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Image of the Day: Acqualina Resort and Spa
May 28, 2015
By: Five Star Alliance
Reminiscent of a Mediterranean villa with its leisure-focused lifestyle and grand design, Acqualina Resort and Spa on the Beach is South Florida's only five-star hotel of its kind to be built completely open to the ocean, free of barriers between the 51-story tower, featuring 98-luxury rooms and 188 lavish residences, and its 400 feet of glistening Atlantic shoreline.
Book a stay at Acqualina Resort and Spa on the Beach >>
Enjoy a Sunshine Escape at Eden Roc Miami Beach
May 14, 2015
By: Five Star Alliance
Located on prime oceanfront property overlooking world-renowned Miami Beach and the spectacular Intracoastal Waterway, the Eden Roc Miami Beach's captivating vibe pulses with a dynamic convergence of modern and classic, delivering the unexpected with a sophisticated and memorable experience. This resort flawlessly blends iconic Miami style with fabled glamour, offering 631 spacious guest rooms and suites, four exclusive pools, an iconic lobby bar, and 70,000 square feet of stylish indoor and outdoor meeting and event space. The resort is home to 15 Steps, the signature farm-to-table dining experience featuring creative and vibrant cuisine, and the first-and-only ELLE Spa. Now, guests, can enjoy special Eden Roc Miami Beach rates for the summer.
The Sunshine Escape Offer Includes:
-Deluxe King Room, some with partial water views. The rooms are 432 square feet, have Wifi, a safe, marble bath, rain shower, iPod docking station, work desk, flat screen TV, Aveda products, mini-fridge, bathrobes, ironing board, individually controlled air condition, balcony, and a lanai terrace.
- 25% off Breakfast for two daily.
- Valet parking
- Nightly rates for this special offer start at $195.
Offer is subject to availability and cannot be combined with other offers, discounts or promotions. Does not apply to existing reservations. Additional terms and conditions may apply, so please read rates carefully and book early to avoid disappointment. Not valid for groups. Book May 1st to June 30th, 2015 for stays June 1st to September 30th, 2015.
Book your Sunshine Escape at Eden Roc Miami Beach >>
Nobu Hotel at Eden Roc Miami Beach to Open During the Second Half of 2015
April 3, 2015
By: Five Star Alliance
Nobu Hotels and Eden Roc Miami Beach plans to open Nobu Hotel at Eden Roc Miami Beach in the second half of 2015. The Nobu Hotel at Eden Roc Miami Beach is part of the multi-million dollar vision to revitalize and re-launch the Eden Roc name and property. Similar to the highly successful Nobu hotel at Caesars Palace Las Vegas, the property will represent the same key principles as Nobu restaurants; a perfect balance of luxury, fun, craft, and theater.
The new Eden Roc Miami Beach Hotel will feature a "hotel within a hotel concept." Nobu Hotel at Eden Roc Miami Beach with feature 210 guest rooms; a signature Nobu restaurant and bar; an additional new beach front restaurant concept; new banquet and catering menus; unique in-room dining offerings; access to the 20,000 square foot spa and fitness facilities; over 70,000 square feet of re-energized and stylish meeting and event space; and one luxurious pool. The 15th floor Penthouse suites will feature panoramic views of the city with inspiration drawn from the Villa at the Nobu Hotel at Caesars Palace. Guests of the Nobu Hotel will enjoy a VIP entrance to the Nobu Hotel Pool and lounge with views over the beach and sea. The guest rooms will be located in the hotel's legendary tower and offer the imaginative, energized and sophisticated environment exclusive to Nobu Hotel.
Book a stay at Eden Roc Miami Beach >>
Say Hello to 1 Hotel South Beach
March 17, 2015
By: K. Clare Johnson
1 Hotel South Beach, the hotel formerly known as The Perry Hotel South Beach, is now
accepting reservations for stays starting the last week in March. The new name follows a
change in ownership and subsequent $100 million investment in the beachfront property.
The hotel sits on several hundred feet of shoreline within the heart of South Beach. The
Main Pool Deck, shown above, is at the center of the hotel. The 30,000 square foot space
offers guests cabanas, day beds, and 180 degrees of ocean view.
The Lobby Lounge, pictured above, was designed to blend the outside and inside together.
This is achieved by lush plants, comfortable designer furniture and unique terrariums and
living green walls.
The range of accommodations include city view, ocean view, pool view, and oceanfront
rooms and suites. Choose from King or Queen bedded rooms or from studio, one, or
two-bedroom suites. A state-of-art fitness center, in-room massage service, and dining
options by Top Chef Tom Colicchio are a few of the other amenities available at the hotel.
Special introductory rates are starting at $664 per night.
Book a stay at 1 Hotel South Beach >>
By: K. Clare Johnson
Your Questions Answered: Does the Metropolitan Miami Beach Offer Connecting Rooms?
February 10, 2015
By: Betsy LeTourneau
We at Five Star Alliance work tirelessly to ensure we bring you the best luxury hotels selection and information regarding amenities, location, and highlights so you are confident that the hotel you choose will exceed your expectations and serve your needs. Sometimes, however, you are left with a few questions that go unanswered, and that's why we're here. Be sure to explore our new blog series Your Questions Answered to find answers to real questions submitted to us by our clients regarding traveling with kids, pets, the best beach hotels, and much more.
QUESTION: We are a family of 3 traveling together and looking for connecting rooms. Does The Metropolitan Miami Beach offer connecting rooms?
ANSWER: Metropolitan by COMO, Miami Beach is a 74-room Miami hotel in the heart of the city's Historic District, overlooking America's most iconic beach. A new hotel poised to become an essential Miami Beach experience, Metropolitan Miami Beach delivers the perfect blend of rest and revelry, urban modernity and period charm by way of light-filled rooms, a COMO Shambhala spa, and leading-edge interiors by the Italian designer, Paola Navone.
Among hotels on Collins Avenue, Metropolitan by COMO, Miami Beach, has one of the best locations: an enviable position for shopping, dining, and entertainment, overlooking the Atlantic Ocean. Guests benefit from easy access to Miami;s city highlights, including the Lincoln Road shopping and entertainment districts, as well as the Miami City Ballet and Bass Museum of Art. Several rooms in the hotel have a water view, be it the Atlantic Ocean or the Intracoastal Waterway and Lake Pancoast.
