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We stayed here as part of botched timeshare exchange at another location. We were only at this property for 2 days but it was a nice change from the place we had originally booked. Looking at some of the recent reviews for this property it just reinforces that expectations are everything.
We come to Hawaii for the weather, beaches and activities. It's true the beach in front of the hotel isn't great but there are so many great beaches on Maui we'd never expect to stay at the hotel the whole time anyway. It is a good beach for watching whales (in season) and green sea turtles. Likewise pools are very low on our priority list. The one time we used the pool it was late afternoon and there were plenty of chairs.
Overall the property is great and well taken care of. There are two pools and a number of sundry shops. We didn't have a chance to try any of the onsite restaurants. The little interaction we had with the staff was very positive.
We had a two bedroom that was perfect for the 3 of us (wife and 12 year old daughter). The bed was okay but the mattress wasn't the most comfortable. The only issue was that some of the living room furniture was a little dated. Nothing horrible just old. The lanai had a good setup for having breakfast and watching the whales.
Our room was on the 7th floor well back from the ocean but we still had a better view than many "ocean front" rooms we've had at other locations.
There was ample parking much of which was in a garage. There's a grocery store and a handful of restaurants just across the street
For a family this place is ideal with all the space, full kitchen and laundry facilities. I'm not sure I'd pick it for a romantic getaway though. We would definitely stay here again.
- Official Description (provided by the hotel):
- Located beachfront on Kaanapali Beach, this magnificent condominium resort combines spacious, well-appointed suites with hotel-caliber services. Enjoy the beauty of expanse ocean views and direct access to the beach. Amenities include complete kitchens, WiFi, onsite cultural programs, an activities desk for off-site arrangements, and a year-round kids program, Camp Kaanapali. This self-contained property boasts 2 swimming pools, a beachfront restaurant and bar, fitness center and spa featuring an indoor/outdoor massage service. All of these great amenities and services combined with an exceptional guest service staff make for an unforgettable vacation and the experience of true Aloha Spirit. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Hotels.com, Sabre Hospitality Solutions, Ctrip TA, TripOnline SA, Hotwire, Cancelon, getaroom.com, HotelQuickly and Evoline ltd so you can book your Aston Kaanapali Shores reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Aston Hotel Lahaina
- Aston Hotel Kaanapali Shores
- Kaanapali Shores Maui
- Maui Aston Kaanapali Shores Condominiums
- Aston Kaanapali Shores Condos
- Maui Resortquest Kaanapali Shores
- Kaanapali Shores Hotel
- Resortquest Kaanapali Shores Maui
- Kaanapali Shores Resort
- Aston Kaanapali Shores Maui/Lahaina


text:
Product code: 27313
Despatched in 3-5 days.
You can trust Simple Baby to care for your baby's delicate skin.This Simple Baby Moisturising Wipes contain no artificial perfume or colour, no unnecessary ingredients and are alcohol free.With a perfect blend of chamomile and moisturising goodness that helps to cleanse and soothe your baby's delicate sensitive skin.Perfect for even the most sensitive skin.
Open pack and remove wipes, unfold and gently cleanse your baby's skin. Ensure that the pack is properly sealed to prevent wipes drying out.After use, dispose of wipes in bin.Do not flush down the toilet.
No artificial perfume or colour.No unnecessary ingredients so it won't upset your skin.No Tears Formula.Dermatologically & Paediatrician Tested and Approved.Suitable for even newborn babies.Alcohol Free.Hypoallergenic.pH Balanced.
Aqua, Phenoxyethanol, Paraffinum Liquidum, Carbomer, Benzoic Acid, Coco-Glucoside, Sodium Hydroxide, Dehydroacetic Acid, Disodium EDTA, Panthenol, Bisabolol, Pantolactone, Anthemis Nobilis Oil.
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subset: commoncrawl


text: Im interested in hear some opinions on where people stand for who they absolutely cannot stand on the current WWE roster. I see a lot of dissatisfaction with how WWE's creative handles things from most of you (including myself) so I want to go into even more detail and see what WWE superstars you feel need to hear the words "YOU'RE FIRED!!!!!" Feel free to elaborate if you wish. For me its Ryback. Cannot stand "The Big Guy"


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City hotel located at Blumenau the economic hub of Vale. The architecture of the German tradition, its flowerbeds, cleaning the streets, meshes, porcelains and crystals appreciated abroad as well as the nature that surrounds the city provide enchanting landscapes. All this combined with the services of a high standard and comfort of the hotel.
This hotel is located in an area full of German tradition. It lies directly in the centre of Blumenau, only a few metres from shopping facilities and a few blocks from the 15 November Street, the commercial centre of the city. The hotel is near the center of commercial and financial activities of the city that promotes Oktoberfest - the second largest beer festival in the world. Blumenau is the economic hub of Value.
The apartments come with an en suite bathroom and are equipped with a direct dial telephone, cable TV, a minibar and centrally regulated air conditioning.
There is a pool and a gym on offer.
Room Facilities:
Direct dial telephone
Double bed
Individual airconditioned


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SCPosted in South Carolina September 30, 2016
The South Carolina Restaurant In The Middle Of Nowhere That's So Worth The Journey
When it comes to remote restaurants, you can't get much further away from civilization in South Carolina than on an island you can only get to by boat. And that's exactly where you'll find one of the most out-of-the-way eateries in the Palmetto State.
Marshside Mama's Cafe is located on the Southwestern side of Daufuskie Island, the next island south of Hilton Head Island, and getting there is only part of the adventure.
Have you heard of Marshside Mama's? Would you take a ferry to get to this amazing eatery on Daufuskie Island? We'd love to know in our comments!


