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The thousands of Central American and Mexican children that have come to the US border in the past few years are not getting here on their own. Here are the 10 things you need to know about so-called ‘coyotes’, the groups of human smugglers that cross migrants in the U.S. border.
1. According to ICE (Immigration and Customs Enforcement), human smugglers are not the same as human traffickers. Human smuggling involves the voluntary intention to get yourself smuggled across international borders, and a smuggler or coyote, is your facilitator. Human trafficking, on the other hand, is the transportation of people with the purpose of selling them or exploiting them in forced prostitution and other forms of labor. In trafficking, people are usually held against their consent.
2. Smuggling is still a crime, and it’s investigated by ICE, part of the Department of Homeland Security. Customs and Border Protection, another Homeland Security Agency, is in charge of enforcement.
3. Smugglers from Central America to the U.S. work as loosely knit groups of people who operate independently in each of the countries along the route. There is no hierarchical structure between them. Each group is in charge of keeping safe-houses, securing lodging and coordinating transportation for migrants in each of the countries. The money charged to migrants is split between the different groups along the route.
4. Human smugglers are not drug cartels or part of the drug cartels. Up to date, there have been no official reports of drug cartels in Mexico or Central America coordinating or taking over the logistics of human smuggling. Mexican cartels, however, do control the territories and the routes along the Mexican portion of the journey. The Zetas Cartel and the Gulf Cartel, for example, control the northeastern region of Mexico that borders Texas. They charge fees to the smugglers and the migrants for usage of the routes. Cartels are known to kidnap and kill smugglers and migrants who fail to pay fees.
5. According to ICE, the current price migrants pay to get themselves smuggled from Central America ranges between $5000 and $12,000. Some reports say the price of smuggling has risen in recent years as U.S. border security has been ramped up and the cartels have carved up routes and standardized fees for smugglers. The more migrants pay, the more they guarantee safe passage by paying off cartels, paying bribes to Mexican law enforcement, and the coverage of travel costs all along the route.
6. Migrants have traditionally seen smugglers as reliable service providers with strong links to the communities where they come from in Central America. Coyotes are well known in Central American townships, and the smuggling business has traditionally been built on trust. Knowing the right coyote is key to securing a safe journey.
7. There have been several reports of human smugglers selling migrants off to the cartels for kidnapping, trafficking, and recruitment. There have also been reports of smugglers abandoning migrants at some point in the journey after charging the fees. Gabriella Sanchez, an anthropologist who studies human smuggling, says these risks are higher when migrants use smugglers they don’t know and that are not connected to their communities. Also, the lower the smuggling fee, the higher the risk of being sold off or targeted.
8. After the child migrant crisis broke out earlier this summer, some reports say that smugglers spread rumors about a possible immigration amnesty. What is clear is that smugglers seem to know that Central American children who make it to the border are not immediately deported back, thanks to a 2008 anti-trafficking law and to the backlog of cases in immigration court.
9. According to a 2012 study by the United Nations Office on Drugs and Crime, a majority of migrants report using the services of a smuggler. In the case of children migrants, Elizabeth Kennedy, a Fulbright scholar and researcher on child migration in El Salvador, estimates that more than half the children are smuggled using coyotes.
10. U.S. authorities began cracking down on smugglers in response to the surge in unaccompanied minors. ICE launched ‘Operation Coyote’ in June, capturing 363 smugglers and seizing over $800,000 in smuggling profits. U.S. law enforcement agencies have cooperated with Central American authorities, making high profile busts. The US is also finding public awareness campaigns to prevent families from sending their children on the journey to the U.S. border.
In this segment, we hear the story of Claudia, a teenager from Honduras who made the journey to the U.S. this year. She used the services of smugglers, and came face to face with many of the risks in the journey.
Photo of alleged smuggler transporting a Central American family to along the border near Mission, Texas in July 2014, by John Moore/Getty Images


