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Hello all,
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Cheeca Lodge & Spa
Islamorada, Florida
Surrounded by the beautiful natural splendor and peaceful tropical ambiance of Islamorada , in the Florida Keys, is the fabulous Cheeca...
Imagine lush tropical gardens, glistening pools, and the gentle rush of waterfalls surrounding your room as you wake to the sun warming the beach. This is a picture of your first morning at Cheeca Lodge & Spa. On Islamorada in the Florida Keys, elegant rooms just steps from the beach offer screened balconies and a peaceful Caribbean style. Spacious and airy, with plush accommodations, all the modern amenities, and luxuries like pillow-top beds with fine linens and high definition televisions, you will relax in splendor. Recline on your balcony and enjoy the tranquil beach atmosphere, and delight in all the wonders of Islamorada, at Cheeca Lodge & Spa.
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Hotfrog Canada provides information regarding Fridge-a-Rama in Winnipeg MB. Fridge-a-Rama is located at 319 Queen St, Winnipeg MB R3J 1K9 and provides Retail services. Contact them on (204) 786-0055.
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121 Irene St WINNIPEG MANITOBA R3T 4C7
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By Ken Barnes, USA TODAY
Britain is busting out with hot new bands and rejuvenated artists. You'll be hearing about tons of them: "new rave" dance rockers The Klaxons, the angular Rakes and these artists:
Jarvis Cocker. The veteran former leader of Brit-pop heroes Pulp releases his first solo album April 4, and it's a musically lavish, lyrically caustic triumph.
The Feeling. Unashamed pure pop band from Sussex, labeled as derivative of '70s pop but actually sounding far more timeless on just-released first album Twelve Stops and Home.
Kaiser Chiefs. Arguably the biggest pop band in the U.K., the Chiefs — from Leeds — failed to establish much of a beachhead with their first album. But new single Ruby is a modern-rock hit and a fine taster for new album Yours Truly, Angry Mob, out Tuesday.
The Fratellis. Glasgow band with a Ramones-like disdain for surnames (instead, they all share the band name), attitude to spare and enoughpunky musical verve to back up their swagger. Flathead is featured in an iPod commercial, and first album Costello Music is just out.
The Kooks. Endlessly clever Brighton band has mastered the art of the insistently catchy pop single, as heard on first album Inside In/Inside Out.
he View. From Dundee, Scotland, they share a producer (and a combative attitude) with Oasis but sound more versatile, ranging from fairly raw rock to almost vaudevillian tunefulness. First album Hats Off to the Buskers arrived Tuesday.
More Brits worth checking out: The Automatic, The Bishops, The Cinematics, Clinic, The Harrisons, Charlotte Hatherley, The Horrors, Little Man Tate, Long Blondes, Maximo Park, Tokyo Police, Club Unkle Bob
Conversation guidelines: USA TODAY welcomes your thoughts, stories and information related to this article. Please stay on topic and be respectful of others. Keep the conversation appropriate for interested readers across the map.


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Backstage 2004 | TV Show Watchlist
Artists discuss their movies and music, and take viewers behind the scenes of their films and recordings.
Premiered: August 20, 2004
Rating: None
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The music students put aside their differences to get through a difficult day. Vanessa makes a promise she might not be able to keep. Denzel must deliver bad news to a friend.
Carly is desperate to cheer up Vanessa after getting her kicked out of the primas. Bianca struggles to come clean with her secret. Denzel must make his mark in the art world before he turns 14.
The fascinating capitals of Finland and Estonia offer a chance to sample each country's history, art, and distinct love of life. We'll start in Helsinki with its Neoclassical old town, modern flair for design, and steamy saunas. Then it's just a two-hour boat ride to Tallinn—with its medieval charms and new-found prosperity—celebrating its freedom and thriving in its post-USSR renaissance.
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- Premiered: August 20, 2004
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- Détails
- Avis
Langues de l'excursion
Points forts
- Vivez une soirée romantique avec dîner
- Explorez les rues de la ville lors d'une visite à pied
- Visite complète de la ville
- Conseils avertis d'une personne de la région
- Escapade romantique, parfaite pour les couples
11am - 12.pm: Hotel pickup
12pm - 12:30pm: Bajra Sandhi Monument
Bajra Sandhi Monument is a monument to admire Balinese people struggle from time to time. The monument is located in a public open space and has very stunning view. From the top of the monument, you can see the city of Denpasar with a 360 view.
1pm - 1:30pm: Museum Bali
A museum of art and history. The museum was built in 1931 by architect P.J. Moojen, near the location of the former royal palace of Denpasar, which had been burnt to the ground during the Dutch intervention in Bali (1906), and used it as a model for its outside walls and courtyards.
1:50pm - 2:20pm: Badung Market
The biggest traditional market of Bali. It is divided into two different market. West building is handicraft and art market and east building is space for daily grocery market. Almost any kind of products can be found here. From food to textile are available with affordable price. Don't forget to bargain if you are entering and shopping into a traditional market in Bali.
3:20pm - 4pm: Water Blow
Great view of soaring sea water, splashing and shaping the sea rock walls. Water Blow is a place where the rock that stands firmly on the beach of Nusa Dua with a beautiful shape swept by the large waves. Because the waves crashing into the walls of the reefs would eventually make the water-sea water is soaring into the air.
5pm - 7pm: Uluwatu Temple and Kecak Dance
This cliff-top temple is one of the most important temple of Bali, dedicated for the God of Rudra. Uluwatu is perfect place for viewing the sunset. Please be aware of the monkeys, sometime they are smart enough to grab your hat or sunglasses and attempt to barter for your food.
A kecak and fire dance performed next to the temple during the sunset time. The dance is the most spectacular dance. The story of the dance is taken from Ramayana story. In general it is a romantic dance of Rama and Sita. A brave and patriotic dance of Hanoman, a loyal white monkey to his adoration, Rama.
7:40pm - 8:40pm: Jimbaran Barbecue Dinner
Enjoy the magical barbecue dinner in an open space while sitting at a table on the beach with your toes in the sand or have a seat on the roofed sea terrace overlooking Jimbaran Bay, colorful fishing boats in front. Sip a cocktail, enjoy the delicious seafood or try Balinese specialties freshly prepared for you.