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Met with Dr. Kaminer for a consult today and was very pleased. I immediately felt at ease and confident that I was in good hands. I have had cellulite since I was 15 years old. I know that its partly genetic as my mother and sister also have it pretty bad. I have tried to lessen my cellulite through diet and exercise with no improvement, which is extremely disheartening. I'm in pretty good shape for 40 yrs old and I'm thin which makes it more frustrating. I learned that really no amount of exercise or diet with truly get rid of the dimples due to the connective bands that pull the skin down. After a comprehensive question and session I have booked my treatment for June 28th. Stay tuned!
Cellulite Despite Diet and Exercise - Chestnut Hill, MA
Met with Dr. Kaminer for a consult today and was...
1 week till procedure!
I have been anxiously counting the days till I get the cellfina procedure. 6 more to go! My husband went and bought a pair of compressions shorts for me when I complained I did not own Spanx. Very sweet! I haven't tried them on but they are extremely soft and I know they'll do the trick because a size medium looks like they're made for a 10 yr old ????.... I've also purchased a two week supply of arnica tablets that I'll start taking two days prior to the procedure. They work really well to help dissipate bruising and swelling. I'm going to BRAVELY post some before pics tonight! Yikes! Keep in mind that although I've had cellulite since I was 16 it's been worsened by a lack of exercise, winter hibernation and my love of wine!
Procedure done!!
I just got home from having the procedure done this morning. I was super excited yet very nervous. My nurse was great. Definitely helped me feel better. She told me my cellulite was the type that would get awesome results. I told her I'd hold her to her words, lol...After changing into a gown the Dr marked all the dimples including ripples that he was going to treat. I was surprised because I was under the impression that ripples were not treated and voiced that to him. He assured me that they could be and that a lot of the info on the web was not current. Yay!!!..... In total, I had 37 areas to be treated. After photos the nurses took me to the procedure room and had me lay on my abdomen. Dimple by dimple they instilled the tumescent (Lactated ringers solution with lidocaine) into each area. Let me tell you, this was PAINFUL!!! I felt like a big baby and let out a couple of swears and tried to keep myself distracted by babbling to the nurses. That took about 15 min. They let the solution sit for another 15 minutes before starting the actual procedure. Once my bum was thoroughly numb, the Dr. started his portion and it took about 20 minutes start to finish. I could feel the pressure of the suction device and hear the buzz of the blade but felt no pain. When it was all over they squeezed out as much solution from my bum and thighs as they could, bandaged me up, stuffed me into the compression garment like a sausage and sent me on my way with a follow up appt in 3 weeks. I live about an hour away from their office and noticed that I was already saturating my bandages on the car ride home. About 45 min into my ride I could feel the lidocaine wearing off. By the time I got home I was in actual discomfort/pain. Fortunately, I have leftover hydrocodone from dental work so I took one of those because I had a feeling tylenol was not going to do the trick. I also started taking liquid probiotics to help prevent a yeast infection from the prophylactic antibiotic I'm taking. So with all that being said, I realize everyones pain tolerance is subjective and everyones experience will differ. I'll update tomorrow on how i'm feeling :)
Feeling really good!
Today is one day post-op and I'm feeling fine. I had a tough time sleeping last night due to the compression garment being so tight around my waist (It's high waist to mid thigh). I unfastened the waist area in the middle of the night and was able to get some rest. I can't say i'm a big fan of this particular garment which is made by the company "Veronique". It's the one they put me in in the office. It has a convenient hole in the crotch to allow you to use the bathroom without having to take it off but it irritates the area between your thigh and labia. I put a ABD pad there to cushion it. TMI?? So overall I don't have any pain but still feel slightly tender. Area also feels a bit numb but Dr said that usually lasts about 24 hrs. He also called me last night to check in on me which I really appreciated. Will take a quick shower today to clean off the area and put new bandages on because I still have spotty drainage. I'll post more pics once the bruising subsides.
Before pic. Yuck!
Verdict is still Out!
It's been 3 weeks since the procedure. My follow up was today but unfortunelty I had to reschedule. The first week was rough after the tumescent fully wore off. Getting on and off the toilet was the hardest. When I look it the mirror Im honestly not sure if I see improvement. Its hard to be objective when you see yourself everyday. I still have a couple areas that seem discolored from bruising as well as marks from where the lidocaine was injected. I know that will take time to resolve.
3 weeks post-op
It's hard to get a great picture when you're taking it yourself. I still have bruising in a few spot and have felt large hard areas in my bum. My guess is it's possible scar tissue. I'm going to use the foam roller at the gym to try to work those spots out.
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text:
- On February 22, 2011, Somaxon announced that it had received from the U.S. Patent and Trademark Office a Notice of Allowance for claims in U.S. patent application no. 11/781,165, which significantly strengthened the Silenor intellectual property portfolio. This patent application generally relates to dosing Silenor at least three hours after a meal to promote faster onset of action and reduce the potential for next-day residual sedation. Because this food effect is described in the Silenor prescribing information, Somaxon will list the patent in the Orange Book once it is issued.
- On February 8, 2011, Somaxon announced that based upon positive commercial trends in the fourth quarter of 2010, it had engaged its contract sales force partner Publicis Touchpoint Solutions, Inc. to recruit and deploy for Somaxon an additional 35 sales representatives that will exclusively promote Silenor. Somaxon expects the majority of these additional sales representatives to be deployed in the field by the end of the first quarter of 2011.
- Somaxon has come to agreement with CVS/Caremark PCS Health, Blue Shield of California and Prime Therapeutics for unrestricted Tier 3 formulary placement of Silenor, and with the U.S. Department of Defense/TRICARE for Tier 2 formulary placement of Silenor. The CVS/Caremark entities and Prime Therapeutics are pharmacy benefit managers that manage commercial healthcare plans covering approximately 50 million lives and 2 million lives, respectively, Blue Shield of California covers approximately 3 million lives and TRICARE covers approximately 10 million lives. Silenor is currently reimbursed on other commercial plans representing approximately an additional 145 million lives, with final formulary positioning subject to final negotiation with such plans.
- On February 8, 2011, Somaxon announced that it had obtained from Comerica Bank a two-year, revolving line of credit for up to $15 million for working capital needs and general corporate purposes. To date no amount of the revolving line of credit has been drawn down.
- On February 3, 2011, Somaxon announced that it strengthened its senior management team through the hiring of Robert Cutler as Vice President of Business Development, responsible for driving Somaxon's corporate growth strategy.
Somaxon Pharmaceuticals, Inc. (Nasdaq: SOMX), a specialty pharmaceutical company, today announced financial results for the full year and fourth quarter ended December 31, 2010. "During 2010, we achieved our key corporate objectives that allowed us to successfully transition from a development stage organization to a commercial specialty pharmaceutical company, including the FDA approval of Silenor®, the commercial launch of Silenor alongside our primary care co-promotion partner, Procter & Gamble, and strengthening our balance sheet," said Richard W. Pascoe, Somaxon's President and Chief Executive Officer. "In 2011, we intend to build upon our progress as we anticipate growing Silenor revenue, leveraging our commercial infrastructure to broaden our business, extending the Silenor franchise and effectively managing our financial and other corporate resources, with the goal of building a sustainable specialty pharmaceutical business dedicated to delivering long-term shareholder value." Recent Highlights


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In order to have the colour of plasticine you want, all you need is to get some yellow wax crayons ready along with the other ingredients in this film brought to you by Science Boffins.
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while i was a xbox owner and modder for a full 2 years before i ever played Halo: combat evolved, i don;t consider my gaming career to have started until i did, and while i quickly moved on to halo 2, CE is still dear to my heart. so Christmas morning when i opened the game, i was extremely excited to see what they had done. i have not played much of the campaign yet nor have i heard much and the revamped and expanded dialogue as the house was crowded and noisy. i played the multilayer maps for a good 3 hours yesterday until my wife kicked me off the couch and loved them. i don't have reach, so these are the only maps i have right now, but damn are they cool. very happy with this game and I really hope they do this same thing with Halo 2.
4 Replies
Dec 27, 2011 at 9:55 UTC
My girlfriend got an Xbox. As soon as I heard the news I ordered the CE Anniversary on Amazon and it's expected to be here on Thursday.
I am too, excited to play good ol' original Halo but with pretty graphics. I still have the original installed on my computer. It's a game that doesn't get old.
I wouldn't be near as excited if there was a remastered version of Halo 2.
Dec 27, 2011 at 9:58 UTC
i wasted weeks of game play on halo 2 and when i would get board, it was either vice city of halo 2 i turned to, i had my favorite levels( pretty much any that you did not play as the alien in) and just went through them over and over. a halo 2 remake would be stellar for me.
Dec 27, 2011 at 2:55 UTC
3 of my friends and I would play the original Halo for hours a night on the original Xbox, of course we had to play 4-player split screen, or occasionally the one of us that owned another copy would bring it over and we'd play system link. When Halo Anniversary came out, we all bought that and have greatly enjoyed reliving the memories of playing those original maps again and we've recreated settings we loved, like playing with everyone invisible on Hang'em High with all rocket launchers, one of our favorites.
Jan 3, 2012 at 8:24 UTC
Playing Halo, 'Rockets Only' on the map Longest' with 3 mates is still one of the best multiplayer experiences I've found :-) Its right up there with Mario Kart Double Dash and Timesplitters2.
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


text:
b-plus GmbH
Ulrichsberger Straße 17
94469 Deggendorf
Phone: +49(0)991.270302-0
Fax: +49(0)991.270302-99
b-plus is an established and recognized partner of the automotive industry. With a strong focus on future technologies, b-plus develops complex hardware and software solutions for applications like advanced driver assistance systems (ADAS) and safety control units. Highly dynamic and innovative future technologies like autonomous driving and others are supported by our portfolio of diagnosis, development and measurement platforms. With a team of 100 employees b-plus supports its customers along their design and development process. Our business units are organized as follows:
--> Hard-, software and development tools for vehicle measurement technology and driver assistance systems, as well as on-board computer systems
(in vehicle applications)
Mobile Automation:
--> Automation and system integration of monitoring and control units as well as diagnosis and display devices for utility vehicles and mobile working machines
(Off Highway applications)
Embedded Systems:
--> Embedded system solutions in the surrounding of the vehicle and harsh environments, e.g. ticketing, surveillance, camera systems, image processing, toll systems…
--> Standard components for modular embedded systems
--> Services for carrier board designs and driver development
With long term experience and significant know-how in project and product areas, various development teams realize sound solutions for sophisticated industrial and safety relevant automotive applications. b-plus sees itself as a reliant full service provider, beginning with the well-founded consulting, the conception and the management of a project up to the realization and is thus the professional partner of the customer, beginning with the development phase up to the series production and system integration.
b-plus GmbH offers you products from these product groups:
embedded world
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text: The sol system asteroid belt is a field of small celestial bodies, usually of metallic composition, which form an elliptical pattern in orbit around the sol star. The asteroid belt is home to several bases. There are four types of asteroid bases located in the asteroid feild. The first type is located inside the asteroid and is used as a mining colony. The second type is located on small craters on an asteroid, they generate a thin atmosphere around the crater due to oxygen generating plants, these colonies serve as residential colonies with an arboritum, school houses, thearters, Cetecean labs, Salons and Art meuseums. The third type are located across the asteroid, the oxygen generating facilities are located in the colonies, these colonies serve as research facilities. The fourth type is also located in craters. Like the second type, they serve as residential colonies. However, there are several kinds of machines that can create water and oxygen in this colony type.