For dining in Miami Beach, Metropolitan by COMO stands tall, with its lobby bar, pool restaurant, main restaurant and bar. Whether you are feasting on Florida's seafood at Traymore restaurant or grazing on crudo in a pool cabana, there remains one common commitment: locally-sourced, quality ingredients, and an old-fashioned service ethic that recalls Miami's golden age.
COMO Shambhala Urban Escape - Metropolitan's luxury wellness spa in Miami Beach - offers a highly personalised approach to wellbeing for both guests and locals. Offering our signature COMO Shambhala therapies for body and face, the spa's light and airy interior presents a clean-lined sanctuary for those looking to escape the city's partying.
For Your Needs, We Recommend:
Among local luxury properties, Miami Beach looks most impressive from Ocean View rooms, with the beach and bright turquoise ocean in sight. These accommodations are available with two (2) twin beds or one (1) king bed, each offering the option to interconnect with an Ocean View Suites. Size: 394 - 436 square feet. Ocean View Suites are ocean-facing, spacious one-bedroom suites that each have a king-size bed, separate living room, and powder room. They all have blue sky views of the Atlantic. The oversized bathroom feature dual vanity, shower, and deep soaking tub.
Book a stay at Metropolitan by COMO >>
All rates are based on double occupancy and extra person charges may apply. Please be advised that the rates, availability, as well as any extra person charges are subject to change depending on the hotel's policies.
By: Betsy LeTourneau
La Savina at Mondrian South Beach is Now Open
January 14, 2015
By: Five Star Alliance
The culinary wait is over. La Savina at Mondrian South Beach is open! The new 140-seat restaurant serves Mediterranean cuisine inspired by the island of Formentera, Spain. The new restaurant features an award-winning culinary team led by Morgans Corporate Executive Chef Tien Ho (Momofuku, Best New Chef Food & Wine). Eliciting the spirit of these magical islands, La Savina offers a simple mix of fresh crudos, grilled meats and fish, artisanal charcuteries and the best seasonal vegetables in South Florida, served alongside Mondrian's unmatched views of Biscayne Bay.
Morgans Hotel Group's Design Team were inspired by the style and relaxed interiors of Formentera beach homes. The indoor, outdoor restaurant boosts a garden overlooking Biscayne Bay with multiple seating options filled with oversized planters, rattan lampshades, Beachwood tabletops, and intimate lantern lighting. The interior dining room brings the Beachwood and bohemian energy inside with concrete colored walls and reclaimed wood throughout the space.
Signature crudo offerings include Hamachi with Serrano chili, crispy shallot and lime; Tuna with romesco and cilantro; Scallop with yuzu, jicama and sorrel; and Crab Salad with espelette, grapefruit and Spanish olive oil. La Savina's beverage menu features a broad selection of Mediterranean wines and cocktails. The comprehensive beer list features American microbrews from local New York breweries and European craft brews. In addition, La Savina's red and white table wine is available by the glass or carafe.
Located just off the entrance of La Savina is Cafe Savina. Open for breakfast, lunch and cocktails, Cafe Savina is a market-driven Mediterranean-inspired cafe and wine bar. As the sun sets, Cafe Savina transforms into a tapas bar similar to those of Barcelona.
View rates, details, and more photos of Mondrian South Beach >>
Spend New Year's Eve in Miami
December 13, 2014
By: Five Star Alliance
Sun, surf, neon, and celebration are the name of the game when you spend New Year's Eve in Miami.
It's not too late for you to join the revelry! We found availability at the following Miami hotels from 1/31/1-1/2/15:
* Sofitel Miami has a Superior Two Double Bedded room for $229 per night.
* InterContinental at Doral Miami has a Club Floor room for $359 per night.
* Ritz Carlton Key Biscayne has a King room for $799 per night.
* The Redbury South Beach has a Redbury King room for $579 per night.
For a beachy splurge, Hilton Bentley has a King Studio for $1728 per night and Ritz Carlton Key Biscayne has a Club Level Executive Suite for $2899 per night.
Book quickly, as these rates will not last long. Be sure to check the rate details carefully to avoid disappointment.
Your Questions Answered: Can You Recommend a Luxury Miami Resort for a Family of Seven?
November 22, 2014
By: Betsy LeTourneau
We at Five Star Alliance work tirelessly to ensure we bring you the best luxury hotels selection and information regarding amenities, location, and highlights so you are confident that the hotel you choose will exceed your expectations and serve your needs. Sometimes, however, you are left with a few questions that go unanswered, and that's why we're here. Be sure to explore our new blog series Your Questions Answered to find answers to real questions submitted to us by our clients regarding traveling with kids, pets, the best beach hotels, and much more.
QUESTION: We are a family of 7 and looking to stay is Miami, FL. What resort offers the best accommodations for our family?
ANSWER: Acqualina Resort and Spa On The Beach epitomizes the sophistication of a Mediterranean villa combined with the beautiful oceanfront location on Florida's Riviera. Located on 4.5 beachfront acres with 400 feet of Florida's stunning Atlantic Ocean coastline, this Miami Beach luxury resort features 188 residences, 98 impeccable hotel rooms and suites and world-class luxury hotel amenities, including three oceanfront restaurants, three oceanfront swimming pools, and an innovative marine biology-based children's program. The luxurious 20,000 square foot ESPA oceanfront spa features 16 treatment rooms including a private hotel spa suite for two, a private spa pool and heated jet pool with a Roman waterfall, Finnish Saunas, Crystal Steam Rooms, Ice Fountains, Experience Showers, and Relaxation Rooms.
The resort is a recipient of the coveted 2013 Forbes Travel Guide Five Star Award, 2014 AAA Five Diamond Award for five consecutive years and 2012 Andrew Harper's Reader's Choice Award for Top 20 Beach and Family Resorts. Acqualina is just five minutes from the world famous Bal Harbour Shops and a short drive to the vibrant daytime activities and nightlife of Miami Beach, South Beach, Hollywood, and Fort Lauderdale.
Acqualina's 54 guest rooms and 44 suites include two deluxe three bedroom oceanfront suites. All are havens of elegance and comfort. The richly appointed accommodations feature classic furnishings with fresh modern elements complete with luxurious fabrics and gracious amenities such as private terraces, flat-screen TVs and bathrooms with imported marble flooring, double sinks, bath tub and glass-enclosed shower. All rooms and suites offer magnificent views and are equipped with fully stocked private bars with gourmet food snacks, goose down duvet and pillows (foam pillows available upon request), Frette sheets, ETRO bath amenities, hair dryer & makeup mirror, luxurious bathrobes, in-room CD players, IPOD docking stations, high-speed Internet access, and umbrellas.