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159 Almore Avenue,
Toronto, ON M3H 2H9.
Join us as the Or Chaim Minyan welcomes another distinguished Scholar in Residence for Shabbat! Rabbi David Silber is the Founder and Dean of Drisha Institute for Jewish Education, the world’s first center for women’s advanced study of classical Jewish texts. He is a sought-after teacher who lectures on biblical narrative and liturgy. Rabbi Silber is the author of A Passover Haggadah: Go Forth and Learn, published by JPS (2011) and the newly released For Such a Time as This: Biblical Reflections in the Book of Esther, published by Koren Publishers (Hebrew).
Friday night, Shabbat Shira: Three Songs of the Torah
Shabbat morning, Shirat Devorah
Devar Torah, Le’Maan Anasenu
After Kiddush, Shirat Hayam and Hallel
Before Mincha, Two Faces of Amalek
Seuda Shlishit, Amalek and Justice

domain: library


I Want It!
Ok, I have to admit. Apple marketing gets to me (or is it that Apple marketing gets me?) Yup, iPhone 4 was announced today and I have to say that soon after I started watching the relevant video I am quickly falling out of love with my iPhone 3GS. Joking aside, this is one cool device but what I am most excited about are two things:
First is FaceTime. That opening scene on the video is me, let me explain. A few weeks back I was working from Europe and I was on Skype video with my wife and kid everyday. Granted I would not have paid roaming charges for a voice call from my cell phone but I would have loved the capability of placing a video call over Wi-Fi without having to carry my laptop around. Now if only Apple would allow a FaceTime call from an iPhone 4 to any other device (laptop, Cisco IP video phone, network connected TV, whatever has a camera and a network connection), but they have phones to sell so I have to respect the network externalities that they are pushing for.
Second is 802.11n. iPhone 4 will support 802.11n in 2.4 GHz only – which is not necessarily great news for enterprise use – as most 802.11n networks are running on the 5GHz band – but is great news for home use. What is more exciting about this development is the battery innovations. Handheld devices stayed away from 802.11n because of the battery life limitations. I bet we will start seeing other handheld type of devices coming out with 802.11n soon, and that can only be good news for everyone.
Isn’t it amazing that a mere 5 days ago AT&T announced the end of unlimited 3G data and today we see a new device introduction that is banking on an extremely bandwidth intensive application (FaceTime) to sell more units? I think my hypothesis about secure and pervasive Wi-Fi hotspots everywhere is gaining some more momentum.
Now, if only I can find an excuse to get the new gadget. When is Father’s day again?


The work will include asphalt repairs, a bitumen spray seal and an asphalt overlay.
19th Jan. 2021 12:01 AM
It is claimed the truck driver failed to observe the speed control signs leading up to the roadworks, failed to operate the...
16th June 2020 12:06 AM
Detours in place and emergency vehicle access will be maintained throughout the works
24th Feb. 2018 1:02 PM