8:40pm - 9:30pm: Back to your hotel
Photos récentes de cette excursion
Calendrier détaillé
Sélectionnez un mois pour afficher le calendrier des disponibilités et le détail des départs.
Nusa Dua, Jimbaran, Kuta, Legian, Seminyak, Sanur, Denpasar, Ubud
10:30am: Ubud
11am: Nusa Dua, Jimbaran, Kuta, Legian, Seminyak
11:30am: Sanur, Denpasar
Le ramassage à l'hôtel commence plus tôt
Informations tarifaires
Cliquez sur le lien ci-dessous pour vérifier le prix et la disponibilité pour la date de voyage qui vous intéresse. Nos tarifs sont constamment mis à jour afin que vous puissiez toujours bénéficier du meilleur prix possible.
Code : DEFAULT
à partir de
Informations supplémentaires
Bajra Sandhi MOnument
- Entrance fees
- Bottled water
- Dinner
- Driver/guide
- Hotel pickup and drop-off
- Food and drink not mentioned in itinerary
- Personal expenses
- Gratuities (optional)
- Confirmation will be received at time of booking
- A minimum of 2 people per booking is required
- Please advise any specific dietary requirements at time of booking
Conditions d'annulation
Si vous annulez de 3 à 6 jours avant la date de départ prévue, des frais d'annulation de 50 % seront appliqués.
Si vous annulez au moins 2 jours avant la date de départ prévue, des frais d'annulation de 100 % seront appliqués.


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Binyamin Netanyahu, Israel's prime minister, has again ruled out accepting a settlement to the Israeli-Palestinian conflict based on the 1967 borders.
Addressing a conference of the American Israel Public Affairs Committtee (AIPAC) in Washington late on Monday, Netanyahu said Israel could not return to the "indefensible" 1967 borders - something that the US president Barack Obama had suggested a few days earlier.
He said "this conflict has raged for nearly a century because the Palestinians refuse to end it. They refuse to accept the Jewish state. This is what this conflict has always been about."
"Peace with Palestinians must leave Israel with security, therefore Israel cannot return to the indefensible 1967 lines," he said.
Yasser Abed Rabboh, secretary-general of the PLO executive committee, said in a interview that Netanyahu's AIPAC speech "was further evidence that Netanyahu is not interested in initiating any serious political process".
"His aim is to buy time and to use forces inside the US Congress to hinder the process at large," he said.
"We welcome Obama's speech and particularly his affirmation of the 1967 lines, and hope this will turn into a UN resolution and a shared stance by the [Middle East] Quartet," he said, in reference to the US president's speech on the Middle East on Thursday.
'Unvarnished truth'
Netanyahu said he would set forth his view of a future Middle East peace in an address to the US Congress on Tuesday, stating again that Israel would never return to its old borders.
"I will outline a vision for a secure Israeli-Palestinian peace ... I intend to speak the unvarnished truth. Now, more than ever, what we need is clarity and events in our region are finally opening people's eyes on the simple truth.
"People in the region are thinking about freedom, yearning for freedom and opportunity, so it's time to stop blaming Israel for all of the region's problems."
Israel and Palestinian peace is "not a panacea for endemic problems of the Middle East. What will change this is one word: democracy, real genuine democracy," he said.
In an address disrupted several times by protesters, the Israeli prime minister condemned the Palestinian group Hamas.
"Imagine keeping a young soldier locked in a dark dungeon for five years without a single visit of the Red Cross," Netanyahu said, referring to Gilad Shalit, the Israeli soldier held captive by Hamas since 2006.
"I think the entire civilised community should join Israel, America and all of us in a simple demand: release Gilad Shalit," he said, describing the soldier's capture as an "outrageous crime".
Israeli anger
Obama drew Israeli anger on Thursday by saying that a Palestinian state in the occupied West Bank and Gaza Strip should largely be drawn along lines that existed before the 1967 war in which Israel captured those areas and East Jerusalem.
On Sunday, Obama presented that blueprint again, in his own address to AIPAC. But he seemed to ease Israeli anger somewhat when he made clear Israel would likely be able to negotiate keeping some settlements as part of land swaps in any final deal with the Palestinians.
Peace talks are frozen, largely over the issue of Israeli settlements in the West Bank. Neither Obama nor Netanyahu have offered a concrete plan to try to revive them.
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Mallin Colorado Eco Lodge
Km 273 de la carretera Austral Sur - General Carrera Lake. See Map
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- Terra Luna Lodge: 5,2 mi
- Hotel Cabañas El Mirador de Guadal: 5,4 mi
Touristic attractions near Mallin Colorado Eco Lodge
Near touristic attractions
|Laguna San Rafael National Park
|3,0 mi
|Capilla de Marmol Natural Sanctuary
|14 mi
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|44 mi
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HM REVENUE & CUSTOMS has managed to recover more than £26m missing inheritance tax in the last year with the aid of new software.
The taxman developed new computer software to cross-reference bank accounts, employment history, company ownership and property transactions, The Times reports.
The technology, known as Connect, can sift through huge amounts of information and looks at about 300,000 people a year who claim their inheritance tax (IHT) is below the £325,000 threshold.
Treasury minister David Gauke (pictured) is expected to say at a conference today: "As [HMRC] finds new ways to exploit technology further, there will no doubt be more that can be done to make data handling even easier, more thorough, and more effective.
"My ambition is for the UK to become the market leader at using data analytics to drive a more effective approach to taxpayer interactions – moving towards a data-driven view of taxpayers that automatically draws on comprehensive data from across the department, wider government and third parties.
"Through harnessing technology to use limited resources more effectively, I see HMRC at the forefront of the nation's deficit reduction ambitions."
Wading into the subject is Labour MP Michael Meacher who is calling for tighter rules to prevent accountants from helping their clients avoid paying tax – categorising evasion and avoidance together, he said: "Tax avoidance and evasion is, I believe, a cancer in Britain's society today".
Margaret Hodge, the chairwoman of the Public Accounts Committee, said: "I think it is a terrible sickness at the heart of our society, that too many well-heeled individuals and corporations simply don't accept they too have a duty from their wealth, their legitimate wealth, of contributing according to their means to a society from which they expect to take according to their needs and their expectations."
Connect was introduced in 2008 but was upgraded last year.