For Your Family's Needs, We Recommend:
Deluxe 2 Bedroom Suite connected to a Deluxe Ocean Front room, with a rolloway added for an additional $50.00 night- The suites offer king-size beds in both the master and second bedroom, premium oceanfront views, separate living room, writing desk, two flat-screen TVs, entertainment unit, mini bar, private terrace, gourmet kitchen (with Sub-Zero built-in refrigerator/freezer, designer granite countertops with full back splash, Italian limited edition cabinets, microwave, dishwasher, stainless-steel sink and garbage disposal), breakfast room and two full bathroom outfitted with imported marble flooring, double sinks, custom vanity area, bathtub and glass-enclosed shower. (2035 sq. ft. of enclosed area; 2379 sq. ft. with terrace). The Deluxe Ocean Front Rooms rooms boast an oceanfront view, king-size bed, foyer area, flat-screen TV, entertainment unit, mini bar, sitting area, writing desk, private terrace and bathroom with imported marble flooring, double sinks, custom vanity area, bathtub and glass-enclosed shower.
Book your family's stay at Acqualina Resort and Spa on the Beach >>
All rates are based on double occupancy and extra person charges may apply. Please be advised that the rates, availability, as well as any extra person charges are subject to change depending on the hotel's policies.
By: Betsy LeTourneau
Happy Opening to Thompson Miami Beach!
November 21, 2014
By: Five Star Alliance
Today's the day! Thompson Miami Beach, formerly the Crown Hotel, opens for business today. Close to South Beach, Thompson Miami Beach is where Art Deco meets mid-century and Miami Modern. The beachfront hotel features a new restaurant by celebrity chef Michelle Bernstein, and a rooftop, outdoor spa.
Be among the first to book a stay at Thompson Miami Beach >>
Did You Know? Fisher Island Hotel And Resort
November 11, 2014
By: Five Star Alliance
Fisher Island was a wedge of land with coconut palms and mangroves that sat in Biscayne Bay. In 1919, Miami Beach founder and developer Carl Fisher bought it from Dana Dorsey, South Florida's first black millionaire. Fisher, when trying to expand the island and build deep-water docks, experienced quite a bit of push back from the County, which delayed his progress. The construction of the Port of Miami served to separate the lush appendage from Miami Beach, creating an entirely distinctive island. In the 1920's, Carl Fisher traded 7-acres of Fisher Island for a yacht from William K. Vanderbilt II.
In 1936, Vanderbilt began building his $1.5 million private island retreat on Fisher Island with a Mediterranean-style mansion. He surrounded this mansion with lush landscaping, gracious guest homes, tennis courts and swimming pools.The main home and the surrounding guest cottages were completed in 1941. Rosemary's Cottage was built for Vanderbilt's stepdaughter Rosemary (Rosamund's daughter from her first marriage.) The studio cottage was built for Rosamund to paint in, as she was an artist. The North and South Cottages were for the their servants. The building now referred to today as the Jr. Suites was built to house the crewman for his yacht. The building now the Spa Internazionale was Vanderbilt's personal airplane hanger.
Book a stay at Fisher Island Hotel and Resort >>


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Bret Ritter's Design Portfolio
Bret, a construction manager from Brooklyn, N.Y., joins the cast of HGTV Design Star season six. Take a peek at a few of the designs that earned him a spot on the show.
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NEW: Live TV
Don't miss your favorite shows. Live stream HGTV now.
Catch Up With Our Blogs
We're serving up the latest news, gorgeous style, crafty DIY projects, clever entertaining tips and more. Consider these your design digests.
Follow Us Everywhere
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subset: commoncrawl


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Sunset Cocktails and (free) Live Jazz
- Music
- 420 Park Avenue South, New York, NY, 10021
Collegiate Bowl Syracuse University, Featuring Sophistafunk
Brooklyn Bowl
Wednesday, February 20, 2013 at 6:00 PM - Thursday, February 21, 2013 at 1:00 AM (EST)
|Collegiate Bowl: Syracuse University Ft.
Earphunk, Pigeons Playing Ping Pong
"Collegiate Bowl" is designed to bring out alumni and friends in the New York Metropolitan Area to Brooklyn Bowl, a 600 capacity multi purpose music venue/ bowling alley/ and restaurant by Blue Ribbon in Williamsburg, Brooklyn, for a night of eating, drinking, socializing, and networking with friends from your school.
Wednesday, February 20, 2013
Doors: 6:00 pm // Show: 8:00 pm
Event Details: http://bkbwl.com/VxJZje
Buy Tickets: http://bkbwl.com/UqckGp
Fans across America are talking about SOPHISTAFUNK, the product of three multi-talented musicians [Adam Gold, Jack Brown, & Emanuel Washington] who combine spoken-word artistry, old-school hip hop, & funky rhythms to create a unique live band experience.
The 4 elements of Sophistafunk:
(1) a 1950s jazz club,
(2) a 1960s poetry den,
(3) a 1970s funk party,
(4) a 1980s hip hop show.
Join millions of people on Eventbrite.
In order to purchase these tickets in installments, you'll need an Eventbrite account. Log in or sign up for a free account to continue.


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Ramsey Liposculpture Doctors and Reviews
Ramsey Liposculpture cost
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Ramsey Liposculpture reviews
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"36 Years Old, One 9 Year Old..."
Dr. Shahine is the most warmest, concerned Doctors I ever met. All my problem areas (back, stomach and buttocks) were addressed to him. He assured me that I could do all three procedures. Everything turned out better than my expectations. Unfortunately, I didn't follow proper instructions, in wearing my palate mat, garment (and most importantly NO TIGHT CLOTHES)! He's actually going to... more
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Lance Armstrong Admits on OWN to Using Performance-Enhancing Drugs
During Oprah Winfrey's interview with Lance Armstrong tonight, January 17, on OWN, the cyclist admitted to using performance-enhancing drugs for more than 10 years and said winning the Tour de France seven times without doping would have been "highly doubtful."
Watch a segment of the interview on Mediaite.com.