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Make no mistake — Israel will not permit Iran to have a nuclear bomb. That is a certainty.
Equally certain is that Iran wants a bomb. Iran very badly wants that nuclear bomb.
And while on either side of the spectrum, Iran, like Israel, is working very hard to achieve their goal. Iran is working to enrich, develop and procure the constituent elements they need to construct a nuclear weapon.
Over the past seven years, the lines in the sand have been moved.
The world has changed. It is not the same world we lived in when the first Nuclear Deal was configured and signed in 2015. During those famous, now infamous, Nuke Deal negotiations Iran had only very limited ability to build a bomb.
And that was why the agreement contained sunset clauses. Why it seemed harmless to include sunset clauses.
The philosophy behind the sunset clauses was that, over time, limitations on Iran’s nuclear development would fade away.
Why? Because, over time, it was believed that Iran would take its place as an upstanding member of the world community. That Iran would no longer be a threat to other nations because of the great benefits it would gain from participating in the world’s economy. That Iran would reap those great profits with the generous assistance of the United States and her economic muscle.
Obviously, something went wrong. Some would venture to say, everything went wrong. From the very outset, Iran did not comply.
The blame, however, does not fall entirely on Iran. Over time, the world also changed — it changed its approach to Iran’s nuclear aspirations.
The lines in the sand were blurred. Iran understood what was happening. And Iranian leadership took advantage of the situation. And why not?
After all, world leadership, including and even championed by the United States seemed committed to coming to a resolution with Iran regardless of the risks.
Israel, too, while not in step with the rest of the Western world, changed their lines in the sand vis a vis Iran, while Israel once fought hard to prevent any Iranian nuclear development especially, enrichment.
Not so long ago, however, the point of no return for Israel was Iran’s enrichment of fissile material. Then the line changed again, this time to how much nuclear fuel Iran had and at what level of purity. And again.
This time the question became not does Iran have enriched uranium, but how much enriched uranium does Iran have. Is it enough to make a bomb and is there enough to create a significant weapon and how many weapons can Iran create.
Israel saw how the world was changing and realized the importance, the necessity, of adjusting her goals on Iran’s nuclear development campaign.
Today, in 2022, Israel’s objective is to slow down the process of Iranian nuclear development. Jerusalem has no delusions about Iran ever becoming a productive member of the world community and this time around.
Israel wants to slow down the Iranian program so that they, Israel, has the time to plan and execute the most efficient and safest strike — an attack that will eliminate Iran’s nuclear threat for the near and distant future.
Israel is mimicking the Western world, following their lead.
You might ask how, why, when did these changes in attitude and in levels of acceptance come about? The answer is that actors on the ground changed as leadership changed. As political objectives changed. As technology changed. And as Iran changed.
Iran is more capable today than they were in 2015. They know more about nuclear fission, and they know more about enrichment. They know more about bomb-making and more about creating bomb-delivery systems.
As Iran changed, as their nuclear capabilities flourished, Israel pursued varying policies aimed setting back their nuclear program.
There were targeted assassinations of scientists as well as targeted explosions in nuclear facilities as well as computer viruses. All part of Israel’s tool chest of smaller and larger attacks on Iran’s nuclear program.
And they worked. They set back Iran’s nuclear progress. But they didn’t work well enough. They did not put a stop to Iran’s nuclear development.
Since 2015, there has been significant Iranian progress in nuclear production capabilities — but they have not yet created a nuclear bomb.
That is because of the Israeli campaign against Iranian nuclear capability. Not yet. But they’re still working hard on it. And Israel is the first target in their sites.
The next logical question is, will Israel’s lines in the sand move again?
No — Unequivocally — NO.
There is no imaginable scenario that Israel will permit Iran to attain a nuclear bomb and delivery system.
There is no Israeli leader, not on the right, the middle or the political left that will permit Iran to attain a nuclear bomb and delivery system.
Micah Halpern is a political and foreign affairs commentator. He founded "The Micah Report" and hosts "Thinking Out Loud with Micah Halpern," a weekly TV program, and "My Chopp," a daily radio spot. Follow him on Twitter @MicahHalpern. Read Micah Halpern's Reports — More Here.
© 2022 Newsmax. All rights reserved.

domain: news


AMD may not have chips capable of challenging Intel's Sandy bridge units right now, but it is only a matter of time before the Bulldozer processors come out for real, four of which were detailed a short while ago.
Several weeks ago, Intel and its partners were forced to halt sales of all Sandy Bridge products because the 6-Series Cougar Point chipset was found to be plagued by a flaw which caused SATA 3.0 Gbps ports to degrade over time.
This left a sort of vacuum on the market for mainstream and high-end motherboards and notebooks, vacuum which, ironically, AMD was unable to fill.
This was because the Fusion APUs already available on the market were only low-end ones, for netbooks and ultraportables.
Now, finally, some exact info has made its way to the web on four of the FX Bulldozer chips, otherwise known as Zambezi.
In addition to a quad-core (FX-4110) and a six-core (FX-6110) model, there will be two eight-core ones, known as FX-8130P and FX-8110.
Unlike the ones mentioned above, they are not based on the Fusion architecture, so they lack built-in graphics.
What they do possess, however, is an integrated DDR3-1866 controller, an unlocked multiplier for overclocking and support for the Turbo Core dynamic overclocking technology.
Additionally, the four-core unit has 4 MB of L2 cache, while the six and 8-core ones have 6 MB and 8 MB, respectively. Finally, their TDP is of 95W, except that of the FX-8130P, which is rated at 125W.
Some may be interested in knowing that the Globalfoundries 32nm process technology will be used in the chips' creation.
All four central processing units should make it to market sometime during the second quarter of the ongoing year (2011), probably in June. They will be compatible with AM3+ sockets and should have clocked of around 3 GHz or more.