Related reading
Edwin Coe recruits HMRC operational head of fraud investigation team
At HMRC, Dmitri Surendran was responsible for leading the London team of the offshore, corporate and wealthy unit of the fraud investigation service
The rising significance of inheritances
Rosamond McDowell looks at key changes to inheritance tax policy, which apply from April this year
Budget is a 'springboard' for tax policy reform, says new report
Report argues that the government must change the way it makes tax and budget decisions
Treasury Select Committee report released on Making Tax Digital
Committee expresses concern about costs to businesses and April 2018 implementation date


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iPhone apps
Violin Solos
Improve your musical and coordination skills with this challenging violin game. Not as challenging as playing a real violin but it can still give your hand and fingers a good workout. Haven't played a violin before? Well, this is your chance to….. Read more
What's New in Version 1.2
- Fixed crash problems for some devices
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Akhilesh Yadav's supporters stage a show of strength
| Updated On : September 17, 2016 7:22 pm
If on Thursday and Friday supporters of Shivpal Singh Yadav created the din when he was stripped of important portfolios, on Saturday supporters of Akhilesh Yadav took to the street.
Mulayam Singh Yadav had never faced such a rebellion in the party. When he was all set to address young party activists, they started shouting slogans demanding Akhilesh Yadav's reinstatement as State president. Mulayam was visibly taken aback. It never happened in his lifetime as he faced a handful of young activists raising slogans.
"I have built this party with my sweat and blood … I will not allow this to happen," he shouted in the microphone amid that din. "All of you keep quiet and listen to me," he said raising his hand like a referee. Volunteers got the message and fell silent. But some of them were still standing on their feet. They were carrying the placard of Akhilesh. "Usse niche karo," (keep it down)", Mulayam screamed.
He then asked the leaders: " How many booth members you people have made. When BJP is working at the booth level you are staging protest over nothing. Where is the conflict? Shivpal and Akhilesh are one."
"When you are shouting slogans here, Akhilesh is having lunch with Shivpal … go and check it out yourself," Mulayam said. It was probably Mulayam's suggestion asking Akhilesh to go and visit his Chacha with family. Later Akhilesh confirmed that he had `chai' with his chacha.
It was a show of strength between Chacha and Bhatija. If on Thursday and Friday supporters of Shivpal Singh Yadav created the din when he was stripped of important portfolios, on Saturday supporters of Akhilesh Yadav took to the street. Presidents of four youth wings of the party– Samajwadi Yuvjan Sabha, Samajwadi Chhatra Sabha, Lohia Vahini and Mulayam Youth Brigade- conveyed to the party president that they won't work under Shivpal Singh Yadav, who was appointed state president.
SP MLC Anand Bhadoria, a close confidant of Akhilesh Yadav , told reporters in front of the CM residence that the party president should reinstate Akhilesh Yadav as the state president immediately. "How can the party change the Senapati in the middle of a war," he questioned.
Bhadoria further said that Netaji should hand over the party to young blood.
Supporters of CM announced that they will continue to protest till Akhilesh Yadav was made the party's state president." When Netaji has returned posts of all the leaders, then why Akhilesh Yadav has been ignored," Sanjay Yadav from Sultanpur said.
Sanjay, along with seven of his friends, was camping here in support for the CM. He further claimed that Akhilesh Yadav has turned into a ' Vikas Purush' for his work during the past over four-and-a-half years.
Even legislators were divided over the raging issue. Around 32 party MLAs were sitting inside the CM's residence in the morning while 21 with Shivpal Singh Yadav at the same time.
Later, Akhilesh asked members of the youth organisation not to raise slogans and put up posters, banners, and hoardings. It is election time. Go back to your respective constituencies and work for mobilising the people," he said.
The Chief Minister also announced that the proposed `Vikas Se Vijay Yatra' from October 3 has been postponed. "On October 4, we will lay the foundation stone of the metro rail in Kanpur and the dates of Yatra will be worked out after consulting the astrologers as to which direction one should go on a particular day''.


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Scavenger receptor class B type I regulates cellular cholesterol metabolism and cell signaling associated with breast cancer development
DOI: 10.1186/bcr3483
© Danilo et al.; licensee BioMed Central Ltd. 2013
Received: 6 March 2013
Accepted: 16 August 2013
Published: 24 September 2013
Previous studies have identified cholesterol as an important regulator of breast cancer development. High-density lipoprotein (HDL) and its cellular receptor, the scavenger receptor class B type I (SR-BI) have both been implicated in the regulation of cellular cholesterol homeostasis, but their functions in cancer remain to be established.
In the present study, we have examined the role of HDL and SR-BI in the regulation of cellular signaling pathways in breast cancer cell lines and in the development of tumor in a mouse xenograft model.
Our data show that HDL is capable of stimulating migration and can activate signal transduction pathways in the two human breast cancer cell lines, MDA-MB-231 and MCF7. Furthermore, we also show that knockdown of the HDL receptor, SR-BI, attenuates HDL-induced activation of the phosphatidylinositol 3-kinase (PI3K)/protein Kinase B (Akt) pathway in both cell lines. Additional investigations show that inhibition of the PI3K pathway, but not that of the mitogen-activated protein kinase (MAPK) pathway, could lead to a reduction in cellular proliferation in the absence of SR-BI. Importantly, whereas the knockdown of SR-BI led to decreased proliferation and migration in vitro, it also led to a significant reduction in tumor growth in vivo. Most important, we also show that pharmacological inhibition of SR-BI can attenuate signaling and lead to decreased cellular proliferation in vitro. Taken together, our data indicate that both cholesteryl ester entry via HDL-SR-BI and Akt signaling play an essential role in the regulation of cellular proliferation and migration, and, eventually, tumor growth.
These results identify SR-BI as a potential target for the treatment of breast cancer.