Oprah Winfrey's exclusive no-holds-barred interview with Lance Armstrong, "Oprah and Lance Armstrong: The Worldwide Exclusive," has expanded to air as a two-night event on OWN: Oprah Winfrey Network. The next episode of "Oprah's Next Chapter" will air on Friday, January 18 at 9 p.m. ET/PT. The interview will be simultaneously streamed LIVE worldwide on Oprah.com.
Cyclist Lance Armstrong speaks exclusively with Oprah in his first interview since he was stripped of his Tour de France titles. He will address the alleged doping scandal, years of accusations of cheating and charges of lying about the use of performance-enhancing drugs throughout his storied cycling career. Winfrey speaks with Armstrong in the only interview since the seven-time Tour de France winner was stripped of his titles and dropped from millions of dollars in endorsement deals after the U.S. Anti-Doping Agency released an extensive report accusing the renowned cyclist of doping throughout his career.
Armstrong was given a Lifetime ban on competing professionally. Late last year, Armstrong resigned as chairman of the foundation he created, Livestrong, which has raised hundreds of millions of dollars in the fight against cancer.
About Author
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suggestions for lodging in Washington,D.C.
Please suggest a good stay in D.C, I like small boutique hotels, do they exist in Washington? I will not have a car.
Please suggest a good stay in D.C, I like small boutique hotels, do they exist in Washington? I will not have a car.
Carole, You posted this in the Italy section.
I've always thought that Italy was a sort of a colony of the US. Now I have confirmation. Washington DC has replaced Rome as its capital.
Maybe she's planning daytrips into Italy. :-) Carol, Not only did you put this in the Italy section, there isn't a US section on this board for you to ask your question. This is all about Europe. Have you tried the TripAdvisors forum for DC?
Wow, I shook up the protocol! This is my first posting and didn't see a U.S.category, but I've had such success with Rick Steve's suggestions, I thought some of his travelers would have lodged in D.C. during their European to and from the States journeys.
That was kinda rude. Considering that we did pretty darn good on some China and Thailand questions a while back. I woulda had a stab at it but boutique just ain't my style. Nuts. Maybe petition the Webmaster to make an exceptions area for nice people ? Sounds like she was a repeat customer too. Probably won't be again. I'll bet that really got into Mr Seves' retirement account. Too bad since he's the guy who keeps the lights on here.
Yes, it is posted on the Italy section but why not take the opportunity to go ahead and respond if you have an appropriate answer. Someone must have
an answer....can't we be flexible?
Maybe Hotel Monaco, Hotel Rouge, or Hotel Helix in DC - they're part of the Kimpton brand (i.e not inexpensive but that's typical of boutique hotels generally)...also Lorien Hotel in Old Town Alexandria, VA (subway accessible to DC). All are in very walkable urban areas. You can PM me if you would like other ideas...I'm pretty familiar with the DC region. http://www.lorienhotelandspa.com/ http://www.rougehotel.com/
There we go. Well done, you two.
I love Ed.
Hay-Adams (very expensive) and Hotel Jefferson are two that come to mind ... BUT both are well beyond my budget. I live in the DC area so have never stayed overnight at these or any other DC hotels (except for Youth Hostel when I used to volunteer there). If you narrow down your choice, I'll be happy to comment on pros and cons of the various locations for getting around, restaurants, etc.
Thanks for all of the responses and not to worry about me not using Rick Steves suggestions. Boutique hotels to me, are not expensive, quite the opposite; small,clean,safe,family owned, sometimes no elevators, elaborate lobbys,or swimming pools, definitely not motels, or Embassy suites. We have them in San Francisco, I've found one in Portland, and of course Europe(Paris and Barcelona).
Maybe check Kalorama Guest House then. It is not a hotel and again, I've only driven by it. It has been around though for a very long time. It is in a very nice neighborhood near the Zoo, walking distance to Woodley Park Metro on the red line. Also check Woodley Park Guest House (same neighborhood).


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First full term pregnancy (FFTP) completed at a young age has been linked to low long term breast cancer risk, whereas late FFTP pregnancy age confers high long term risk, compared to nulliparity. Our hypothesis was that proteins linked to breast cancer would be differentially expressed in human milk collected at three time points during lactation based on age at FFTP.
We analyzed breast milk from 72 lactating women. Samples were collected within 10 days of the onset of lactation (baseline-BL), two months after lactation started and during breast weaning (W). We measured 16 proteins (11 kallikreins (KLKs), basic fibroblast growth factor, YKL-40, neutrophil gelatinase-associated lipocalin and transforming growth factor (TGF) β-1 and -2) associated with breast cancer, most known to be secreted into milk.
During lactation there was a significant change in the expression of 14 proteins in women < 26 years old and 9 proteins in women > = 26 at FFTP. The most significant (p < .001) changes from BL to W in women divided by FFTP age (< 26 vs. > = 26) were in KLK3,6, 8, and TGFβ2 in women < 26; and KLK6, 8, and TGFβ2 in women > = 26. There was a significant increase (p = .022) in KLK8 expression from BL to W depending on FFTP age. Examination of DNA methylation in the promoter region of KLK6 revealed high levels of methylation that did not explain the observed changes in protein levels. On the other hand, KLK6 and TGFβ1 expression were significantly associated (r2 = .43, p = .0050).
The expression profile of milk proteins linked to breast cancer is influenced by age at FFTP. These proteins may play a role in future cancer risk.
Keywords:Lactation; Breast cancer risk; First full term pregnancy; Milk proteins Funded in part by Avon Foundation for Women
Immediately following parturition there is an increased risk of breast cancer observed for all age groups [1]. This is not surprising, since the growth factors required to allow breast glandular proliferation, as well as the extensive remodeling required during involution, may stimulate the growth of already present neoplastic mammary epithelial cell(s). Over the long term, parity is protective for women whose first full term pregnancy (FFTP) was completed at a young age (variously defined as < 20 up to age 26), and increased in parous women whose FFTP occurred after 35 years of age [2]. Rodent models suggest that early FFTP leads to changes in the pattern of breast lobular development and differentiation, cell growth, and gene expression [3]. Early FFTP imprints a specific genomic signature on the breast [3]. Most studies demonstrate a protective effect of breast feeding [4], though the influence of breast feeding duration on cancer risk is less clear, with a recent large study demonstrating no effect on risk of premenopausal breast cancer [5]. We think that breast cancer associated proteins can provide clues to how early FFTP confers a protective effect on the breast.