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ALL APPOINTMENTS MADE ARE TELEPHONE CALLS FIRST
‘Help Us to Help You’
PLEASE DO NOT COME TO THE SURGERY
PLEASE ENSURE THAT YOU LEAVE YOUR CORRECT CONTACT DETAILS
PLEASE KEEP YOUR PHONE ON YOU ON THE DAY OF YOUR APPOINTMENT AS CLINICIANS WILL PRIORITISE CALLS IN ORDER OF CLINICAL URGENCY
IF YOU NO LONGER NEED YOUR APPOINTMENT PLEASE DO CANCEL SO IT CAN BE OFFERED TO SOMEONE ELSE
Click below to watch how the NHS allows you to access your GP Surgery safely during the Covid 19 Pandemic. Details of appointments offered at Watford Health Centre are detailed below.
Routine Appointments (Please call after 2pm or book online)
During the current COVID pandemic appointments can be made routinely up to 2 weeks in advance. Appointments are initially by telephone and may involve photos or video consultation and if the clinician feels that a physical assessment is required then this will be arranged within a COVID secure environment. If you feel your problem is more urgent please book an on the day appointment.
We have remained open since the start of the pandemic and do remain open to provide the best care possible whilst ensuring we keep all our patients safe.
48 Hour release Appointments (Please call from 8am or book online)
These are for patients who do not require an emergency on the day appointment but feel it cannot wait for a routine consultation.
On the Day Appointments (Please call from 8am or book online)
The remaining appointments are kept free for booking the same day and these can be booked from 8.00am via telephone and also via online booking. We will try very hard to accommodate you in your first phone call. Consultations may be carried out by the most appropriate member of the practice team.
Click here to BOOK AN APPOINTMENT ONLINE.
**An appointment time will be offered when booking but during the pandemic telephone calls are being prioritised in order of clinical urgency. PLEASE keep your phone on you on the day of your appointment**
Extended Hours Appointments (18.30-20.00)
These are available Monday to Thursdays and can be booked 3 weeks in advance. We also on occasions provide Extended Access appointments on Saturdays and Sundays. Please do ask our staff regarding these.
Online consultations (Non urgent)
You can submit an online consultation using the following link: https://www.online-consult.co.uk/org/watford-health-centre.
This will be reviewed by a clinician with a response within 2 working days. A clinician will decide the appropriate management having reviewed the history. Please do not use this for anything urgent.
Please contact us on 07402404258 so we can book your appointment.
Parking will be available on Rosslyn Road and if you have a disabled badge you can park temporarily on the double yellow line on Upton Road. Please ensure you wear a MASK.
For any queries regarding the vaccine do contact us on 7402404258. We will be happy to discuss any concerns.
More information can be found on our Covid & Flu Support page.
Please click here for Dementia UK -Useful Tips on the Covid-19 Vaccine for people with Dementia

domain: medical


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Wild Destinations Photography
Digital Photography Tours and Workshops of Nature and Wildlife
INTRODUCING – Our unique workshop formats
Tailored for You – Individual or Very Small Group Location Photography Workshops
Create your own custom, individual or very small group (2-6) Nature Photography Workshop and Wild Destinations Photography owners Rick and Jeree’ Waller will make it happen.
Choose the site and the dates.
Invite a friend or two, spouse, partner, relative or very small group.
Designate your photography level and what you wish to learn.
Fully Accessible workshops for People with Disabilities/Physical Limitations.
Women Only small groups.
Church small groups.
Business/Non-Profit small groups.
Dog Friendly small groups.
LGBTQ small groups.
Small groups with a language interpreter.
Workshops for up to 10 people
Locations for Tours / Workshops to the following Locations:
Utah National Parks
and more !
Home of the unique hands-on Location Photography Tours, Workshops and Classes in inspiring Nature and Wildlife settings.
For Everyone –
- Never more than 10 people!
- Photograph and Experience some of the most Beautiful and Pristine locations in the World
- Enhance your Vacation experience with a small group of people who share your sense of photographic adventure
- Enjoy the industries smallest groups and highest instructor-to-client ratio.
- Be inspired by our premier “Location Lighting & Scouting” photographers and instructors
For the Intermediate and Advanced/Professional Photographer –
- Expand your Imagination and Imagery!
- Define and Refine your Signature Style
- Break through Technical Barriers
- Expand your Expertise
For the Novice Photographer –
- Begin your Photography path with nurturing, patient and knowledgeable Instructors who love to share their insights and experience
- Build lifetime friends and photography travelers
- Learn the capabilities of YOUR equipment and how to use it to create captivating compositions