text:
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Conceived and designed the experiments: DJ WS. Performed the experiments: DJ WS. Analyzed the data: DJ WS. Wrote the manuscript: DJ WS.
The success of cisplatin (CP) based therapy is often hindered by acquisition of CP resistance. We isolated NSC109268 as a compound altering cellular sensitivity to DNA damaging agents. Previous investigation revealed an enhancement of CP sensitivity by NSC109268 in wild-type Saccharomyces cerevisiae and CP-sensitive and -resistant cancer cell lines that correlated with a slower S phase traversal. Here, we extended these studies to determine the target pathway(s) of NSC109268 in mediating CP sensitization, using yeast as a model. We reasoned that mutants defective in the relevant target of NSC109268 should be hypersensitive to CP and the sensitization effect by NSC109268 should be absent or strongly reduced. A survey of various yeast deletion mutants converged on the Rad5 pathway of DNA damage tolerance by template switching as the likely target pathway of NSC109268 in mediating cellular sensitization to CP. Additionally, cell cycle delays following CP treatment were not synergistically influenced by NSC109268 in the CP hypersensitive rad5Δ mutant. The involvement of the known inhibitory activities of NSC109268 on 20S proteasome and phosphatases 2Cα and 2A was tested. In the CP hypersensitive ptc2Δptc3Δpph3Δ yeast strain, deficient for 2C and 2A-type phosphatases, cellular sensitization to CP by NSC109268 was greatly reduced. It is therefore suggested that NSC109268 affects CP sensitivity by inhibiting the activity of unknown protein(s) whose dephosphorylation is required for the template switch pathway.
Since its discovery three decades ago, Cisplatin (CP) has been widely used as an effective anticancer agent against a wide variety of solid tumors like tumors of ovary, testis, head and neck, cervix and lung [1,2]. However, treatment success by platinum agents is diminished by both intrinsic and acquired resistance, necessitating a dose escalation that is limited by side effects like nephrotoxicity, ototoxicity, peripheral neuropathy and myelosuppression [1,3,4]. Acquired resistance is often multifactorial in nature, with common mechanisms attributed to decreased cellular drug accumulation through reduced influx or increased efflux [5,6], elevated thiol content and increased ability to repair or tolerate platinum DNA adducts [3,4]. A non-toxic compound used in combination with CP that potentiates sensitivity may increase the therapeutic index of CP, especially in the case of otherwise CP resistant cancers [7-10].
A two-hybrid yeast assay was used by us to screen the National Cancer Institute's Diversity Set for compounds that can modify the checkpoint response elicited by the topoisomerase I inhibitor camptothecin [11]. Initially, NSC109268 was isolated as such an agent that reduces the checkpoint response to camptothecin. On further study by quantitative survival analysis, it was revealed that NSC109268 increased cellular CP sensitivity [12]. This was in marked contrast to its general tendency of decreasing sensitivity to other DNA damaging agents like nitrogen mustard [12]. CP and nitrogen mustard both create common lesions, namely interstrand crosslinks but with different yield.
NSC109268 had been shown to inhibit the chymotrypsin-like activity of the 20S proteasome in both Jurkat T cell extract and rabbit purified 20S proteasomes using an in vitro assay [13]. In addition, inhibition of phosphatases by NSC109268 had been suspected following molecular modeling, using the human PP2Cα structure in a virtual ligand screening of the Diversity Set of compounds [14]. Using biochemical assays of enzyme activity, NSC109268 was indeed found to strongly inhibit PP2Cα and, less severely, the PP2A group of serine-threonine protein phosphatases [14].
Described initially in budding yeast, we further confirmed cellular sensitization to CP by NSC109268 in the CP-sensitive ovarian carcinoma cell line 2008 and, even more pronounced, in its CP-resistant counterpart, 2008/C13 [12]. Cellular sensitization to CP by NSC109268 was consistently correlated with a slower S to G2/M phase progression in both yeast and the CP-resistant carcinoma cell line [12]. Although NSC109268 enhanced CP-induced p53 levels, its effect on cell death following CP (i.e. apoptosis and necrosis) was not dependent on p53 [15].
Given the similarities of the effect of NSC109268 on mediating cellular sensitization to CP in yeast and cancer cell lines, given also the high degree of conservation of DNA repair pathways and the availability of a collection of deletion mutants of non-essential yeast genes, yeast must be considered a valuable model to study the targeted pathway(s) of NSC109268 that are relevant for CP sensitivity [16]. The major target of CP is chromosomal DNA, with the majority of CP adducts comprising of DNA intrastrand crosslinks, mainly diguaninyl crosslinks [17]. Albeit much less frequently, CP also induces the relatively much more lethal interstrand crosslinks [18]. Nucleotide Excision Repair (NER) is the major pathway for bulky platinum adduct removal and thus error-free repair of DNA damage by CP [4]. Consequently, defects in the NER pathway result in hypersensitivity to platinum agents and restoration of NER integrity correlates with reversal of CP sensitivity [19]. Increased expression of the NER gene ERCC1 (RAD10 in budding yeast) is frequently associated with CP resistance in ovarian and gastric tumors [1].
Interestingly, among various predictors of CP sensitivity examined, such as increased platinum accumulation, decreased glutathione levels, decreased adduct removal or decreased tolerance to platinum-DNA adducts, decreased tolerance was the strongest predictor of CP sensitivity in ovarian cancer cell lines [20]. Furthermore, 2008/C13 cells have been described as being more efficient in replicative bypass of CP lesions than their CP-sensitive counterparts [21]. Therefore, inhibitors of specific DNA repair or tolerance pathways might prove especially efficacious when used in combination with CP.
Activities of polymerase ζ, polymerase η and RAD18, all involved in control of various modes of DNA damage tolerance are required for replicative bypass of CP intrastrand crosslinks [22]. In response to DNA damage, yeast Proliferating Cell Nuclear Antigen (PCNA) is monoubiquitinated or polyubiquitinated at K164. Monoubiquitination of PCNA by RAD18, a ubiquitin ligase, promotes translesion synthesis mediated by Polζ or Polη [23,24]. During translesion synthesis, low fidelity DNA polymerases replicate directly past the lesion in either an error-prone or error-free fashion. Human cells expressing no Polη or reduced levels of REV3, an essential component of the translesion polymerase DNA Polζ, are more sensitive to CP [25-27].
K63-linked polyubiquitination, extending monoubiquitinated K164 of PCNA, depends on Ubc13–Mms2 (forming the E2 enzyme) and Rad5 (the E3 enzyme) and is required for an error-free damage tolerance pathway, most likely mediated by template switch (TS) (Figure S1) [24,28-31]. In contrast to translesion synthesis, the lesion is avoided by a "copy choice" mechanism using an alternate, undamaged DNA template. Upon DNA damage, a lesion in the leading strand template can lead to uncoupling of leading and lagging strand synthesis and single-stranded DNA gaps can be found in both leading and lagging strands. Subsequently, strand invasion mediated by certain Homologous Recombination (HR) factors that include Rad51, Rad52, the complex of Rad55-Rad57, promote the formation of TS intermediates that are dissolved by the action of the Sgs1-Top3 complex (Figure S1) [28,30,31].
In this study, using survival and cell cycle analyses, we identified the Rad5 pathway of damage tolerance by TS as the target pathway of NSC109268 relevant for CP sensitization. Inhibition of this pathway appears to result in the observed delayed S phase traversal of CP + NSC109268 treated yeast cells. Furthermore, our data suggest a critical role of NSC109268 as a PP2Cα and PP2A phosphatase inhibitor in influencing cellular sensitization to CP.
Cisplatin (CP) was purchased from Sigma Aldrich. NSC109268 was initially provided as part of the Diversity Set compound library by the Developmental Therapeutics Branch of the National Cancer Institute and later synthesized by Omm Scientific, Dallas, Texas. NSC109268 was dissolved at a stock concentration of 1 mg/ml in dimethylsulfoxide, CP at 10 mg/ml in dimethylformamide.
S. cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was the parental haploid wild-type strain used throughout the study unless indicated otherwise. Haploid mutants, each deleted for a defined open reading frame of a non-essential gene, were from the systematic gene deletion collection (purchased from Open Biosystems). BY4741 rad5::HIS3 and BY4741 sml1::KanMX4 rad53::URA3 were constructed in our laboratory. Strains YJK17 (MATα hoΔ hmlΔ::ADE1 hmr::ADE1 arg5,6::HPH::MATa-inc ade1-100 leu2,3-112 lys5 trp1::hisG ura3-52 ade3::GAL::HO), YJK24 (ptc2Δ::URA3MX ptc3Δ::NatMX. YJK17 isogenic), YJK26 (pph3Δ::KanMX, YJK17 isogenic) and YJK70 (pph3Δ::KanMX ptc2Δ::URA3MX ptc3Δ::NatMX, YJK17 isogenic) were kindly provided by Dr. James Haber [32]. Diploid strain D7 (MATa/α ade2-119/ade2-40 trp5-12/trp5-27 ilv1-92/ilv1-92 CYH/cyh2) was originally from Dr. Fritz Zimmermann [33]. Note that this strain does not require adenine and forms white colonies due to intragenic complementation.
All strains used in this study were grown to early logarithmic phase in YPD (1% yeast extract/2% peptone/2% dextrose). Subsequently, cells were washed, resuspended in phosphate-buffered saline (PBS) and treated with CP and NSC109268 at 30°C with constant shaking, typically for 2 h unless indicated otherwise. For strains YJK17, 24, 26 and 70 5 mM phosphate buffer/5 mM sodium chloride (pH 7.0) was used instead of PBS. After treatment, cell suspensions were appropriately diluted and plated onto YPD plates and, in case of D7, also synthetic media plates lacking tryptophan where appropriate. Published protocols and recipes were followed [34]. Plates were incubated at 30°C to allow colony formation. Surviving fractions after drug treatment were calculated by dividing the titer of macrocolony-forming cells in the drug-treated sample by the titer of macrocolony-forming cells in the untreated control sample. Standard deviations are indicated if experiments were repeated at least three times. Otherwise, data of representative single experiments are shown, but in all cases data points were reproduced at least once. Dose Enhancement Factors (DEF) were calculated by determining the ratios of CP doses resulting in 50% or 10% survival, without vs. with inclusion of NSC109268. For DEF calculations, survival values were corrected for killing by NSC109268 alone (usually 10-30%).
Wild-type yeast (BY4741) and isogenic mutant strain rad5 were grown in YPD overnight at 30°C to early logarithmic phase. Cells were then synchronized in G1 using the yeast mating pheromone, α factor, at a final concentration of 10 µg/ml as previously described [12]. After resuspending cells in PBS, 80 µM CP was administered along with 5 µg/ml of α factor for 1 h at 30°C with shaking. Next, cells were washed, resuspended with PBS and α factor and treated for 20 min with 0.7 µM NSC109268. Control samples, containing the appropriate solvent instead of the drug, were incubated in parallel. After treatment, cells were washed and resuspended in fresh YPD to allow for synchronous reentry into the cell cycle. At the indicated time points, samples were collected, fixed in ethanol, sonicated and stained with SYBR Green I (gel staining solution, Lumiprobe) for FACS analysis of DNA content as described [35]. Cellular fluorescence was measured in a FC500 Flow Cytometer (Beckman Coulter Corp.). CXP and ModFit software were used to obtain histograms depicting DNA content versus cell number (of a total of 10,000 cells) and to calculate the fractions of G1, S and G2/M cells.
CP uptake is largely mediated by the plasma membrane copper transporter CTR1 in both yeast and mammals [36,37]. Following internalization, both copper and CP were shown to cause downregulation of CTR1 in ovarian cancer cells by the proteasome-mediated pathway [38] contributing to CP resistance. Since NSC109268 may prevent the degradation of CTR1 through its 20S proteasome inhibitory activity [13], we tested CP uptake as a target of NSC109268 in CP sensitization. Haploid wild-type yeast cells were treated with CP for 1 h, then washed to remove CP and treated with NSC109268 for another hour. Following this regimen, NSC109268 clearly sensitized yeast cells to CP (Figure 1) synergistically, as with simultaneous administration of NSC109268 and CP [12] or pretreatment with NSC109268 before adding CP (data not shown). Since NSC109268 sensitized previously CP-treated cells even in the absence of external CP, we conclude that mechanisms other than increased CP uptake, such as inhibition of DNA repair, must be responsible for sensitization to CP.
In order to identify the pathway targeted by NSC109268 in mediating cellular sensitization to CP we reasoned that, if the critical pathway is already inactive, such mutant cells will be CP sensitive and no sensitization that exceeds an additive effect will be achieved by combination treatment. Several functional screens in S. cerevisiae and Schizosaccharomyces pombe had already identified genes that upon deletion confer sensitivity to CP [39-41]. Genes surveyed by us included those operating in NER (RAD10), DNA damage tolerance (RAD5, MMS2, REV3 and RAD18), interstrand crosslink repair (PSO2) and HR (RAD51). In general, the published relative CP sensitivities of the mutants investigated were confirmed (Figure S2, data not shown). Figure 2 summarizes the effect of NSC109268 on CP sensitivity of all tested deletion mutants, expressed as the factor by which the CP dose resulting in 50% or 10% survival can be reduced in the presence of NSC109268 (dose enhancement factor, DEF), see also Table S1 for data and gene product function. In Figure 3, selected dose response curves are shown.
In surveying a large number of isogenic mutants of haploid strain BY4741, we ruled out several pathways as primary targets of NSC109268 in causing cellular sensitization to CP. For example, PSO2 is a gene known to be specifically involved in interstrand crosslink repair in yeast. CP is known to induce interstrand crosslinks eliciting cell killing in yeast [42] and as expected, we found the pso2Δ mutant strain to be hypersensitive to CP alone. The combination of NSC109268 and CP was highly synergistic in inducing cell death (Figure 2, Figure 3A) which ruled out the affected interstrand crosslink repair pathway as the target (and possibly also interstrand crosslinks as critical lesion). When testing rad10Δ mutant strain, defective in a key endonuclease component of NER, we found the strain to be hypersensitive to CP, as previously reported [39], and additional sensitization to CP by NSC109268 was still observed (Figure 2, Figure 3A). Similar results with rev3Δ and rad30Δ mutant strains argued against damage tolerance by translesion synthesis as the target of NSC109268 in mediating cellular sensitization to CP (Figure 2). Rtt101, a component of a novel ubiquitin ligase complex promoting replication through damaged DNA at stalled replication forks [43] could also be excluded (Figure 2). Similarly, by testing the CP-hypersensitive rad50Δ mutant, we concluded that the MRN/X (Mre11-Rad50-Xrs2) complex and its activity in DNA double-strand break repair is an unlikely target (Figure 2).
As representative examples of the cell cycle checkpoint pathway, we selected mutants deleted for the DNA damage recognition protein Rad24 and the Rad53 kinase. A deletion of the latter is viable in an SML1 deletion background. Both sml1 and sml1 rad53 strains can be sensitized towards CP by NSC109268 to a similar extent (Figure 2, Figure 3B). Since rad24 retained synergism as well, we did not obtain evidence for the checkpoint pathway being a critical target.
In revisiting CP uptake, we found combination treatment to remain synergistic in inducing cell killing in CP-resistant ctr1Δ, a CP uptake mutant (Figure 2). Similar results were obtained for ccc2Δ, deleted for the yeast ortholog of ATP7B, a P-type ATPase that mediates cellular CP efflux and counteracts CP lethality in mammals [5]. Together, these results confirm our previous notion that modification of CP transport is unlikely to be a mechanism by which NSC109268 mediates cellular sensitization to CP.
As previously reported [39], mutant strain rad5Δ, defective in damage tolerance by TS, was found to be CP hypersensitive. Remarkably, we did not detect additional sensitization upon the administration of NSC109268 during CP treatment of rad5Δ cells (Figure 2, Figure 3C). This result was confirmed by detecting a similar absence of synergism in deletion mutants of other components of the Rad5 mediated TS pathway – RAD18, UBC13 and MMS2 (Figure 2, Figure 3C). As compared to wild-type, no or only a low degree of sensitization by NSC109268 beyond an additive effect was also found for deletions of the RAD51, SGS1 and PSY3, encoding HR components that all are likely to play a role in damage tolerance by TS [28,44] (Figure 2, Figure 3D).