There is good evidence that during involution, which is initiated by weaning, the breast microenvironment becomes tumor promotional [6]. Our strategy was to identify protein biomarkers in breast milk that are associated with the protective effect of early FFTP vs. the cancer promoting effect of late FFTP. We have identified cancer associated proteins in breast nipple aspirate fluid, fluid from the milk ducts of nonlactating women, including kallikrein related peptidases (KLKs) [7,8], basic fibroblast growth factor (bFGF) [9], and YKL-40 [10].
The KLK family consists of 15 highly conserved serine proteases. Several members of the family have been reported as potential cancer biomarkers [11], and many KLK genes are differentially expressed in hormone-related malignancies [12]. It has previously been demonstrated that multiple KLKs are detectable in breast milk [12]. Furthermore, the epigenetic regulation of KLK6 expression has been demonstrated in hormone sensitive (T47D) and insensitive (MDA-MB-231) malignant breast cells [13].
bFGF [14] has been detected in human milk, while YKL-40 (and its bovine homologue, mammary gland protein (MGP)-40) levels in breast milk rise during the time of breast involution, as the breast returns to the prepregnancy state [15]. Neutrophil gelatinase-associated lipocalin (NGAL, a.k.a. lipocalin 2) is a small secreted glycoprotein which binds matrix metalloproteinase-9 to protect it from degradation [16]. NGAL is expressed in breast carcinomas, inhibition of NGAL impairs breast tumorigenesis and metastasis [17], and increased NGAL expression has been associated with decreased disease-specific survival [18]. The homolog of NGAL has been identified in cow's milk [19].
Transforming growth factor (TGF)β is linked to breast cancer in preclinical models [20] and to prognosis in human breast cancer [21]. In mouse models, TGFβ is induced during mammary gland lactation and involution [20], and it mediates proapoptotic effects during involution [1]. Transgenic mice that overexpress TGFβ show increased apoptosis in the mammary epithelium throughout mammary development [22]. KLK6 is thought to act on the TGFβ1 signal transduction pathway and thereby influence cell migration and motility, and KLK6 alters the expression of TGFβ1 in breast cancer cells [23].
The time of weaning, a period of breast involution and remodeling, appears critical to future breast cancer risk [6]. The analysis of wean milk will provide essential information regarding the biology of markers related to breast involution. We investigated 16 proteins: 11 KLKs, bFGF, YKL-40, as well as NGAL and two isoforms TGFβ, which, like bFGF and YKL-40, have been associated with breast cancer [1,18]. This study was conducted based on the hypothesis that lactation exerts a protective effect by upregulating cancer preventing and downregulating cancer promoting proteins in the breast based on age at FFTP.
Healthy women were prospectively recruited prior to or soon after delivery. They were eligible if they delivered a ≥ 37 weeks gestation infant and were planning on breast feeding. After agreeing to enroll in an Institutional Review Board approved project, three milk samples were requested from each participant: baseline (BL), defined as within 10 days of the initiation of lactation, two months after lactation started, and when the woman is weaning (W). The participant was asked to provide the wean sample once they had decided to stop nursing, and had started to decrease the number of daily feedings. At least two samples from a given woman were required in order for the samples to be included in our analyses. Each sample was collected from the same breast.
Subjects were recruited in Columbia, MO, Grand Forks and Fargo, ND, after IRB approval. Mothers were asked not to breast feed their infants for at least two hours before milk collection, which involves draining the breast using manual extraction or a breast pump. Samples were immediately frozen after collection. Collected milk was thawed, centrifuged (1500 × g, 20 min, 4°C), and the fat and cellular layers separated. The aqueous phase was then centrifuged at 12000 g for 15 min at 4°C, the second lipid layer removed and stored at -80°C prior to analysis.
Assessment of protein biomarkers
Total protein
25 μL of standard and milk samples were added in duplicate to each microplate well (Pierce, Rockford, IL), followed by 200 μL of working reagent with mixing. The microplate was then incubated at 37οC for 30 minutes and absorbance measured at 562 nm.
The concentration of KLKs was measured with a highly sensitive and specific non-competitive immunoassay. Each assay incorporated two KLK specific monoclonal and or polyclonal antibodies (Ab), raised in mouse and rabbit respectively. One Ab was used for antigen capture and other for detection (Table 1) in a sequential two site immunometric format with time resolved fluorescence detection. KLK2 and KLK3 antibodies were obtained from MedixBiochemica (Kauniainen, Finland). The remaining KLK antibodies were developed in the laboratory of Dr. Eleftherios Diamandis Director of Clinical Biochemistry, University of Toronto. The KLK assays have a detection limit ranging from 0.005 μg/L for KLK3 to 0.2 μg/L for KLK7, 8 and 11 and a dynamic range of 10-20 μg/L, depending on the KLK (Table 1). These assays have been used in multiple prior publications [8,12,24-27]. Standards (recombinant KLK) and samples were analyzed in duplicate with inclusion of three quality control samples in every run.
Table 1. Demographics and Kallikrein Assay Information
bFGF was analyzed using an enzyme linked immunosorbant assay (ELISA) kit from R&D Systems (Minneapolis, MN), following the manufacturer's instructions. The kit utilizes a quantitative sandwich enzyme immunoassay technique. The detection limit of the kit is 10 ng/L.
Samples were analyzed by immunoassay as per the manufacturer's instructions (Quidel Corporation, San Diego, CA). The kit uses a monoclonal anti-YKL-40 antibody conjugated to biotin which binds to streptavidin and captures YKL-40 in the standard or sample. The detection limit is 10 ng/mL.
NGAL content in milk was measured by immunoassay (BiPorto Diagnostics, Denmark) according to the manufacturer's instructions. Briefly, 100 μL of standard and diluted milk samples were added to wells coated with a monoclonal antibody against human NGAL. Bound NGAL was detected with a second antibody labeled with biotin and the signal developed with horseradish peroxidase-conjugated streptavidin and substrates, the reaction ended with stop solution and absorbance measured at 450 nm. The detection limit of the NGAL kit is 4.0 pg/mL.