domain: artistic


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Dr. Uchiyama Course 03
Microscope (All Videos)
Jan 19, 2023
This video includes all of the following videos from Course03 Microscope.
[#1 Advantages of Microscopes]
Do you think that the microscope is a tool for specialists who use it for treatments performed by specialists? According to Dr. Uchiyama, it is a necessary tool for generalists who can use it for any kind of treatment.
He told us about his encounter with the microscope and the changes that have occurred in his practice and body since he started using it in his clinical practice.
He also talks about the method he actually uses in his clinical practice from each point of view on the issue of whether the mirror technique is better or the direct view is better.
[#2 Vision Control in EXTARO 300 ]
Although we tend to focus on the ease of use of microscopes, such as magnification and handling, it is also necessary to know about the type of light source. He explained in detail the differences in visibility between xenon, halogen, and LED light sources.
He said, "When you use a microscope, you can simply see and fill the root canal there and it will be cured. That's all there is to it. That's all there is to it," said Dr. Uchiyama. You should not miss the actual clinical video of a root canal, which is difficult to see in the dark, taken with a microscope.
He also talks about a difficult case of root canal filling of a mandibular molar.
[#3 How to record movies with EXTARO 300]
When recording photos or videos of a microscopic examination, not just any camera will do. It is necessary to understand the features of the microscope and the features of the camera equipment, and to choose a combination that makes the best use of both.
In this session, we talked about the functions and features of microscopes, as well as the minimum camera functions you should know.
[#4 Microscopes and rubber dam]
Don't you think that rubber dams are just for performance and don't affect your clinical practice with or without them?
There was a time when Dr. Uchiyama himself actually did not use them. However, he now uses rubber dams in all cases when he can. He summarized the advantages of using rubber dams and how to prepare them in advance.
He also discussed how to form the abutment teeth with a microscope and how to create a provisional restoration. Please also pay attention to Dr. Uchiyama's focus on how to eliminate bleeding from the gingiva.
[#5 Advantages of rubber dam 1]
When rubber dam protection is used in restorative treatment, it is necessary to drill holes with a certain degree of vigor. Dr. Uchiyama has made it more efficient by using the same drilling method for anterior teeth, molars, and root canal treatment. In the first half of the lecture, he talked about the drilling and fixing methods and the use of floss to fix the rubber dam firmly in place.
In the second half of the lecture, he discussed a difficult case in which the gingiva was black and transparent due to root staining, and esthetically restored the tooth using a microscope and a rubber dam.
[#6 Advantages of rubber dam 2]
In the first half of the presentation, he discussed tips for molar rubber damming to ensure a good target tooth while maintaining a good field of view. The method of fixation without the use of clamps is very useful and improves operability and field of view.
In the latter part of the lecture, he taught us how to directly construct the abutment without grinding or extracting the tooth for anterior teeth with low crown height diameters due to living teeth. The best results can be achieved by understanding the characteristics of the materials used and selecting and treating the appropriate cases.
[#7 Bonding with microscope]
This is a CR restoration using a microscope and rubber dam. Using actual microscopic cases, he taught us how to do plaque out and how to remove excess resin, and he explained each step in detail, mentioning recommended instruments and materials.
When extraction is performed under the microscope, it is easy to see the difference between the bone surface, soft tissue, and even the defective granulation during curettage of the extraction socket.
This video shows the importance of preoperative CT diagnosis and the importance of anticipating post-extraction treatment.