Breast cancer is estimated to have affected >200,000 women in the United States in 2012 and will be responsible for approximately 40,000 deaths, making it the second leading cause of cancer deaths [1]. Recently, attention has focused on examining the role of components of the diet, such as cholesterol, in breast cancer development. Plasma cholesterol levels are often lower in patients with advanced lung [2] and breast cancers [3–5], a phenomenon known as the "preclinical effect of cancer" [6]. This effect is believed to be consequential and not causal [6] but does suggest a role for cholesterol in cancer. Several epidemiologic studies have shown a positive correlation between elevated high-density lipoprotein cholesterol (HDL-C) levels and breast cancer risk [5, 7–9], and recent data have corroborated this claim [10–16]. In vivo works have suggested that hypercholesterolemia induced by diet and/or genetic background leads to increased tumor burden and metastasis in murine breast cancer models [10, 12]. In vitro analyses have shown that human breast cancer cell lines exhibit increased proliferation and migration in the presence of HDL [11, 13, 15–17]. The effect of cholesterol on breast cancer may be attributed to several of its properties and functions. Cholesterol is the precursor of bioactive steroid hormones such as estrogen. It is also necessary for the formation of plasma membrane microdomains known as lipid rafts [18]. Lipid rafts are believed to organize signaling molecules in the plasma membrane and, as a result, have been implicated in the development of human cancers [19]. Therefore, cholesterol may play an essential role in the regulation of tumor growth [20, 21].
The HDL lipoprotein is an important carrier of plasma cholesterol and can function as a signaling molecule by initiating MAPK and AKT signaling pathways and stimulate migration in endothelial cells [22–24]. The activation of these signaling pathways is dependent on HDL binding to the HDL receptor, the scavenger receptor class B, type I (SR-BI), and subsequent lipid transfer to the cell [25–27]. SR-BI functions as the HDL receptor and has been shown to mediate the selective transfer of cholesteryl ester from HDL molecules to cells in a process known as the selective HDL-cholesteryl ester uptake [28]. Its role in the development of atherosclerosis has been well documented [28], but its role in cancer has not been extensively investigated. Nevertheless, SR-BI has been implicated in prostate [29] and breast cancer [15, 30]. In the case of breast cancer, SR-BI protein levels were found to be increased in malignant tissue samples compared with the normal surrounding tissue [30].
In the present study, we have examined the role of HDL and SR-BI in the regulation of cellular signaling pathways in breast cancer cell lines and in the development of tumors in a mouse xenograft model. Our data show that HDL can stimulate migration and can activate signal-transduction pathways in the two human breast cancer cell lines, MDA-MB-231 and MCF7. Furthermore, we also show that knockdown of the HDL receptor, SR-BI, attenuates HDL-induced activation of the MAPK and PI3K/Akt pathways in both cells lines. A more detailed analysis reveals that SR-BI regulates signaling pathways via Akt activation, and the regulation of SR-BI expression or activity can limit tumor development in a mouse model.
The following antibodies were used: SR-BI was from Novus Biologicals, Inc. (Littleton, CO, USA). CD31 antibody was from Abcam, Inc. (Cambridge, MA, USA). Phospho-Erk1/2 (T202/Y204), Erk1/2, Phospho-Akt (S473), and Akt were from Cell Signaling Technology, Inc. (Beverly, MA, USA). GAPDH was from Fitzgerald Industries International (Acton, MA, USA), and β-Actin was from Sigma-Aldrich Corp. (St. Louis, MO, USA). Anti-mouse secondary antibody was from Thermo Fisher Scientific, Inc. (Rockford, IL, USA), and anti-rabbit secondary antibody was from BD Biosciences (San Jose, CA, USA). The signaling inhibitors U0126 and LY294002 were from Cell Signaling Technology and Sigma-Aldrich, respectively. BLT-1 was from EMD Millipore (Billerica, MA, USA).
Cell culture
MCF7 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), and MDA-MB-231 cells were as previously described [31]. MDA-MB-231 and MCF7 cells were grown in Dulbecco modified Eagle media (DMEM) containing 10% fetal bovine serum (FBS) in an incubator kept at 37°C with 5% CO2.
Purification of lipoproteins
Human plasma was obtained from adult female volunteers. Approval for the use of human plasma was obtained from the Office of Human Research at Thomas Jefferson University, and consent was obtained from the volunteers for the use of their plasma samples. Lipoproteins (LDL (1.019-1.063 g/ml), HDL2 (1.063 to 1.125 g/ml), HDL3 (1.125 to 1.21 g/ml] were separated by density-gradient ultracentrifugation, as previously described [32]. HDL3 was dialyzed against PBS, 0.5 mM EDTA, 1% NaN3. In experiments with MCF7 cells, lipoproteins were stripped of estrogens with activated charcoal to remove endogenous steroid hormones, as previously described [16].
Migration and invasion assays
Migration was assayed by using modified, noncoated Boyden chambers from BD Biosciences (San Jose, CA, USA). Both MDA-MB-231 and MCF7 cells were cultured in 10-cm dishes in DMEM containing 10% FBS. Cells were harvested, counted, and washed 3 times with PBS. Cells were resuspended in DMEM containing 1% BSA, and 5.0 × 104 cells were added to the upper chambers. The bottom chambers contained 1% FBS or 100 μg/ml lipoprotein in 1% BSA. Cells were incubated at 37°C overnight. At the end of the experiment, upper chambers were swabbed with a cotton swab to remove nonmigrating cells, stained with crystal violet, and quantified by using phase-contrast microscopy. Migration was quantified by counting the number of cells in five separate fields at 10× magnification. Invasion assays were performed as described earlier for transwell-migration assays, but used Matrigel-coated Boyden chambers (BD Biosciences). The upper chambers contained cells in 1% BSA, and the bottom chambers contained 1% FBS or 100 μg/ml lipoprotein in 1% BSA.
Immunoblot analysis
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors from Roche Applied Science (Indianapolis, IN, USA) and Sigma-Aldrich Corp., respectively, and prepared and analyzed as previously described [33]. In brief, proteins were separated by sodium dodecylsulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Membranes were blocked in 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). Primary antibodies were diluted in 5% BSA in TBS-T and incubated for either 1 hour at room temperature or overnight at 4°C. Membranes were washed 3 times in TBS-T, and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Membranes were washed 3 times in TBS-T, and visualized by using Pierce Chemiluminescent Substrate from Thermo Fisher Scientific, Inc.
Knockdown of SR-BI
MDA-MB-231 and MCF7 stably transfected cell lines were produced by lentiviral transduction. Lentiviral particles containing a pool of three short-hairpin RNA (shRNA) constructs targeted against SR-BI were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Control lentiviral particles containing scrambled shRNA sequences were also purchased from Santa Cruz. Cells were transduced with the lentiviral particles according to manufacturer's protocol. Two days after transduction, target cells containing either control shRNA (shCTL) or shRNA against SR-BI (shSRBI) were selected by using 2.5 μg/ml puromycin. Successful knockdown was verified by immunoblot analysis.