TGFβ isoforms
The protein expression levels of TGFβ1 and β2 in milk samples were determined by immunoassay (R&D Systems) following the manufacturer's instructions. Briefly, 50 μL of standard and activated milk samples were added to wells in duplicate, incubated for 2 hours at room temperature (RT), washed and 100 μL of TGFβ1 or β2 conjugate added. Following another wash, 100 μL of substrate solution was added for 30 min at RT, the reaction ended with 100 μL of stop solution and absorbance measured at 450 nm. The detection limits of the TGFβ1 and β2 kits are 4.61 and 7.0 pg/ml, respectively.
Assessment of KLK6 methylation
DNA Isolation and bisulfite treatment
DNA was isolated from all 42 breast milk cell pellets that were available from women who underwent KLK6 protein analysis women using the QIAamp DNA mini kit (Qiagen, Germantown MD) with the following modifications: 10 μl of RNAse A (1 mg/ml) was added to each DNA isolation at the beginning of the protocol; at the end of the protocol an ethanol precipitation was performed to clean and concentrate the DNA. DNA quantity and quality were assessed using the NanoDrop 8000 spectrophotometer (Thermo Scientific, Wilmington DE). Up to 500 ng of high quality DNA were bisulfite converted using the EpiTect Bisulfite Kit according to manufacturer's instructions (Qiagen, Germantown MD).
KLK6 PCR and pyrosequencing
Two μl of bisulfite converted DNA were used in each PCR reaction to amplify the promoter region of the KLK6 gene using the PyroMark PCR kit (Qiagen, Germantown MD). KLK6 primers were designed to amplify an area of the promoter region upstream of the transcriptional start site (TSS). This region includes 4 CpG sites (-72, - 64, -56 and -53 upstream of TSS) that have been shown to be methylated in breast cancer cell lines and unmethylated in normal cell lines [13]. Primer sequences are as follows: sense primer: 5'- GTAAAGGAGGATTGTTAGATAGGG-3', antisense primer: 5'-biotin-CCAACACCCCAATACCAT-3', sequencing primer: 5'-GATAAAAGGAAGTTATTGATG-3'. Pyrosequencing was performed using a PyroMark Q24 pyrosequencer (Qiagen, Germantown MD) [28] using PyroGold Reagents (Qiagen, Germantown MD). Pyrosequencing using mixtures of in vitro methylated and unmethylated DNA demonstrated a high degree of linearity (R2 = 0.99) for this KLK6 DNA methylation assay.
Statistical analysis
Expression levels of proteins were first logarithm-transformed using natural base prior to analysis. Changes in protein expression over three time points (BL, 2 months, W) were tested with longitudinal analysis using a linear mixed model. P values were adjusted for multiple tests with Holm's step-down procedure. For proteins that showed a significant change over the three time points, a paired t test was performed to detect the change between each pair of time points. Regression analysis was carried out to identify the association between the ages at FFTP with BL protein levels or change of protein expression between the three lactation periods. In order to identify the maximum number of possible associations with FFTP, we did not adjust p values for multiple tests. The effects of nursing time on total protein and individual proteins were assessed using regression analysis. Levels of KLK6 methylation were compared among the three time points using one-way analysis of variance. The association of KLK6 methylation and protein levels was tested using regression analysis at each time point and at each CpG site. The interaction of KLK6 and TGFβ1 was analyzed using regression analysis.
Breast milk samples were collected from 72 women, 35 women for whom it was their FFTP and 37 for whom it was not (Table 1). One woman enrolled in the study after breastfeeding for 2 months and no BL milk was available. A 2 month milk sample was not collected from two women because they weaned prior to the 2 month time point. Milk was collected at all three timepoints from 47 women. Wean milk samples were not collected from 25 women (14 have not yet weaned, 9 weaned but did not provide a wean sample, and 2 were lost to follow-up).
There was considerable variability between samples in total milk protein concentration. For this reason, all biomarker results are listed and all statistical analyses were conducted controlling for total protein concentration in the milk sample. Total protein expression was not influenced by either age at FFTP or whether the samples were collected from a primiparous vs. a multiparous woman. FFTP age was not found to interact with parity status.
The expression of multiple milk proteins varies during the course of lactation
For our two milk collection time points, overall (considering all sample collections) change in individual protein expression was compared in women with young (< 26 years) vs. older (> = 26 years) age at FFTP (Table 2). Of the 16 proteins evaluated, there was an overall change in the expression of 14 (for women < 26) and 9 (for women > = 26) proteins, respectively.
Table 2. Mean Natural Log (ng/g) Values of Baseline (BL), 2 Month (2 Mo) and Wean (W) Samples vs.
For proteins with a significant global change, we determined (Table 2) the change for each of three lactation periods (BL to 2 months, BL to W and 2 months to W). Mean expression significantly decreased for 8 (for women < 26) and 8 (for women > = 26) proteins, respectively, between BL and 2 months, and increased for 13 (for women < 26) and 11 (for women > = 26) proteins, respectively, between 2 months and W. Between BL and W there was a highly significant (p < .001) increase in the expression of KLK6, 8, and TGFβ2 in both age groups. KLK3 expression was lower in W than BL milk of women < 26 but not those in the > = 26 group.
Expression (and expression change during lactation) of biomarkers linked to breast cancer are related to age at FFTP
For each of the 16 proteins, we evaluated the association of expression with age at FFTP. At BL, bFGF (p = .0116) and NGAL (p = .012) expression were significantly higher in women who were older at the time of their FFTP (Figure 1a,b). There were no significant associations at 2 months or W. There was a larger increase in KLK8 expression (p = .022) from BL to W (controlling for total time that a woman nursed) with younger age at FFTP (Figure 1c).
Figure 1. Natural log a) bFGF and b) NGAL baseline milk expression; or c) change (wean--baseline) in natural log KLK8 milk protein expression based on age at FFTP. For KLK8, only matched samples are represented.
DNA methylation of KLK6 varies among women and with breast feeding duration but does not explain protein expression pattern
To determine the extent to which the changes observed in KLK6 protein levels were controlled by epigenetc events, we isolated DNA from a total of 42 breast milk cell pellets (eight at BL, 13 at 2 months and 21 at W) obtained from 32 women. Methylation analysis by pyrosequencing revealed high mean levels of KLK6 promoter methylation at all three time points 57% (BL), 34% (2 months) and 48% (W), and no significant differences across time points (F = 2.27; p = .117). Among the ten women for whom methylation was assessed at two time points, there was an average absolute difference of 37% methylation between the two sampling times. The greatest differences (Figure 2) occurred between BL and 2 months (mean change for the 6 women = 44%) and between 2 months and W (mean change for the two women = 42%) with little change between BL and W (mean change for the two women = 10%). The methylation level was not associated with KLK6 protein level (p = .35).
