck was conceptualized by a legend who grew up switching between salt and freshwater carves - Parks Bonifay.
Koal With Technora//
Another new creation for this year - we added the response of Technora to our standard surf core. A more reactive laminate than a traditional fiberglass construction. A blend of our Koal Classic surfer layup, along with a new lively material, bridging the gap with our ultra responsive new Hex Shell construction. Designed for a rider looking for a blend of a smooth response, while still being able to bury the rail, or air off the lip.
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Ronix 2016 Preston Boot
An amazingly well rounded boot sits on top of our state of the art Brainframe hardware. Support is focused below the ankle to create a solid connection with hollowed ankle cutouts that allow you to move freely in any direction, while still feeling locked in. Our most affordable high performance Brainframe boot. A perforated softer liner creates enough side to side mobility for a well rounded riding journey. KEY FEATURES + Brainframe Technology + Stage 3 Liner + 3-D Molded Tongue + Built In J-Bars + New Lace Lock + Shota Tezuka Pro ModelLearn More
Ronix 2016 Super Sonic Space Odyssey Powertail Wakesurf (Pre Order)
A new line of surfers giving kids a reason to unite behind the boat. 2 shapes with the same technology as our most popular boards,The Powertail and The Fish, packed down to a 3'9" size. Designed and approved by the Wifi generation of surfers.
Combining the stable aspects of a Longboard, the bottom turn drive of a Thruster,and the ability to air out like no other kid's board out there.There has never been a board with this much surface area in the tail for bigger airs off the top deck, and can easily be ridden behind smaller size boat wakes. Learn More
Ronix 2016 Network Boot
High end support and the acclaimed Ronix fit in an open toe construction. The most supportive, yet still adjustable performer in our line-up built with our Stage 3 foam. Footwear that delivers more confidence to the rider and control to the board - yet still has the versatility of fitting multiple feet sizes. With a molded 3-D tongue and using the finest high end foam ingredients this open toe boot proves that you deliver performance and adjustability into one. You'll never need to roam outside of your Network again. KEY FEATURES + The Original Baseless Hardware + Stage 3 Liner + 3-D Molded Tongue + Built In J-Bars + Chad Sharpe Pro ModelLearn More


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Thank you so much for visiting our site! We would love to hear from you if you are interested in booking a session or just getting more information please send us a contact form from you!
When people write stories of two people fitting, I feel like Chandler and Jesse would be the leading roles to play this script. The heart they share for one another is uncanny. It illuminates from their heart and shines so brightly in the way they smile, laugh and love one another so deeply & unconditionally. When you watch them you kind of just stand in awe of not only their zeal for life, but the way they give of themselves to the ones who surround them.
It is never about Jesse & Chandler.
© 2016 Unveiled Radiance Photography|ProPhoto Photography Theme Design by Northfolk & Co.


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Top Definition
a contraction of the words "it is all"
Dun worry! Sall good, bro!
by RoboSpy March 12, 2004
Trending Now
It is all
S'all good mate.
by beanlord32 March 13, 2011
past tense of see often used instead of saw.
"did you see that" "yeah I sall that"
by bluebug22 December 22, 2006
meaning it's all good used when you messed up on something
ex: daym yo i juezs messed up buh itsz s'all good!
by dabastashytupinhere May 13, 2003
The worst feeling in the world created by the devil himself, when you get sand stuck on the side of your balls and you want to roll over and die when you attempt to walk.
I was playing in the sand and got a serious case of salls.
by Polish chicken July 13, 2016