Cholesterol-content determination
Cells were grown to confluence in 10-cm dishes in the presence of complete media (DMEM, 10% FBS). In half of the plates, cholesterol was extracted with isopropanol. The extract was dried down in glass tubes under nitrogen and resolubilized in a smaller volume of isopropanol. Cholesterol content was determined by using the Cholesterol E kit from Wako Chemicals USA, Inc. (Richmond, VA, USA) as per the manufacturer's instructions. In the other plates, cells were lysed with 0.5 NaOH, and lysates were collected. Protein concentration was determined with the bicinchoninic acid assay (BCA) from Thermo Fisher Scientific, as per the manufacturer's instruction. Total cellular cholesterol levels were calculated by dividing the total cholesterol by the total protein per dish.
3H-Thymidine incorporation proliferation assays
Cell proliferation was measured with [3H]-thymidine incorporation to assess DNA synthesis and proliferation. MDA-MB-231 cells were seeded (5.0 × 104 cells/well) in 12-well plates in 1 ml of DMEM containing 10% fetal bovine serum (FBS) and grown overnight. The following day, media was aspirated, cells were washed twice with PBS, and were serum-starved with 1% fatty acid free bovine serum albumin (BSA) for 1 hour. Media containing FBS, 1% BSA, or 1% BSA with lipoproteins (100 μg/ml) and 1 μCi/ml of 3H-thymidine from Perkin Elmer (Waltham, MA, USA) was added to cells. Cells were incubated for 6 hours, at which time media was removed, cells were washed twice with PBS and incubated in 10% trichloroacetic acid to precipitate DNA. Cells were solubilized in 0.1 M NaOH and 1% SDS. Radioactivity was measured by liquid scintillation counting. Protein concentration was determined by using the BCA assay.
Tumor studies
All mice were housed and maintained in a barrier facility at the Kimmel Cancer Center at Thomas Jefferson University. Mice used in this study were athymic nude mice obtained from Taconic (Hudson, NY, USA). Animal protocols used for these studies were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University. MDA-MB-231 (106) cells containing either shRNA targeted against SR-BI (shSRBI) or control shRNA containing scrambled shRNA (shCTL) were subcutaneously injected in the flanks of 7- to 9-week-old nude mice. MCF7 cells (5 × 106) were orthotopically injected into the mammary fat pad of 9-week-old athymic nude mice implanted with slow-release 17β-estradiol pellets (0.36 mg/pellet, 60 days) from Innovative Research of America (Sarasota, FL, USA). Four weeks after injection, tumors were excised, weighed, and the volume was determined by using the formula (width2 × length)/2. Half of each tumor was flash frozen and stored at -80°C and subsequently homogenized and lysed in RIPA buffer for immunoblot analysis, as previously described [33]. The other half was fixed in formalin for 24 hours and then used to prepare paraffin-embedded sections.
Immunohistochemical analysis
Paraffin-embedded tumor sections were deparaffinized in xylene and rehydrated. Antigen retrieval was performed in 10 mM citrate buffer pH6 for 10 minutes by using a pressure cooker. Endogenous peroxidase activity was blocked with 3% H2O2, and sections were blocked in 10% goat serum obtained from Vector Laboratories, Inc. (Burlingame, CA) and incubated with primary antibody overnight at 4°C. Sections were washed 3 times with PBS, incubated with biotinylated secondary antibody for 30 minutes, followed by HRP-conjugated streptavidin for 30 minutes by using a Streptavidin-HRP kit from Dako North America, Inc. (Carpinteria, CA, USA). After three washes in PBS, the presence of bound antibody was visualized by using 3,3′-diaminobenzidine (DAB). Slides were counterstained with hematoxylin, dehydrated, and mounted with coverslips.
TUNEL assay
Apoptosis was measured with TUNEL assay by using the TUNEL-based ApopTag Peroxidase In Situ Apoptosis Detection Kit from Millipore (Temecula, CA, USA), as per manufacturer's instructions. In brief, paraffin-embedded tumor sections were de-paraffinized and rehydrated. Sections were treated with 20 μg/ml proteinase K from Roche Applied Science (Indianapolis, IN, USA) for 15 min at room temperature and washed, and peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 5 minutes. Sections were then incubated with equilibration buffer, followed by incubation in TdT enzyme for 1 hour at 37°C. After washing, sections were incubated with HRP-conjugated antibody directed again digoxigenin for 30 minutes at RT, washed, and apoptotic positive cells were visualized by using DAB. The percentage of apoptotic cells was quantified by dividing the number of TUNEL-positive cells by the total number of cells observed in four distinct fields per section.
Statistical analyses
All values are expressed as the mean ± standard deviation (SD). The Prism 4.0 program (GraphPad Software, Inc., San Diego, CA, USA) was used for statistical analysis. Statistical significance was examined by using the Student's t test or ANOVA when appropriate.
HDL3stimulates migration and activates Akt and Erk1/2 in MCF7 and MDA-MB-231 cells
These results indicate that HDL3 can function as a signaling molecule in these two breast cancer cell lines. LDL had a modest effect on Akt activation, and no effect on Erk1/2 activation in either MDA-MB-231 or MCF-7 cells was observed (Figure 1D).
Knockdown of the HDL receptor, SR-BI, attenuates the effects of HDL3on signaling in MDA-MB-231 and MCF7 cells
To determine the role of SR-BI on the regulation of signaling pathways, both shCTL and shSRBI MDA-MB-231 and MCF7 cells were serum starved overnight and then incubated in media containing 10% FBS for 30 minutes or 100 μg/ml of HDL3 for 0, 5, 15, and 30 minutes, as indicated. We found that the activation of Akt was greatly reduced in the shSRBI cells compared with the shRNA control cells. Similar results were obtained with both MDA-MB-231 and MCF7 cell lines in the presence of FBS (lanes 1 and 6 in Figure 2A and B). Consistent with the results presented in Figure 1C, HDL3 was able to stimulate the activation of Akt in both cell lines in a time-dependent manner. However, activation of Akt in shSRBI MDA-MB-231 cells was greatly reduced when stimulated by HDL3 for 15 and 30 minutes (Figure 2A, lanes 9 and 10), compared with the Akt activation observed in shCTL MDA-MB-231 cells when stimulated by HDL3 for the same periods (Figure 2A, lanes 4 and 5). Similar results were obtained in MCF7 cells. In that case, Akt activation was reduced in the shSRBI MCF7 cells when stimulated by HDL3 for 15 and 30 minutes (Figure 2B, lanes 9 and 10), compared with shCTL MCF7 cells stimulated by HDL3 for the same periods (Figure 2B, lanes 4 and 5).