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"Prefer the mint flavor of Tums just for personal preference- but they definitely serve their purpose and do the trick. Helpful for short term relief of heart burn after eating trigger foods (tomato, "
Want it? Need it? Or just considering? Keep track of your shopping list all in one place.

domain: health


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US Coast Guard Chief retires in Gowanda
Chief Machinery Technician Joseph J. Lipp IV retired after 20 years of active duty service in the U.S. Coast Guard.
He entered the Coast Guard in May 1999 and attended Basic Training in Cape May, NJ. Upon graduation, his first duty assignment was Machinery Technican “A” School. His subsequent assingments were: Coast Guard STANT Portage MI, USCGC Reliance (WMEC 615) homeported in Kittery ME, Engineer Petty Officer (EPO) for Coast Guard Station Portsmouth NH, EPO for Station Point Allerton, MA, and Coast Guard Sector Buffalo as part of the Ready for Operations team.
Chief Joseph Lipp retired in Gowanda. The ceremony was held at Gowanda Moose Lodge #1382. The hall’s rental was donated by the moose lodge, and presided over by Commander Kenneth Blair, Deputy Commander Sector Buffalo. Chief Lipp was awarded the Coast Guard Achievement Medal during the Ceremony for his role in establishing the Eastern Great Lakes Chief Petty Officer Association, a local chapter of the Chief Petty Officer Association, of which he has been the president since its charter in 2016.
The retirement ceremony also noted the contributions of his family: his wife Heather Lipp from Sanford, ME, and his three daughters, Ceili age 18, Fatih age 21, and Vanessa age 23. His son Joseph, age 16, was unable to attend. Chief Lipp also used this opportunity to give his family gifts to recognize the efforts.
He looks forward to retirement, continuing to support those in the service through the local chapter of Chief Petty Officer Association, and the Gowanda Community where he was a co-coach for the Gowanda Wave Runner swim team.


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The population of Newfoundland and Labrador continues to grow, with Statistics Canada reporting a trend of sustained population growth for the fifth consecutive quarter. Statistics Canada’s latest quarterly population estimates released Wednesday, June 22, show that the Newfoundland and Labrador population increased by 422 persons from January 1, 2022 to April 1, 2022.
This latest report from Statistics Canada confirms that the population growth trend that began at the start of 2021 has continued into 2022. Since January 1, 2021 the population of Newfoundland and Labrador has grown by 3,211 people.
This continued population growth trend remains primarily fueled by immigration; a key priority area for the Provincial Government with the goal of welcoming 5,100 newcomers per year by 2026. Migration from other parts of Canada also continues to contribute to population growth, with migration remaining concentrated from Ontario and Alberta.
“Through immigration and attracting people from other parts of Canada, the population of Newfoundland and Labrador continues to grow. Our government understood the importance of growing our population and strategic investments we have made are paying off. Newcomers are incredibly important for our success as a province and Newfoundlanders and Labradorians continue to welcome them with open arms.
Honourable Andrew Furey
Premier of Newfoundland and Labrador
“It’s official – Newfoundland and Labrador’s population growth is near a ten-year high. And it’s because newcomers, from abroad and mainland Canada, continue flocking to our shores. They are being attracted by job opportunities, like those in our fast growing high-tech and resource sectors, and a quality of life that is the best in the nation. The word is out, Newfoundland and Labrador is growing!”
Honourable Gerry Byrne
Minister of Immigration, Population Growth and Skills
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Immigration, Population Growth and Skills

domain: geography


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Prairie Lane Elementary
- The Prairie Lane Design Advisory Committee met for the first time this week to begin discussions for the extensive remodel with additions planned for the school. They will work with TACKArchitects and Project Advocates on design concepts, meeting throughout the fall. The project is estimated to cost $10.8 million; work will begin in the summer of 2019.
Sunset Hills Elementary
- Students and staff have moved in to a brand new Sunset Hills Elementary School! The new building is a 2-section facility, approximately double the size as the previous school, and was completed for an estimated $13.8 million as part of Westside 2015 Facilities Master Plan. Click here to see photos and video from the Ribbon Cutting, Open House, and 1st Day!
- Work continues as crews build a brand new Swanson Elementary school! Footings in classroom areas are near completion. Steel will be installed soon. The new Swanson is scheduled to open in the Fall of 2019.
- Construction of a new Westside Boys & Girls Club Honoring the Hawkins Family continues near Westbrook Elementary. Work on walls for the addition have begun, and crews are working their way around the perimeter. The East parking and drive thru lanes are now complete and functional; sod and landscaping will be added soon. This project, funded by the Boys & Girls Club of the Midlands, is scheduled to open in the Fall of 2019.
Westside High School
- Students and staff started the school year with a beautiful new Little Theater space, thanks to the generosity and support of the Westside Foundation. The District is now accepted Statements of Qualifications for Construction Manager at Risk services to construct improvements to an expansion of the WHS Welding Lab/Engineering Tech space, a project funded by private donors and the Westside Foundation.
Westside Middle School
- Work at Westside Middle School is also complete on time and on budget. Watch for an upcoming Open House sometime this fall!