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Ratings based on 92 Verified Reviews
They paid and stayed. We double-checked.
4.2 Room cleanliness
4.1 Service & staff
4.0 Room comfort
4.2 Hotel condition
Expedia Verified ReviewsThey paid and stayed. We double-checked.
by Princesita
Posted Apr 28, 2016 on Hotels
The bed was great but smaller that I expected, I don't know if their queen is smaller.
by Laurie
Posted Apr 26, 2016 on Hotels
by A verified traveler
Posted Apr 6, 2016 on Hotels
I basically just slept there while working all day elsewhere. It was great for that. Neighborhood was not so great. Decent restaurants were scarce in the area. But, I ate my meals where I was working at. Very comfortable and quiet which is most important for me.
by A verified traveler from M
Nice hotel near the airportPosted Apr 5, 2016
Last minute booking. Staff was accommodating and price was beyond reasonable. Shuttle was fast and the driver was kind. Facility was very nice
by A verified traveler
Posted Mar 9, 2016 on Hotels
by A verified traveler
2nd Year to stay.Posted Feb 13, 2016
Pros: 24 hour shuttle, full kitchen, location to the airport.
Location: Not much close by, use the hotel van to get to restaurants or by food and cook at the hotel.
This was my second year to stay a week in this hotel for work related training. Last year I had quite a bit of trouble with the hotel shuttle and almost didn't stay here again because of it. I decided to give it another try again this year and was much happier with the hotel shuttle. The shuttle was much more reliable and on time. I had no issues over the week scheduling it or having to wait an excessive amount for it to arrive, like last year. The hotel staff is very nice and accommodating.
by Graham E.
Room are not cleanPosted Dec 29, 2015 on Orbitz
Rooms are clean only once a week and male staff rude , don't respond to needs when required.
by A verified traveler
Smoking room.Posted Nov 16, 2015 on Hotels
I booked a room with a single bed and was given a smoking room with two beds.
by Darrell from Fort Worth, Texas
Low Quality MotelPosted Aug 12, 2015 on Hotels
Cost not reflective of hotel you would expect! Neighborhood not safe, high fence with bobbed wire at top gives prison feel, room not reflective of one purchased it was small and cheap looking. Afraid to leave anything in car, sleeping under the covers was not even a consideration as health issues were a major concern. Hole in wall with picture frame barely covered the hole. I purchased this room to come to a funeral of family member at last minute and had no time to change hotels which I surely would have done!!!!!!......not a guilty hotel at all!
by A verified traveler
Poor value for money, subpar servicePosted Jul 20, 2015 on Hotels
I am not the pickiest hotel guest. All I generally ask is that a room be clean and as advertised, the facilities work, hotel staff at least appear interested in doing their jobs, and that the wifi works (even if it's slow.) First, I booked a queen bed suite, which said it came with a sleeper sofa. It comes with a recliner. Second, the room was small, and not a "suite." It's a studio with a kitchenette. Again, deceptive room description. Third, one of the front desk staffers was rude and seemed annoyed at being asked for anything. Fourth, getting your (very small) trash can emptied is an ordeal. Set it outside at night, or good luck getting it emptied during the day. Fifth, one day the water turned brown and stayed that way all day. Don't know why, but unacceptable nonetheless. Finally, spotty and inconsistent wifi which persisted throughout my stay. Often unusable even for basic web browsing. I didn't try any streaming--what would be the point? Overall, hotel was a poor value for the money. Could've paid half as much for this kind of service. The free laundry (washer and dryer) was a nice amenity. I will give them that. The location is also very close to the airport with relatively direct access to downtown Houston.


text:
Randy Mayor
4 servings (serving size: 3 ounces)
Nutritional Information
Calories 165
Caloriesfromfat 27 %
Fat 5 g
Satfat 1.5 g
Monofat 2.1 g
Polyfat 0.9 g
Protein 24.7 g
Carbohydrate 3.9 g
Fiber 0.2 g
Cholesterol 79 mg
Iron 1.5 mg
Sodium 269 mg
Calcium 14 mg
1 (1-pound) pork tenderloin
2 teaspoons brown sugar
1/2 teaspoon five-spice powder
1/4 teaspoon salt
1/8 teaspoon ground red pepper
Cooking spray
1 tablespoon hoisin sauce
1 tablespoon orange juice
1/2 teaspoon dark sesame oil
Preheat oven to 400°.
Trim fat from pork. Combine sugar and next 3 ingredients (sugar through pepper). Rub pork with spice mixture.
Place pork on a broiler pan coated with cooking spray; insert a meat thermometer into thickest portion of tenderloin. Bake at 400° for 10 minutes. Combine hoisin, orange juice, and oil in a small bowl; brush over tenderloin. Bake for an additional 15 minutes or until thermometer registers 160° (slightly pink).
July 2000