We also tested a deletion of Rad59, an HR protein that stimulates single-strand annealing in complex with Rad52 [45]. Rad59 has been proposed to only participate in the canonical HR pathway but not in the TS pathway of damage tolerance [28]. The combination of CP and NSC109268 remained synergistic in inducing cell death in the rad59Δ strain (Figure 2, Figure 3D). This result implies that HR pathway per se may not be a crucial target of NSC109268 in mediating cellular sensitization to CP.
In a diploid strain (D7, unrelated to BY4741), we tested the influence of CP and NSC109268 on survival and genomic instability events related to interchromosomal recombination. Strain D7 contains two detection systems for such events, the ade2-119/ade2-40 and trp5-12/trp5-27 heteroalleles [33]. Scoring red or pink sectored or pure colonies detects a wider range of events than the trp5 gene conversion system, including chromosome loss events.
For this diploid strain, synergistic interaction of CP and NSC109268 was verified for colony survival (Figure 4A). Frequency of CP-induced gene conversion in the trp5 system and aberrant colony formation in the ade2 system were increased in a dose-dependent manner (Figure 4B,C). Whereas NSC109268 had little effect on its own, it synergistically enhanced CP-induced recombination/aberrant colony frequency in either system. Since gene conversion and mitotic recombination events are dependent on HR [46], an inhibitory effect of NSC109268 on HR per se appears to be unlikely.
G1-synchronized haploid wild-type and isogenic rad5Δ strains were treated with CP alone or sequentially with CP and NSC109268 before release into nutrient medium to allow for cell cycle progression. By itself, the chosen low dose of NSC109268 caused only a brief delay of G1/S phase transition. To facilitate interpretation, we used a low CP dose that induced only a small effect on cell cycle kinetics in wild-type cells on its own. However, confirming previously published data [12], the combination of NSC109268 and CP resulted in a notable slow-down of S phase progression, as measured by a delayed increase in G2/M phase cells (Figure 5A, see Figure S3 for FACS profiles). Using the same CP dose, synchronized rad5Δ cells were delayed for G2/M entry as compared to untreated cells (Figure 5B). However, NSC109268 did not lead to any enhancement of this CP-dependent delay beyond the small effect attributable to NSC109268 alone, thus being additive at best (Figure 5B). Preliminary studies suggest that the same is true for the later occurring extensive G2/M arrest (data not shown). This is in marked contrast to the synergistic effect on cell cycle kinetics of NSC109268 in CP-treated wild-type cells (Figure 5A). Thus, cell cycle studies supported our survival analysis hinting at the Rad5 pathway of damage tolerance by TS as a target of NSC109268 in mediating cellular sensitization to CP.
We wished to determine if the known activities of NSC109268 as 20S proteasome inhibitor [47] or as phosphatase 2Cα and 2A inhibitor [14] critically affect sensitization to CP by NSC109268. Bortezomib, a known proteasome inhibitor possibly mimicking NSC109268's proteasome inhibitory activity, was not synergistic in inducing cell death (Figure S4A). Furthermore, NSC109268 and CP combination remained synergistic in ump1Δ (Figure 2, Table S1), deleted for a DNA-damage inducible chaperone involved in 20S proteasome maturation and required for UV resistance [48]. These results argue against direct proteasome inhibition by NSC109268 as critical for mediating sensitization to CP.
Polyubiquitination of PCNA is an essential signal for activation of the Rad5 pathway. As an indirect consequence of proteasome inhibition by NSC109268 due to reduced ubiquitin recycling, we considered that free ubiquitin levels in NSC109268 + CP treated cells may be too low for PCNA polyubiquitination to occur efficiently [49]. This possibility, however, was unlikely since NSC109268 and CP combination remained synergistic in inducing cell death even if cells were overexpressing plasmid-encoded ubiquitin [50] (Figure S4B). Therefore, we found no evidence that proteasome inhibition by NSC109268 leads to cellular sensitization to CP, directly or indirectly through impairment of free ubiquitin levels.
Next, we considered the phosphatase inhibitory activity of NSC109268. To test for the role of PP2C or PP2A inhibition by NSC109268 in CP sensitization, we first treated single mutant strains defective in Ptc2 or Ptc3, both classified as Ser/Thr protein phosphatase 2C family members, or Pph3, a type 2A-like protein phosphatase A. The combination of NSC109268 and CP was found to remain synergistic in inducing cell death in all single gene deletion mutants of haploid strain BY4741 (Figure 2).
Next, combinations of phosphatase mutations were studied in a different genetic background (YJK). As compared to BY4741, it should be noted that we were able to increase the dose enhancement effect in this strain background while reducing the lethality of NSC109268 alone. At 50%/10% CP survival, DEF between 3 and 5 were determined for WT, pph3 and ptc2ptc3 (Figure 2, Figure 6). However, whereas CP sensitivity without NSC109268 was notably enhanced in the triple mutant ptc2Δptc3Δpph3Δ synergism was greatly diminished, with DEF reduced to less than 2 (Figure 2, Figure 6). While confirming the known redundancy between the studied phosphatases [32] these results indicate the involvement of both phosphatase 2C or 2A inhibitory activities of NSC109268 in mediating CP sensitization.
Among the platinum family of anti-cancer compounds, CP has been a very effective agent against a host of tumors such as those of the ovary and testis. However, its utility is severely restricted by dose limiting side effects and cellular resistance, either intrinsic or acquired during CP administration. We identified NSC109268 as a compound increasing cellular sensitivity to CP in Saccharomyces cerevisiae [12]. Enhancement of cellular sensitization to CP by NSC109268 was confirmed for CP-sensitive ovarian carcinoma 2008 cells and the derived CP-resistant line 2008/C13 [12]. Shown in both yeast and 2008/C13 cells, the increase in cellular sensitivity to CP by NSC109268 correlated with inhibition of S phase progression [12]. In in vivo studies with xenografted human head and neck carcinomas treated with CP alone, others had shown that a hindrance of S phase traversal correlated with CP responsiveness better than G2/M arrest [51].
In this study, we explored the possible targets of NSC109268 responsible for enhancing CP sensitivity using the systematic yeast gene deletion collection. We were guided by the concept that absence of the target should cause CP sensitivity but preclude additional sensitization by NSC109268. A similar strategy resulted in the identification of HR as the target of synergism between CP and camptothecin in yeast and human cancer cells [52]. Alterations in uptake or efflux of CP appeared unlikely to be the cause of sensitization since synergism was not diminished if CP was administered first, prior to treating with NSC109268 in CP-free buffer. This notion was further confirmed by mutant analysis.
After investigating various DNA repair and tolerance pathways such as NER, DNA translesion synthesis or interstrand crosslink repair, we identified a DNA damage tolerance pathway, the Rad5 pathway, as critical determinant of the synergism between CP and NSC109268 in budding yeast. This pathway is believed to bypass DNA damage at replication forks, that have been stalled in S phase due to DNA adducts, by using the newly synthesized strand of the sister chromatid as a template (Figure S1) [28-31,53]. Fork regression or recombinative sister strand junction formation have been discussed as the underlying mechanisms in this process [29]. CP primarily causes bulky DNA adducts which lead to a slow down of the replication fork, eventually leaving behind single-stranded gaps to be filled by Rad5 mediated TS pathway, involving certain HR components [28,31]. Inhibition of Rad5 mediated lesion bypass activity by NSC109268 may also result in fork collapse leading to genetic instability – as shown here with reporter strain D7 – and finally cell death, thus mediating cellular sensitization to CP. It has been debated if the Rad5 mechanism is critical for S-phase progression in the presence of DNA damage, as suggested here, or acts predominantly in G2-phase to fill remaining single-stranded gaps [30,54-56]. The discrepancies in timing found in the literature may very well depend on level and kind of DNA damage.
Our findings seem to fit particularly well with the model of filling of single-stranded gaps by template switch using a subset of recombination functions (Figure S1) [28,31]. We found proteins involved in initial PCNA monoubiquitination, such a Rad6 or Rad18, and in subsequent polyubiquitination, such as Rad5, Ubc13 or Mms2, to be required for CP+NSC109268 synergism. But we also identified HR proteins such as Rad51 or the junction-resolving enzyme Sgs1 as participants of the targeted mechanism(s). This includes Psy3, a member of the Shu complex proposed to participate in TS following strand invasion [44]. All of these proteins that are required for synergism of CP and NSC109268 are exclusively or non-exclusively involved in the TS pathway.
Interestingly, the mutant group showing no or very reduced synergism did not include Rad59 which has been shown by others to play a role in canonical HR but not in the TS pathway [28]. We also demonstrated that CP-mediated and HR-dependent gene conversion events [46] are increased by NSC109268 and not decreased. (Current models of the Rad5 pathway do not involve the homologous chromosome in a template switch mechanism, so this result does not contradict the inhibition of the Rad5 pathway.) Based on these observations, we do not favor that NSC109268 acts as a general HR inhibitor to exert its function on CP sensitivity, however, a more rigorous demonstration is still required.
A screen for single gene deletion mutants of budding yeast conferring exquisite CP sensitivity had previously revealed RAD5 and other key players of the same pathway as top hits [39]. In humans, this pathway was initially regarded as a tumor suppressor pathway since the essentially error-free bypass should counteract genetic instability [29,57]. On the other hand, the human ortholog of the yeast Rad5, HLTF, was reported to be overexpressed in radiation resistant recurrent human cervical carcinoma and a knockdown of HLTF in HeLa cells lead to a decrease in cellular proliferation [58]. Conceivably, targeting HLTF in chemotherapy-resistant cancers may lead to increase in cellular CP sensitivity.
Upon investigating the known activities of NSC109268 as a 20S proteasome and phosphatase 2C and 2A inhibitor [13,14], we found no evidence for the importance of proteasome inhibition in mediating CP sensitivity in yeast. In contrast, our survival studies indicated 2C and 2A phosphatase inhibition by NSC109268 to be responsible for mediating CP sensitization. Analysis of the yeast single mutants and double mutants ptc2Δ, ptc3Δ (each one defective for functionally redundant PP2C phosphatases), pph3Δ (defective for PP2A-like phosphatase) compared to the triple phosphatase mutant ptc2Δptc3Δpph3Δ revealed a marked defect in the viability of ptc2Δptc3Δpph3Δ cells in the presence of CP, while neither of the single mutants were similarly sensitive to CP. These results suggested redundancy of phosphatase PP2C and phosphatase PP2A-like mediated pathways in CP damage responses, as was already reported for other agents such as hydroxyurea or methyl methanesulfonate [32]. If phosphatase redundancy is removed in the triple mutant, sensitization of CP-treated cells by NSC109268 is greatly reduced suggesting phosphatases as the relevant target.
Phosphatases Psy2 or Ptc2 and, in a redundant fashion, Ptc3 have been shown to be required for turning off the DNA damage-induced cell cycle checkpoint by dephosphorylating kinase Rad53 [59,60]. However, upon treatment of rad53Δ mutant (sml1Δ background), Rad53 was excluded as a major target of NSC109268 in mediating CP sensitization. Thus, a reduced ability to dephosphorylate Rad53 checkpoint kinase and to release checkpoint arrest is not responsible for the cellular sensitization to CP by NSC109268.
Although the actual protein target(s) remain to be determined, this study underlines the critical importance of protein phosphatases in DNA damage responses that goes beyond recovery from checkpoint arrest. Even in a model organism such as yeast, significant gaps in our knowledge will need to be filled. For the first time, this study shows an influence of phosphatases on the Rad5 tolerance pathway. Additionally, the notion of intertwined HR and TS pathways is supported [28,30,31]. Interestingly, the triple phosphatase mutant pph3Δptc2Δptc3Δ used by us was reported to be defective in early steps of HR [32] that may also be necessary for sister chromatid junction formation and processing within the Rad5 TS pathway [28,30,31]. Interestingly, Rpa2, a subunit of the single-strand DNA binding complex Replication Protein A, represents another target candidate since its phosphorylation status, which may be enhanced by NSC109268-mediated phosphatase inhibition, is inversely correlated with RAD51 foci formation [61].
Taken together, our studies with NSC109268 in budding yeast demonstrate its potential as a cellular chemotherapy sensitizer, most likely by acting through the inhibition of dephosphorylation of critical protein(s) of the Rad5 pathway mediating CP damage tolerance during S phase. NSC109268 and related compounds may thus be especially valuable in CP combination therapy of the subset of cancers that developed CP resistance due to alterations within the Rad5 pathway. It remains to be seen if this is a mechanism of resistance that is widespread among cancer patients.
Model for DNA damage tolerance by template switch. Provoked by a bulky lesion (star) in the leading strand template, the proposed interplay of recombination proteins, PCNA ubiquitination and DNA polymerases in filling a single-stranded gap is depicted. Adapted from [28].
Relative cisplatin sensitivity of haploid yeast mutants in agar diffusion assays. For these semiquantitative streak tests, early-logarithmic phase BY4741 wild-type or mutant cell cultures were concentrated to 4x107 cells/ml. Of these suspensions, 10 µl samples were streaked on YPD plates in a radial fashion, and 250 µl of 1.5 mM CP were pipetted in the circular center hole (0.5 inch diameter). Plates were kept at 4°C for 3 hours to allow for CP diffusion before incubating at 30°C. Streaks were photographed after 30 hours.
Flow cytometric DNA profiles of G1-synchronized cells released into fresh medium after treatment with cisplatin, NSC109268 or both. Wild-type (A) or RAD5-deleted cells (B) of BY4741 were synchronized with α-factor and then sequentially incubated with CP and NSC10268 before release into fresh YPD medium, as described in Material and Methods. Samples were taken for a period of 100 or 120 min past treatment. Proper control regimens (mock treatment, CP alone, NSC10268 alone) were applied for comparison and are indicated in the figure.
Absence of evidence for direct of indirect influence of inhibition of protein degradation on sensitization to cisplatin by NSC109268. (A) The absence of a non-additive effect of the combination of bortezomib and CP is shown by determining the survival of wild type cells (BY4741) after simultaneous treatment with CP and bortezomib (75, 338 or 375 μM) for 2 h. Surviving fractions of colony forming cells were plotted as a function of CP dose. (B) The absence of an influence of ubiquitin overexpression on the synergism of. CP and NSC109268 in inducing cell killing is shown. The dose responses of wild-type cells overexpressing ubiquitin after treatment with CP and with or without NSC109268 (3 μM) for 2 h are shown. Wild-type cells (SX46A MATa RAD ade2 (ochre) his3-532 trp1-289 ura3-52) had been transformed with a plasmid carrying wild-type ubiquitin gene under the control of the copper inducible CUP1 promoter (YEp96-CUP1-UB) [50], kindly provided by Dr. Mark Hochstrasser. Strains transformed with YEp96-CUP1-UB or vector plasmid were grown to early logarithmic phase in Trp-dropout medium. To overexpress ubiquitin, CuSO4 was added at 100 μM to the medium for 3 h before treatment and plating onto –Trp dropout plates.
Mutants studied for influence of NSC109268 on cisplatin sensitivity, ranked by dose enhancement factor (DEF) at 50% survival. See also Figure 2.
We would like to thank Dr. James Haber for providing yeast strains and Dr. Mark Hochstrasser for providing plasmids. We also thank Xiangle Sun for assistance with flow cytometry and data analysis.
NSC109268 was initially provided by the Developmental Therapeutics Branch of the National Cancer Institute. These studies were supported by a grant from the Joe and Jessie Crump Fund for Medical Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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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Today, I told my boyfriend that I didn't want to go out with him because I was having a fat day. After ten minutes of fighting, he threw a ring box on the floor and stormed out. I basically refused his proposal because of my body issues. FML
By henley / Monday 23 July 2012 01:33 /