Finally, Erk1/2 appeared to be constitutively active in MDA-MB-231 cells (Figure 2A, lanes 1 to 10). However, almost no change in Erk1/2 activation was detected in shSRBI MDA-MB-231 cells treated with HDL3 for 30 minutes (Figure 2A, lane 10) compared with shCTL MDA-MB-231 treated with HDL3 for 30 minutes (Figure 2A, lane 5). This effect was in contrast with observations made with MCF7 cells (Figure 2B). In shCTL MCF7 cells, HDL3 rapidly stimulated Erk1/2 activation, reaching a peak at 5 minutes (Figure 2B, lane 3) but maintaining a sustained effect at 30 minutes (Figure 2B, lanes 4 and 5). Activation of Erk1/2 in shSRBI MCF7 cells followed a similar pattern, but the intensity of activation was greatly reduced (Figure 2B, lanes 7–10). These results suggest that downregulation of SR-BI in MDA-MB-231 and MCF7 cells attenuates signaling via the AKT and MAPK pathways. Additionally, our results show that the interaction between HDL and SR-BI regulates activation of these signaling pathways.
Finally, the effect of LDL was also tested in these cell lines. Results presented in Figure 2C and 2D demonstrate that the downregulation of SR-BI in MDA-MB231 and MCF7 cells had no effect on the regulation of Akt and Erk1/2 activation by LDL.
Knockdown of the HDL receptor, SR-BI, inhibits proliferation and migration of MDA-MB-231 cells
Previous studies have shown that a mutant of SR-BI inhibits proliferation of the luminal B subtype of human breast cancer cells, MCF7, in the presence of HDL [30]. Further, to investigate the role of SR-BI in a triple-negative (lacking the estrogen receptor (ER), progesterone receptor (PR), and Her2) basal B subtype breast cancer cell line, we determined the effect of knocking down SR-BI on the proliferation of MDA-MB-231 cells. The proliferation of shSRBI MDA-MB-231 cell was reduced by twofold compared with the proliferation observed with shCTL MDA-MB-231 cells (P < 0.01) (Figure 3B). Knockdown of SR-BI also significantly reduced cellular migration (P < 0.05) by 1.65-fold (Figure 3C). Finally, a reduction of SR-BI protein levels was associated with a marginally significant reduction of the ability of MDA-MB-231 cells to invade (Figure 3D) (P = 0.0517).
Pharmacologic inhibition of SR-BI reduces proliferation and signal transduction in MDA-MB-231 cells
We also examined the effect of BLT-1 on signal transduction in these cells. In agreement with the finding described in Figure 2A, Akt activation in shSRBI MDA-MB-231 cells treated with FBS for 30 minutes was reduced compared with shCTL MDA-MB-231 cells (Figure 4B). Similar results were obtained with shCTL MDA-MB-231 cells with treated BLT-1. Akt activation was reduced in the treated shCTL MDA-MB-231 cells compared with untreated control cells. Finally, SR-BI knockdown or pharmacologic inhibition had no effect on Erk1/2 activation (Figure 4B) compared with the control cells. Collectively, these data suggest that Akt activation may be mediated, in part, by SR-BI, and the downregulation of SR-BI is responsible for the observed reduction in the cellular proliferation.
Inhibition of PI3K, not MEK1/2, inhibits growth of shCTL MDA-MB-231 cells
Knockdown of SR-BI results in decreases in in vivotumor growth of MDA-MB-231 and MCF7 cells
To elucidate the mechanism by which SR-BI regulates tumor formation, tissue immunohistochemical analyses (Figure 6C) and immunoblot analyses of homogenized tumors (Figure 6D) were performed. Immunohistochemistry analyses demonstrated the reduction in SR-BI protein expression in shSRBI MDA-MB-231-derived tumors compared with shCTL MDA-MB231. Results also revealed that levels of the proliferative marker, pErk1/2, were decreased in shSRBI MDA-MB-231 tumors, compared with those observed in control tumors. Consistent with in vitro findings, pAkt levels were decreased in shSRBI MDA-MB-231 tumors compared with those observed in control tumors.
Finally, SR-BI has been shown to activate Akt [23], which may inhibit apoptosis, thereby promoting cell survival [39]. Therefore, we assessed apoptosis with TUNEL staining in tissue sections obtained from shCTL and shSRBI MDA-MB-231 xenograft tumors (Figure 7B). As anticipated, we observed a significant increase in apoptosis in shSRBI MDA-MB-231 tumors compared with shCTL MDA-MB-231 tumors.
In the present study, we examined the role of HDL and its receptor, SR-BI, in breast cancer development and progression. We found that HDL3 stimulates migration and activates signaling pathways such as MAPK and PI3K in two breast cancer cell lines. Inhibiting selective HDL-cholesteryl ester uptake by knocking down or pharmacologically inhibiting SR-BI resulted in an attenuation of cell-signaling events induced by HDL. Additionally, loss of SR-BI resulted in decreased proliferation, migration, and tumor growth of MDA-MB-231 cells. These findings suggest that regulating cholesterol metabolism and cellular signaling pathways via SR-BI may be linked and may additionally identify new targets associated with tumor progression.
HDL, signal transduction, and cellular migration
HDL has a well-established role in the etiology of atherosclerosis, particularly in reverse cholesterol transport, whereby HDL removes excess cholesterol molecules from peripheral tissues and returns them to the liver for excretion or recycling [40]. In addition, HDL functions in a number of other cellular processes, including inhibition of apoptosis in macrophages [41], induction of migration in endothelial cells [24], and the initiation of cell-signaling events in multiple cell types [22, 23, 42]. Although clinical studies have suggested that plasma HDL levels may be correlated with increased breast cancer risk [5, 7–9, 12], the mechanisms by which HDL exerts its effect have yet to be elucidated. HDL has been shown to activate Erk1/2 in fibroblasts [23, 42], Chinese hamster ovary cells [22], endothelial cells [23], and prostate cancer cells [35, 43]. Studies have also shown that it can activate Akt in endothelial [23] and prostate cancer cells [35, 43]. Interestingly, the activation of Erk1/2 [44] and Akt [39, 45] has been implicated in several human cancers, including breast cancer.