Figure 2. Methylation of KLK6 in DNA isolated from cells obtained from breast milk. The average of the percent of methylated DNA at each of 4 CpG sites in the proximal promoter of KLK6 is shown for milk samples collected at Baseline (n = 8), Two Months (n = 13), and Wean (n = 21).
Association of KLK6 with TFGβ1
KLK6 has been reported to interact with the TGFβ1 signal transduction pathway and influence TGFβ1 expression in breast cancer cells [23]. We determined the association of these proteins in breast milk, considering all samples for which there were paired values. The two proteins (Figure 3) were significantly associated (r2 = .43, p = .0050).
Figure 3. Association of natural log TGFβ1 and KLK6 protein expression in breast milk.
One approach to assess the influence of a specific process such as age at FFTP is global (proteome wide) assessment. Unfortunately, this approach has limited sensitivity [29] even with sample concentration approaches [30], and lack reliable quantitation of individual protein concentration. We elected to use immunoassays which are highly sensitive and quantitative, to compare individual protein expression during the course of lactation.
A striking feature of the data is that the expression of most proteins varies during lactation, even after controlling for total protein concentration. A similar observation in a smaller sample was made by another group in the assessment of milk immunoglobulins and lysozyme [31]. The most common trend that we observed was for the concentration of proteins to decrease from BL to 2 months after starting nursing, and then to increase between 2 months and W. Although the reason(s) for this are uncertain, a possible explanation is that the breast secretes a given amount of each protein daily, with greater milk volume during the 2 month lactation period compared to BL or W periods leading to a lower protein concentration. Indeed, total milk protein concentration has been shown to negatively correlate with 24 hour milk volume [32], and gradual weaning over a period of three months has been shown to increase total protein as milk volume decreased [33].
The most significant changes in expression from the start to the end of lactation in both FFTP age groups were in KLK6, 8 and TGFβ2. There was a significant decrease in KLK3 in women < 26 at the time of FFTP, but not in women with a later age of FFTP. The significance of the change in one but not the other group is uncertain. For KLK6, 8 and TGFβ2, there was a small change early (between BL and 2 months), with a larger change later (between 2 months and W) in lactation. For all three proteins there was an increase in mean expression at the time of weaning compared to BL.
KLK6 and 8 expression is downregulated in breast cancers [34]. As such, it has been speculated that these genes may function to suppress tumors [13]. KLK6 [13] is epigenetically regulated with gene silencing in tumors. KLK6 may play a protective role against tumor progression by inhibiting the epithelial-to-mesenchymal transition [13]. KLK8 has been shown to function as a serine protease [35]. The mechanism of KLK8 gene silencing in breast cancer has not been reported, though alternative splice variants of the gene which influence prognosis have been reported in lung cancer [36]. The increase in KLK8 expression from BL to W was greatest in women who whose FFTP occurred at a young age, which is consistent with known protective effect of early age at FFTP [2] and a tumor suppressive effect of KLK8.
The significant changes in KLK6 protein expression during lactation and the report of regulation of KLK6 by methylation in breast cancer cells [13] prompted us to examine KLK6 promoter methylation in cells obtained from breast milk. We detected surprisingly high levels of KLK6 promoter methylation with moderate differences among the three sampling times that did not explain the observed changes in protein expression. Indeed, the lowest mean level of methylation, 34%, occurred at 2 months. While we expected low methylation to be associated with the highest protein expression, KLK6 expression, on average, was highest in wean milk for all age categories (Table 2). Whereas methylation scores were relatively high at all sampling times, repeated measures from ten women revealed average methylation changes during lactation of 37%.
To assess the likelihood that the observed variability in KLK6 methylation throughout lactation was an artifact, we examined the promoter methylation of p16 (data not shown), which we would not predict to vary with lactation duration. In contrast to KLK6, there was little methylation of p16 at each point, ranging from 1 to 5%, indicating that the variability in KLK6 methylation is not likely to be an artifact. It is important to note that, unlike the prior report associating KLK6 methylation with KLK6 expression in cultured malignant breast cells [13], our clinical investigation evaluated breast cells collected from the breasts of healthy women. Regarding the variability in KLK6 methylation which we observed, we believe that this is at least partly due to differing percentages of epithelial cells and leucocytes that were present in the milk at the three sampling times, since DNA was isolated from the total cell pellet. Nonetheless, we confirmed that epithelial cells present in milk from healthy breasts have high levels of methylation by examining several epithelial-enriched cell fractions (data not shown).
Whereas we did not find a association between KLK6 methylation and protein expression, we did observe a highly significant association between KLK6 and TGFβ1 protein expression. TGFβs regulate normal breast development, apoptosis and matrix remodeling during breast involution induced by breast weaning, with biphasic effects on tumor progression, acting as tumor suppressors in early stages of cancer and promoting invasion and metastasis at later stages [37]. Our observation that the expression of these proteins are directly correlated, whereas they are inversely associated in breast cancer cells in culture [23], is consistent with the pleiotropic effects of these molecules to suppress cancer initiation and promote later stages of cancer invasion and metastasis.
This report has limitations. Most notable is the limited sample of women whose FFTP was age 35 or greater, as well as the limited BL and 2 month sample sizes for the methylation analyses. We may have missed associations with age at FFTP due to this. A second limitation is that there was some variability between when the wean milk sample was collected and when the woman stopped nursing entirely. A third limitation is that we cannot say with certainty that the proteins identified which are upregulated at the time of weaning would put these women at lower risk of future breast cancer, since we do not have sufficient follow-up to address this. On the other hand, as previously discussed, we know from other reports that higher expression of KLK 6, 8, 10 and TGFβ2 have been associated with a lower risk of breast cancer [34,38].