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Healthcare Economics Committee | ASCRS
Responsible for presenting information on issues affecting the colon and rectal surgeon to Congress and agencies of the federal government. The Committee's responsibilities include keeping members informed on these issues and tracking developments in managed care, regulatory, legislative and other issues that impact coding, billing and reimbursement for colorectal services.
What's New?
With the new ICD-10 coding taking effect this fall, the Healthcare Economics Committee took on the task of simplifying the new codes for members. A crosswalk between ICD-9 and ICD-10 was developed for popular colon and rectal surgery codes and shared with the membership via the website to help provide a smooth transition.
"Even though many surgeons may be using their electronic medical record to find the appropriate ICD-10 diagnostic code, the ICD-9 to ICD-10 crosswalk was important to provide so that all of our members can see the magnitude of the coding changes for common colon and rectal diseases," said Stephen Sentovich, MD, chair.
The goal of the committee is to keep members informed on legislative issues and to track developments in managed care, regulatory, legislative, and other issues that impact coding, billing, and reimbursement for colorectal services.
Chair: Walter Peters, Jr., MD
Vice Chair: Jeffrey Cohen, MD
Advisor: Guy Orangio, MD
Council Rep: Sonia Ramamoorthy, MD
|Jennifer Ayscue, MD
|Ali Mahmood, MD
|H. Randolph Bailey, MD
|Jeffrey Manchio, MD
|Amir Bastawrous, MD
|John Park, MD
|Valerie Bauer, MD
|Jay Redan, MD
|George Blestel, Jr., MD
|Irfan Rizvi, MD
|John Byrn, MD
|Rudolph Rustin, MD
|George Chang, MD
|Stephen Sentovich, MD
|Donald Colvin, MD
|Jenny Speranza, MD
|Gary Dunn, MD
|Steven Stryker, MD
|James Fitzgerald, MD
|Charles Ternent, MD
|Eric Haas, MD
|Kelly Tyler, MD
|William Harb, MD
|Michael Worsey, MD
|Srinivas Ivatury, MD
|Ronald Zelnick, MD
|Amit Khanna, MD
|Deepak Kumar, MD