In the present study, we established a role for HDL as a mediator of signal transduction in two breast cancer cell lines. Consistent with the results obtained in other cell types, we found that, in both MCF7 and MDA-MB-231 cells, incubation with HDL3 induces a rapid activation of both Erk1/2 and Akt signaling pathways. These novel findings in breast cancer suggest that HDL may regulate various signaling pathways and may therefore alter tumor progression.
In the present study, we found that HDL can induce migration of two breast cancer cell lines, MCF7 and MDA-MB-231, suggesting that HDL may play a role in the early stages of metastasis. This finding is consistent with previous studies showing that HDL can stimulate migration of endothelial cells [24, 46]. Interestingly, the observed migration was shown to be mediated by SR-BI [24]. By contrast, a recent study reported that HDL inhibits migration of MDA-MB-231 in Boyden chamber assays [14]. However, in this study, the investigators used serum as the chemoattractant, and HDL was added to the upper chamber, thereby measuring the ability of HDL to prevent cellular migration induced by serum. By contrast, our method allows the analysis of the role of HDL in the regulation of cellular migration and therefore allows a direct measurement of the capability of HDL to induce migration of MDA-MB-231 cells. As a result, our results indicate that HDL may play a role in the pathogenesis of breast cancer, especially in the later stages.
SR-BI, signal-transduction regulation, and tumor formation
SR-BI has been implicated as a mediator of several cell-signaling events in the context of atherosclerosis [22, 23, 25–27]. Previous studies have shown that HDL binding to SR-BI and subsequent lipid transfer are sufficient to activate Src, which subsequently activates the PI3K/Akt and MAPK pathways [25, 27]. In endothelial cells, one of the downstream effectors of Akt is eNOS, which catalyzes the production of NO. The results obtained in the present study are consistent with the hypothesis that SR-BI may also play a role in signal transduction in the context of cancer. In agreement with this hypothesis, upon knockdown or pharmacologic inhibition of SR-BI in MDA-MB-231 cells, Akt activation was significantly reduced, suggesting that SR-BI may be mediating this response. In addition, downregulation of SR-BI was accompanied by a reduction of total cholesterol levels in MDA-MB-231 cells. These results are consistent with reports that indicate that the cholesterol flux mediated by SR-BI plays a role in the regulation of signal-transduction initiation [26]. In our model, decreased total cholesterol levels may represent a reduction in SR-BI-mediated cholesterol flux and therefore significantly reduce signal-transduction activation. SR-BI also binds LDL, which can, like HDL, promote the cellular entry of cholesteryl ester. Although LDL, may promote the entry of cholesteryl ester via SR-BI, it is not sufficient to induce migration of breast cancer cells, and it does not appear to alter Akt activation (Figure 2C, D). Taken together, our data suggest that both cholesteryl ester entry via HDL-SR-BI and Akt activation are required for cellular proliferation and migration, and, eventually, tumor growth.
Activation of the PI3K/Akt pathway promotes growth, survival, and proliferation [45] and has been implicated in a variety of human cancers [39]. Importantly, Akt is aberrantly hyperactivated in approximately 40% of breast cancers [39]. We observed a reduction in proliferation and migration in the SR-BI-knockdown cells compared with control cells in association with reduced Akt activation. These results suggest that SR-BI may mediate the activation of Akt and its downstream effects in the presence of HDL. Mechanistically, we showed that the inhibition of the PI3K/Akt pathway results in significantly reduced proliferation of shCTL MDA-MB-231 cells, similar to the reduction in proliferation observed in shSRBI MDA-MB-231 cells. Importantly, no further reduction in proliferation of shSRBI MDA-MB-231 cells was detected upon inhibition of the PI3K/Akt pathway. Taken together, these data suggest that reduced Akt activation observed in the shSRBI MDA-MB-231 cells may be responsible for reduced proliferation of these cells compared with shCTL MDA-MB-231 cells.
Previous studies suggested a role for SR-BI in the etiology of breast cancer. Cao et al. [30] showed that expression of SR-BI is increased in human breast tumors compared with the normal surrounding tissue. They also demonstrated that recombinant expression of a mutant form of SR-BI, which lacked the carboxyl-terminal tail of the protein, could inhibit proliferation of breast cancer cells. Their study further suggested that this effect was possibly due to reduced Akt activation. Our study is the first to demonstrate directly that Akt activation is reduced when SR-BI is knocked down or pharmacologically inhibited. Furthermore, in agreement with the previously mentioned study [30], we showed that proliferation of MDA-MB-231 cells was significantly inhibited by downregulation of SR-BI protein levels and by pharmacologic inhibition of SR-BI. In addition, we observed that SR-BI knockdown inhibits migration. This finding may suggest a role for SR-BI in the initiation of metastasis. Finally, we demonstrated that knockdown of SR-BI in MDA-MB-231 cells can lead to reduced tumor growth in vivo accompanied by increased activation of Erk1/2 and Akt, and an increase in cellular apoptosis. In MCF7 cells, knockdown of SR-BI also led to reduced xenograft tumor growth.
Previous studies have shown that Akt can inhibit apoptosis through a variety of mechanisms, including the phosphorylation of BAD, thus preventing cytochrome c release from mitochondria and the direct inhibition of the caspase activation cascade [39]. Increased cellular cholesterol levels have been shown to increase Akt activation and decrease apoptosis in prostate cancer cells [47]. Consistent with these observations, a recent study showed that inhibition of xenograft tumor growth could be achieved with colon cancer cells that re-express the ATP-binding cassette transporter A1 (ABCA1) [48]. ABCA1 is a lipid transporter that mediates the efflux of cellular cholesterol to lipid-free apolipoprotein A-I [49]. Furthermore, in this study, re-expression of ABCA1 resulted in decreased mitochondrial cholesterol content and increased release of cytochrome c, which ultimately led to increased apoptosis. Our work also indicated that SR-BI knockdown can significantly reduce apoptosis in xenograft tumors, as shown by TUNEL staining. Consequently, a reduction in the levels of cellular cholesterol content may be responsible, at least in part, for the decreased apoptosis observed in our model. Taken together, these data also suggest an important role for cholesterol in the regulation of cellular signaling pathways and tumor formation. Importantly, excess cellular cholesterol accumulates in the form of esterified cholesterol. Previous works and ours suggest that the accumulation of esterified cholesterol may lead to a modification of signaling pathways associated with proliferation and migration in tumors. Consistent with this hypothesis, increasing cellular esterified cholesterol levels have been shown to induce cellular proliferation and enhance invasiveness of tumor cell lines [50]. Conversely, the inhibition of cholesterol esterification has been shown to have the reverse effect [51, 52].