Asian eVTOL developer XPENG AEROHT has conducted the first flight of its XPENG X2 electric flying car in Dubai.
It the first public display of the X2 since the company completed the specific operations risk assessment and achieved a special flying permit from the Dubai Civil Aviation Authority (DCAA).
This first flight was witnessed by over 150 attendees, including representatives from the Chinese Consulate in Dubai, the Dubai International Chamber of Commerce, the DCAA, the Dubai Department of Economy and Tourism, the Dubai World Trade Centre and global media.
The flight took place at Skydive Dubai, located adjacent to The Palm, offering excellent airspace conditions and panoramic views of the cityscape to provide the historic flight with a breathtaking backdrop. It followed a keynote introduction to the X2 and the Future of Mobility by Vice Chairman and President of XPENG, Brian Gu, on the opening day of GITEX at the Dubai World Trade Centre.
Brian Gu, Vice Chairman and President of XPENG, commented: “XPENG X2’s public display in Dubai represents a significant milestone for XPENG AEROHT and the international achievement of flying cars. Dubai is a world-renowned ‘City of Innovation’, which is the reason we decided to hold the X2 first public flight event here. Today’s flight is a major step in XPENG’s exploration of future mobility.”
The X2’s first public flight was highly supported by the Dubai International Chamber of Commerce. The Dubai Chamber has been deeply involved in the whole process since the launch of the project, providing strategic consulting to XPENG AEROHT. Acting President and CEO Hassan Al Hashemi attended the event and cited in his speech: “The first global public flight of the X2 flying car will change the game of future mobility. The flying car is the epitome of ambition, innovation and future-oriented vision that has always inspired Dubai and its leadership. Today, we witnessed a historic moment that will define the next 50 years.”


Celebrate the iconic life of a Mexican-American singer with a Selena Impersonator! This beautiful artist will sing the best Selena songs and dance to them!
London Jae PreciseNY’s #1 Beyoncé Impersonator
London Jae has been an established performer since 2003. She is the headliner at rocks bar & nightclub in Albany, Ny, The Stonewall Inn, NYC. She is also the CEO of the house of precise. London Jae is a drag star, live vocalist, production manager and stage director.


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The Tool in the Pool is back. Ryan “Blame it on Rio” Lochte successfully returned from his 10-month suspension by winning the 200-meter medley in record time Sunday at the U.S. Open in Long Island. Of greater importance though is that the gas station bathrooms in and around the East Meadow N.Y. area are still...
NYS Entity Status
NYS Filing Date
MAY 01, 2014
NYS DOS ID#
NYS Entity Type
DOMESTIC LIMITED LIABILITY COMPANY
2014 - B FUNDS, LLC
Around the Web
- Clueless in East Meadow
By Stitches - Wednesday Aug 9, 2017
- Hamptons Property Asks $150 Million
Thursday Jul 13, 2017
The 14-acre beachfront spread on Meadow Lane in Southampton, N.Y., was assembled from four parcels of land and includes several homes.
- The Look: New York City Parks in the Summer: Romance, Games and a Performance for a Dying Tree
By DANIEL ARNOLD, JOANNA NIKAS and EVE LYONS - Saturday Sep 2, 2017
Daniel Arnold spent the last two months photographing parks in all five boroughs. The experience showed him “a very different pulse of the city.”
- Sallie Krawcheck’s Ellevest just landed a big new round of Series C funding
By Connie Loizos - Monday Sep 18, 2017
Ellevest, a nearly three-year-old, New York-based digital investment platform built for women and led by former Wall Street titan Sallie Krawcheck, has raised $34.6 million in fresh funding.The round — which was led by Rethink Impact, and includes participation from PSP Growth, Salesforce Ventures, CreditEase Fintech Investment Fund, LH Holdings, SK Impact Fund, Morningstar, Khosla… Read More
- Third Point, a Hedge Fund, Sets Its Activist Sights on Nestlé
By MICHAEL J. de la MERCED - Sunday Jun 25, 2017
The fund, run by Daniel S. Loeb, has called on the Swiss food giant to sell its stake in L’Oréal and move more quickly to adapt to changing consumer tastes.
- Shareholders Demand More Drastic Shifts at Nestlé
By STEPHANIE STROM - Tuesday Jun 27, 2017
The changes requested by the Third Point hedge fund underscore the idea that legacy food brands must radically shake up their portfolios to remain profitable.