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If you have been vaping herbs, it is time for you to try out the waxes and oils as well. Vaping oils is extremely fun and give you an enchanted vaping experience. As I assume that you have not yet tried oils for vaping, I am going to tell you my personal experience for vaping cannabis oil.
Cannabis oil is extracted from the vegetable matter and the bud of the flowering female cannabis plant. The oil is specifically extracted from the THC (Tetrahydrocannabinol) from the matter obtained from the flower.
Cannabis oil is available in the market in different types and textures. You can also find cannabis wax in dark molasses like tar and you can also find cannabis oil in a pure golden yellow honey like oil. The color and texture of the end product mainly depend upon the process used to extract the cannabis oil. Cannabis oil is a 60% to 90% concentrated extract of the THC.
So Why Vaping Cannabis Oil?
Vaping cannabis oil can be quite tasteful and fun, but just like burning cannabis herb, it does produce free radicals as like the burning of tobacco and other herbs. So the best approach is to use a method so that we can get the tasty stuff from the cannabis oil and not the nasty stuff.
The common method of vaping cannabis is usually by using a glass bubbler or bong using a wand and nail setup. The nail is super-heated, and the cannabis oil is dabbed with a wand into the nail which results in vapors that can be inhaled by the person.
But a bong is way hotter for the oil which results in undesirable compounds and chemicals. Technically the song is not vaporizing the oil rather it combusts the oil resulting in compounds which are dangerous and not at all needed. Therefore, using a vaporizer is a better approach.
Some cannabis oil vaporizers are found in the market that is used specifically for vaping cannabis oil. Using the cannabis vaporizer, you can rub the oil on the foam pad which is then used to produce the vapors.
The first thing that comes under your consideration is the dense flavor of the cannabis oil, and the next thing is surely high. I recommend you to start off with real small amounts as the stuff is quite strong.Read More