In summary, our results suggest that HDL and SR-BI have pro-oncogenic activity and can induce migration and activate signal-transduction pathways responsible for cellular proliferation and tumor formation in two breast cancer cell lines. Additionally, knockdown or pharmacologic inhibition of SR-BI could attenuate signaling mediated by HDL and inhibit proliferation, migration, and tumor growth. Taken together, these findings identify SR-BI and HDL as potential therapeutic targets for the treatment of breast cancer.
ATP-binding cassette A1
- Akt:
Protein kinase B
Blocks lipid transport-1
Bovine serum albumin
- eNOS:
Endothelial nitric oxide synthase
Estrogen receptor
- Erk:
Extracellular-signaling-related kinase
Fetal bovine serum
High-density lipoprotein
High-density lipoprotein-cholesterol
Low-density lipoprotein
Phosphatidylinositol 3-kinase
Scavenger receptor class B, type I.
PGF was supported by grants from the Susan G. Komen Foundation and the National Institutes of Health (R01-CA-098779). This project was funded, in part, under a grant with the Pennsylvania Department of Health (to M.P.L.). The Department specifically disclaims responsibility for any analyses, interpretations, or conclusions.
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21st of November 2016
The latest exhibition at which STAX network took part was held in the beginning of November in Chicago, USA. Our satisfaction for participation at such a great event is not only in the approach to the large North American market, but also in the recognition and confirmation of our own quality and competitiveness. On our stand E-10136, visitors could get familiar with the whole line of Read more... conveyors, that exceed all expectations by its characteristics and capacity, but also to get acquainted with some of our future plans talking to representatives of STAX network. Chicago exhibition, as the largest one, is a great opportunity for STAX network to round off its presence in all meridians, but also incentive to further work on improving our products and maintain our competitiveness. Less...
18th of November 2016
Although STAX network has already been present at the market of the Russian Federation, PAP-FOR 2016 exhibition held at the end of October in St. Petersburg, was a great opportunity for us to confirm the existing partnerships, but also to present a wide array of our products to new potential partners. Among 190 companies from 25 countries that exhibited at the exhibition, STAX network is proud of Read more... the number of visitors on its stand F01 and the interest expressed by clients for production programs that our teams were presenting on the spot. Less...
21st of November 2016
If the number of visitors to some exhibition space held worldwide were benchmark for the success of a company's presentation at an exhibition, it would put STAX network on a high position. Among 270 exhibitors, with more than 5000 visitors, visitors of STAX Technologies stand No 132 had the opportunity to get familiar with our latest production and technological innovations. Being satisfied Read more... with the outcome of October presentation in Italy, STAX Technologies teams have already been engaged in planning presentation at the MIAC 2017. Less...
10th-12th of April 2017
Meet us at TISSUE WORLD Milan, Italy at Fieramilanocity Hall 3 from 10.04. till 12.04. 2016. STAX network team will be hosting you at booth D100.
31st of May-02nd of Jun 2017
Meet us at TISSUE WORLD São Paulo, Brazil at Transamerica Expo Center (Hall E) from 31.05. till 02.06. 2016. STAX network team will be hosting you at booth 180.
11th-13th of October 2017
Meet us at MIAC 2017 - International Exhibition of Paper Industry that takes place in Italy every year in the city of Lucca. In 2017 MIAC will be held from 11.10. till 13.10. 2017.


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Passion Flower Was First Discovered By Monks In The Late Middle Ages. Native To The USA It's Now Cultivated Elsewhere.The Plant Is A Climber, Reaching Upto About 9m In Length With Ovate Or Cordate Leaves. Main Constituents Include: Alkaloids, Harman, Harmine, Harmaline, Harmol And Harmalol. Main Medical Use Is As A Sedative, Hypnotic, Antispasmodic, Hypotensive. Passiflore Have Extracts Of CNS Depressant Activities
Navchetana Kendra Health Care Pvt Ltd
E-138/a,shastri Nagar (opp.metro Piller No.176), Delhi - 110052, India
We are counted amongst the major Frangipani Oil Manufacturers, Exporters, and Suppliers in India. Our Frangipani Oil is known for its characteristics such as long lasting, alcohol free, and uncut. Due to its awesome aroma, Frangipani Oil is mostly used in massage oils, scented candles, etc. The Frangipani Oil can be availed from us at the most competitive prices. Common Name : Plumeria Blends With : Ylang-YlangSandal
Katyani Exports
325, 3rd Floor, Vardhman Big V Plaza, Plot No.- 12, Near M2k, Road No.- 44, Rani Bagh, Pitampura, Delhi - 110034, India
We are offering Frangipani Oil. The frangipani oil is extracted from the flowers of the frangipani plant. It is universally known as plumeria. Plumerias are beautiful, tropical plants and are native to warm, climatic areas. These flowers are also used for decorative purposes such as in flower arrangements and for medicinal purposes. It is preferably in dry, sandy soil with full exposure to sunlight. There are about 1 more... less...
Kazima Perfumers
B-47/c, Joshi Colony, I.p. Extension, Patparganj, Delhi - 110092, India
Deals in flower extract, natural substances, ayurvedic body care product, Essential Oils, ayurvedic skin care product, fruit extracts, flower extract, ayurvedic hair care product, fruit extracts, fruit extracts, flower extract
Shahnaz Group Of Companies
Shahnaz Husain Group Of Companies 901-903, 9th Floor, International Trade Towernehru Place, Delhi , India
Deals in Melissa Lemon Balm Extract, Lycopene Chemical
Navchetana Kendra Health Care Private Limited
E- 138/a, Shastri Nagar, Delhi - 110052, India