We report the upregulation of KLK 6, 8 and TGFβ2 at the time of breast weaning and involution. All four proteins have a tumor suppressive effect, consistent with the known protective effect of pregnancy in younger women. KLK8 levels were generally lower at BL in women older at the time of their FFTP, but the change was greater in these women, suggesting a protective correction toward a higher KLK8 expression level by the end of lactation. bFGF and NGAL expression was higher in BL lactation samples among women with an older age of FFTP. Both are tumor promotional, consistent with the association of late FFTP with increased breast cancer risk. Finally, we observed a significant association between KLK6 and TGFβ1, which may be one mechanism by which these molecules interact during lactation to influence future breast cancer risk. We plan to investigate these proteins in the nipple aspirate fluid of women with and without breast cancer, to see if their expression is associated with disease.
BL: Baseline; bFGF: Basic fibroblast growth factor; ELISA: Enzyme linked immunosorbant assay; FFTP: First full term pregnancy; KLK: Kallikrein related peptidase; MGP: Mammary gland protein; NGAL: Neutrophil gelatinase-associated lipocalin; RT: Room temperature; TGF: Transforming growth factor; TSS: Transcriptional start site; W: Wean.
Competing interests
Dr Sauter is a consultant for Atossa Genetics, Inc.
Authors' contributions
WQ conducted total and individual protein assessment on all samples. KZ performed statistical analysis for the study. BK managed and provided quality assurance of the data analyzed. RLR assisted with preliminary data analysis and provided critical review of the manuscript. EPB performed methylation analysis and manuscript review. KFA oversaw methylation analysis and provided manuscript review. ERS initiated the study, oversaw the conduct of the entire project, enrolled participants, and prepared the manuscript for publication. All authors read and approved the final manuscript.
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Pre-publication history
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Paper bags with sand in the bottom and a candle are put out in rows at churches, missions and on private properties throughout the Southern USA and other parts of the World on Christmas Eve.It is a lovely sight to see and to photograph. Here the luminaria/farolitos are set out in Tularosa, New Mexico. Luminaria means "Festival Lights" and Farolito means "Little Lantern". Catholics hope that the lights will guide the Spirit of the Christ Child to their homes.
Bob Christopher is a photographer based in Calgary, Alberta, Canada. He travels extensively throughout the world and, as a result, has the opportunity to capture a host of beautiful images from all parts of the globe. Bob's photographic ability, passion, creativity and attention to detail make his images very desireable for the collector and the home decorator alike. Bob is interested in photographing a wide number of different subjects and has compiled an impressive collection of images for you to choose from to decorate your home or office or business areas. All of his images have a special look that are the result of his commitment to making photographs of the highest calibre. Every aspect of each image is completely the...
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Show some class and dignity you fool.
Type: Posts; User: sagemania
Show some class and dignity you fool.
Are you stupid or are you acting this way?. In the last quarter the Rockets ran with Cannan, Covington, Casspi, Brewer and DMO. Cannan is a rookie who wil be relegated to the D as will Covington,...
He is just as good if not better then the current broken down version of Wade. Kobe is another case.
KD is not staying around in OKC if Presti sticks around. He is still furious of the highway robbery the Rockets pulled on his team.
On what planet will Asik be guarding Lee?. Why would the Rockets force Asik to guard Lee? It will probably be Jones and Montijunes. On defense Lee is a turnstyle so Houston can easily take advantage...
How did the Rockets get exposed in the playoffs? No one gave them a chance and they pushed the Thunder all the way.Also Dwight was coming off a back surgery and played with a sever busted shoulder....
Houston's game plan was that they will out score you and for the most part it worked well.Now with the addition of Howard, that will almost mean 48 minutes of rim protection/elite rebounding with...
Considering Houston led the league in fast break points, led the league in 3pt shot mades and had the second best offense in the entire leage, good luck with that.
The Rockets have 48 minutes of elite rim protection. Ezeli and Bogut are nowhere near Howard/Asik combo interms of interior defense/rebounding.
Parsons was one of the Rockets best players and Asik played quiet well. WTF are you talking about?
Houston had the second best offense last season with just shooting 3's. Now with Dwight who can score himself or create double teams(great open looks for the shooters) will make the Rockets even...
I'm not a Lakers fan. I'm a Rockets fan. Aldridge will be a unrestricted free agent in 2015 when the Lakers will have a ton of cash. LBJ and Melo aren't going to La so the only realistic options are...
Chicago(if rose is healthy)
Lakers will endup with someone like Love or Aldridge(If the Rockets don't trade for him)
OKC: Healthy Westbrook and Durant will be a must.Someone needs to fill in martins points off the bench
Clippers: bes PG in the game and having two scrappers and great 3pt shooters in Dudley and...
Dwight 21 points 14.7 boards 3 blocks 56% fg
Harden 25 points 6.8 assists 5.3 boards 2 steals 46% fg
For now we haven't achived squat and it's all names on paper. If we don't trade asik for a pf, i'm confident Montinues who is a stretch big with a sold 3 point shot who btw is a 7 footer and...
no one wants lin so were stock with him but I like the idea of keeping asik. 48 minutes of animal defense and rebounding.
We'll have his bird rights by 2014 vso he'll get paid in the future. That's the reason he re-signed
lets not forget despite the iggy signing, they've lost landry and jack two key pieces.
anybody who can say who will be better with any confidence whom will be better is kidding themselves. Once we shipout lin and Asik and get a legit PF we'll see.
I'm more excited that we got Garcia back then this guy.
Now we need to go out and get a PF.Especially a stretch 4 that can balance it wth D12 parked in the key
Houston has the biggest foothold in the Asian Market and China in particular. Plus the Rockets are in the top 7 biggest Markets in the entire NBA


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Watching De Niro and Stallone piss all over their most iconic roles provides no pleasure. It made me feel – Sad. Sad. Sad. show more
Grudge Match at its core is an affront to the cinema gods, an attempt to capitalize off two iconic films for a few cheap laughs. show more
Grudge could have saved itself with a rousing finale, but the buildup is so tedious you just want the fight to end. show more
January 19, 2014 yoliewin
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Enjoyed the movie and seeing both actors get into shape for the finish. I felt that there was enough humor, and good banter among the story to keep me interested and moving the story along to the finale. Enjoyed the show.
January 08, 2014 o2optional
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Lough out loud hilarious for raunchy humor!
January 04, 2014 fjpmsw
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I don\'t know who the \"critics\" are, but they are wrong! The acting is superb, the movie is very funny and even the story is very entertaining! I didn\'t even get up to get my snacks! I would see this movie again. Robert De Niro still has what it takes! His facial expressions alone will make you laugh


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