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'Pretty Little Liars' Star Divorcing
November 12, 2011 by staff
'Pretty Little Liars' Star Divorcing, The actress and her husband David Donoho met on the set of "Charmed."
Pretty Little Liars and Charmed star Holly Marie Combs has filed for divorce from husband David Donoho after seven years of marriage, TMZ reports.
Combs, 37, who is best known for playing witch Piper Halliwell on Charmed, married Donoho in 2004 after meeting on the set of the WB supernatural drama when he was a key grip on the show.
The actress cited irreconcilable differences as the cause for the separation and their relationship has been unstable for "quite a while."
Combs, who currently stars in ABC Family's Pretty Little Liars, and Donoho have three young children together and she is reportedly seeking joint custody. According to the report, there was no prenup. An active Twitter user, Combs has not yet commented on her split.
Please feel free to send if you have any questions regarding this post , you can contact on
Disclaimer: The views expressed on this site are that of the authors and not necessarily that of U.S.S.POST.


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Hi, folks. Are you ready for more live blues music? The Boise Blues Society is bringing you a whole flock of shows in the coming weeks. Come out and dance!
If you haven't yet seen or heard the BBS Band, you have TWO chances this week:
- Monday evening (October 20) we'll be playing during the KRBX Radiothon Fall fundraiser. Tune in to 89.9 or 93.5 at 6pm and dance at home.
- Wednesday evening (October 22) we open the 2014 Pre-Holiday Benefit for the Idaho Food Bank, 6pm at The Bouquet (10th and Main), followed by British invasion rock music by Gerry & the Dreambenders.
Several fabulous bluesy events will heat up your November:
– Saturday Nov 8 at 7:30pm, catch the 20th Anniversary Blues Brothers Rock 'n Soul Revue in the Mardi Gras Ballroom.
– Nov 9 from 3-6pm, spend your Sunday afternoon at The Drink enjoying a wide variety of local musicians in our Blues Jam Sessions.
– Do you love the smooth sounds of brass? Thursday, Nov 13 at 6:30pm, we are bringing the award-winning, funky, neo-soul blues band Josh Hoyer and the Shadowboxers to the Sapphire Room for one night only! This is a ticketed all-ages event; members get a discount on advance orders with the emailed discount code. Click here for more info and to purchase your tickets.
Finally, we are thrilled to report that BBS has received a $1000 grant from the City of Boise's Arts & History Department to support our Blues in the Schools (BITS) program. We plan to continue bringing blues music into schools throughout the Treasure Valley, as well as to bars, festivals, and other events for people of all ages to enjoy.
Thanks so much for your support, and we hope to see you soon at a live blues show.
BBS Board of Directors


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Mark Harrell was a three-star prospect out of Charlotte (N.C.) Catholic High School and Scout's No. 52 rated offensive tackle in 2012. He was the second-leading vote-getter among offensive linemen on the 2011 Associated Press North Carolina all-state team.
During Harrell's junior season (2010), he helped lead Charlotte Catholic to a 14-1 record. He would go on to participate in the Semper Fidelis All-American Bowl and was chosen to play for the North Carolina-South Carolina Shrine Bowl.
Harrell spent his early years with the Irish playing center before transitioning to right tackle, where he finished the spring of '16 No. 2 on the depth chart behind junior Alex Bars.
Harrell's best-case scenario likely would require a physical setback or two along the Irish offensive line in order for him to see his first extended action in an Irish uniform. Notre Dame is light on offensive tackles, although two (Tommy Kramer and Liam Eichenberg) arrive this fall. Harrell is the fourth offensive tackle on the roster (prior to the arrival of the freshmen) behind Mike McGlinchey, Alex Bars and Colin McGovern.
Harrell completes his five-year run with the Irish as a backup with incoming freshmen Kramer and/or Eichenberg ascending above him. Harrell still should see time on Notre Dame's blocking unit up front on placekicks.
It's somewhat rare that a non-major impact offensive lineman through four years returns for a fifth season. But with Notre Dame's numbers crunch at tackle, Harrell's return gives the Irish depth at tackle. Brian Mattes was another lightly-used guard/tackle at Notre Dame who preserved a year of eligibility during his rookie season in 2002. Virtually all of his snaps came on special teams. He did not start a game or play significant minutes, but was brought back for a fifth season in 2006 and was a backup to Bob Morton at right guard.
Harrell was a three-star prospect and the No. 52-rated offensive tackle in the country coming out of high school. The fact he's yet to play a meaningful minute in his first four years for the Irish is a testament to the talent Notre Dame has accrued along the offensive line.
Harrell has participated in seven games since his arrival to Notre Dame in 2012. He did not see game action his first two seasons. Five of his seven appearances came in 2015 against Texas, Georgia Tech, UMass, Pittsburgh and Wake Forest.
"We're going to move Mark Harrell out to tackle. He's got the experience that he can go out there and do some things. (Colin) McGovern can play some tackle for us as well. We feel pretty good out there."
-- Brian Kelly prior to the start of '16 spring drills


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Category:Mosques converted from churches by the Ottoman Empire
From Wikipedia, the free encyclopedia
This category contains church buildings that were converted into mosques under the Ottoman Empire (14th century – 1923), regardless of their current use.
This category has only the following subcategory.
Pages in category "Mosques converted from churches by the Ottoman Empire"
The following 32 pages are in this category, out of 32 total. This list may not reflect recent changes (learn more).


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What evidence was provided by Darwin?
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An extremely catchy pop riff and an iconic music video helped make a-ha's first #1 single 'Take On Me' a classic and much-loved song for millions around the world. The video has been viewed over 180 million times on YouTube and the song remains on daily airplay on radio stations in many markets, nearly 30 years after its release.
Over the years, we've seen many great cover versions and tributes to 'Take On Me' and have been collecting links, media and facts about the song and now this page is a place on a-ha.com where all this information will be presented as a celebration of the legacy of the song.
Help us build our 'Take On Me' archive! We have collected a lot of information about the song over the years, but we need your help to make this the best archive of information about the song available online!
Please share links to your favourite 'Take On Me' covers and tributes in the comments below. Links to articles and other media are very welcome too!
"I think matt hales is a very talented writer and performer, and have personally been much inspired by his aqualung project.
of the many covers done of take on me over the years, this one is perhaps the furthest from the original, and in my view therefore the most interesting." – Magne Furuholmen, about the Aqualung cover of 'Take On Me'
Source: a-ha.com
"This recognizes something that has been going on now for almost 30 years. It's a funny thing. It's a resiliant bugger. It's wonderful." – Morten Harket, about the BMI 3 Million Award
Source: YouTube
"I am continuously surprised how well something you wrote at 15 could continue to be relevant at 50. Thanks to BMI for reminding me of that." – Magne Furuholmen, about the BMI 3 Million Award
Source: YouTube
"It lives it's own life, we can't really touch it." – Morten Harket, about the 'Take On Me' riff as part of the 'Feel This Moment' song by Pitbull and Christina Aguilera
Source: YouTube
"When we were recording 'Take On Me', I had no idea it was going to be the hit it turned out to be", Gerry Kitchingham admits. "Maybe that's because I was making records every day, but it didn't really jump out at me. Only later on did I really get to appreciate it."
Source: Sound On Sound
"I have no doubt that the video made the song a hit," Furuholmen says. "The song has a super catchy riff, but it is a song that you have to hear a few times. And I don't think it would've been given the time of day without the enormous impact of the video."
Source: Rolling Stone
"Notable for using the combination of pencil-sketch animation and live action called rotoscoping (whereby real footage is traced over), 'Take On Me' wasn't just groundbreaking but genuinely nerve-racking […] More than 25 years on, it remains as thrilling as ever to watch."
Source: Time.com
"It was unbelievable," Paul says. "It was as if my whole life flashed through my mind at once when I heard it." Paul Waaktaar-Savoy, about hearing that 'Take On Me' went to #1 on the US Billboard chart in October 1985.
Source: a-ha.com
"I knew that it was going to look good but I had no idea it was going to be in heavy rotation on MTV for a year," says Mike Patterson, who, with wife Candace Reckinger over 16 weeks, sketched some 3,000 drawings over individual frames.
Source: bbc.co.uk
"I can go anywhere on the earth, I've been in so many different continents, and wherever I go, everybody goes, 'I love that video'." – Mike Patterson.
Source: bbc.co.uk
Billboard Hot 100
Below is a selection of links covering "Take On Me", but it is by no means exhaustive. Please share the links you find in the comments below and we will add to this list. Thanks!
- a-ha´s video Take On Me ranked as #8 as best music video ever
- a-ha receive Multi-Million Performance Award for "Take on Me"
- a-ha presented with a BMI Million-Air Award
- Aqualung covers 'Take On Me'
- Back to the beginning
- Celebrating the Legacy of "Take On Me"
- Contest: Win a copy of the new book 'Mad World'
- Harry Hill's "Take On Me" video for Children in Need
- Live Q&A with Magne today on Facebook
- Morten at the Billboard Music Awards
- Morten will perform at the Billboard Music Awards!
- New Volkswagen ad inspired by "Take On Me"
- "Take On Me" (adoption, addiction and a-ha)
- 'Take on Me' back in the US Top 20!
- "Take on Me" chosen as the greatest Norwegian hit of the last 50 years
- 'Take on Me' featured in 'Spectacle' exhibition in Brazil
- "Take On Me" – Greatest Video Ever?
- "Take On Me" – The video success
- The Washington Nationals 'Take on Me' phenomenon
- Vote for "Take On Me"
- The 30 All-TIME Best Music Videos
- The Girl in the Video: "Take On Me"
- AC Newman covers a-ha's 'Take On Me' for Starbucks
- Pitbull + Christina Aguilera 'Feel This Moment' With A-Ha Frontman Morten Harket at 2013 Billboard Music Awards
- VW redoes A-ha's 'Take On Me' music video
- Taking on a-ha classic
- Billboard Music Awards push big sales gains
- Pitbull and Christina Aguilera Sample A-ha's 'Take On Me': Hear Song Snippet
- Pitbull and Christina Aguilera 'Feel This Moment' with A-ha on Billboard Awards
- En fantastisk følelse
- Take On Me' by a-ha
- Giants' Michael Morse uses 1980's pop song for his A-ha moment
- "Take On Me" – Videosuksessen
- The secret history of a-ha's smash hit 'Take On Me'
- Classic Tracks: a-ha's 'Take On Me'
- New A.C. Newman – "Take On Me" (A-ha Cover)
- Volkswagen pays homage to A-ha's 'Take on Me' video in new ad
- Chuck Finale Postmortem
- Video: Volkswagen gets inspired by A-ha's 'Take on Me'
- Harket på scenen med Aguilera og Pitbull
- «Take on me» i sesongfinalen av «Grey's Anatomy»
- This Week in Billboard Chart History: 30 Years Ago, a-ha's 'Take on Me' Topped the Hot 100
- Talking Away: A-Ha On The Making Of Take On Me
- A-ha unveils alternate version for music video 'Take on Me'
- A-ha och 30 år med Take On Me
- A-ha: how we made Take on Me
- Watch an Alternate Version of A-ha's Classic 'Take on Me' Video
- A-ha reunite on 30th anniversary of Take On Me single success
- Take On Me video has career on its own, A-ha singer Morten Harket admits
- 30 år siden a-ha toppet Billboard: Hvor er deres etterfølgere?
- Tidsvitne: Take On Me til topps i USA 4:6
- 'Take On Me': a-ha's one American hit still a wonder
- a-ha celebrates 30th anniversary of 'Take On Me'
- "Take On Me til topps I USA"
- "Take On Me" video featured on Snapchat
- Celebrating 30 years since a-ha's breakthrough with "Take On Me"
- "Take On Me" 30th anniversary picture disc
- Record Store Day 2015
- "Take On Me" at #8 in the UK Vinyl singles chart
- Kygo to remake "Take On Me" for streaming on Apple Music
- Kygo releases remix of "Take On Me"
- Hydro and a-ha collaboration leads to Kygo's "Take On Me" remix
- Alternate version of the iconic "Take On Me" video
- 30th anniversary of "Take On Me" topping the Billboard chart


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4 Min Read
WASHINGTON (Reuters) - Group of 20 member nations must avoid beggar-thy-neighbor currency policies and the richest advanced economies need to stick to their long-standing rule to let market forces set their exchange rates, a senior U.S. official said on Monday.
Treasury Undersecretary Lael Brainard, the top U.S. official for international economic affairs, said fiscal and monetary policies should be aimed at achieving domestic objectives, as opposed to targeting a weaker currency to bolster exports.
"The G20 needs to deliver on the commitment to move to market-determined exchange rates and refrain from competitive devaluation," she told reporters at a briefing outlining U.S. priorities for a G20 finance ministers meeting on Friday and Saturday in Moscow.
Free exchange rates also help support fair global growth, Brainard said, adding that the meeting needed to focus on ways to strengthen the fledgling global economic recovery and avoid an undue tightening of fiscal policy that could hurt growth.
"Global growth is weak and vulnerable to the downside. Strengthening global demand must be at the top of the G20" agenda, Brainard said. "We must avoid jeopardizing the recovery with a premature shift to restraint."
She added that Europe in particular could consider "recalibrating" the pace of its fiscal consolidation, as unemployment remains high and the euro area as a whole is stuck in recession.
In recent weeks, currency concerns have jumped to the top of the agenda for the Moscow meetings of the G20 group of advanced and emerging economies.
U.S. and European officials privately have been concerned about comments from Japanese officials that suggest Tokyo is targeting a specific level for the yen.
Japan's new government has pressed for aggressively expansionary monetary policies, which have prompted the Japanese currency to weaken sharply. The yen has lost 15 percent against the U.S. dollar since October, and last week hit a near three-year low against both the dollar and the euro.
The G7 is considering issuing a statement this week reaffirming its commitment to "market-determined" exchange rates, two G20 officials said earlier on Monday.
"For the market process to work, exchange rates must be allowed to reflect market forces," Brainard said. "The G7 has long committed that exchange rates should float, except in rare circumstances where excess volatility or disorderly movements might warrant cooperation."
Brainard declined to say whether the United States has expressed its concerns to Japan in particular.
"We support the effort to reinvigorate growth and to end (deflation) in Japan," she said. "And of course, we also know Japan is an important member of the G7, and we'll continue to work with Japan in the G7 to continue to adhere to our important agreements in that forum."
Market participants interpreted her comments as suggesting the United States would not officially criticize Japan's currency policy, sending it even lower on Monday. The dollar rose to its highest level since May 2010 versus the yen, and the euro gained more than two percent against it.
Brainard will be attending the G20 meetings for the United States. President Barack Obama's Treasury nominee, Jack Lew, has not yet been confirmed by the Senate, and the acting secretary, Neal Wolin, is not able to attend as he is running the agency.
Reporting by Anna Yukhananov; Editing by Andrew Hay and Paul Simao


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