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We have all heard that some parts of the world are “cheap” to visit while others are “expensive” but most people have a hard time quantifying any more precisely. That’s why Price of Travel launched our Backpacker Index numbers for cities all over the world in late 2010. Now totally updated in its 4th year, below you’ll find all major destinations around the world ranked by price for backpackers. More specific information can be found by clicking on the regional articles just below, or by clicking on the individual city names in the main list. Price of Travel - Make a budget at a glance « Compare over 115 destinations around the world by price
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Findings from two African trials, presented February 22 at the Conference on Retroviruses and Opportunistic Infections (CROI), showed that a vaginal ring containing the antiretroviral drug dapivirine can reduce women's HIV risk by as much 60 percent in women aged 25 and older. Microbicide proponents and HIV activists heralded these findings as a breakthrough that offers women greater self-protection against HIV. But some conference participants labeled the results “disappointing," pointing to the much lower levels of protection found among younger women.
Over 4,500 women in four sub-Saharan African countries participated in the two studies, ASPIRE (A Study to Prevent Infection with a Ring for Extended Use) and The Ring Study. The studies also found an overall reduction in HIV risk of 27 percent and 31 percent, respectively. For women under age 21, however, the ring offered minimal protection--a finding believed to result from inconsistent use.
Despite these mixed results, researchers, activists, and women themselves hail the possibility of a woman-controlled option for self-protection as “revolutionary.” Unlike condoms and other HIV prevention methods, women themselves can opt to use the vaginal ring, without informing or seeking approval from their partner. The device can be left in place for a month.
Researchers anticipate further research on the ring before its broader release--including studies on combining microbicidal and contraceptive functions, and on longer-lasting protection. While research and licensing will likely take a year or more, advocates look forward to better understanding of the potential of dapavirine vaginal rings as an exciting tool for women’s self-protection against HIV.
Check out AIDSFree’s prevention activities here.


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Metody kontroli procesu projektowania i dokumentacji projektowej systemów informatycznych
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The article deals with problems concerning the control of the process of designing information systems and of the design documentation connected with it. Such controls aim at increasing the reliability of information systems. There have been described, among others, the following methods: source code review, comparison of a program with on attested version, program tracing, reprocessing, parallel simulation.


No, I am not wrong on this. You are making the mistaken assumption that the value of UOA is all or nothing,
No, I think he's stating that for the use of determining engine WEAR, which a LOT of people on this site do, it is basically useless. And he would be correct in that statement.
AND you are mis-characterizing what I'm saying about UOA.
I think his statement is meant to broadly address those using $30.00 UOA's to find the "holy grail" of engine oil... Not to you specifically.
You are also overlooking the fact that I usually order particle counts with UOA.
You would be in the minority though. You are not the one being discussed, as your technique, since you use things like particle counts, steps outside the scope of the $30.00 UOA crowd....
I have never assumed, nor argued, that basic UOA is the be all and end all answer to what's happening in an engine. As I've already noted, you have to look at all the available information, in combination, before drawing any conclusions.
It is a good indication of contamination level, oil life depletion, and can act as a great indicator for things like coolant leaks, fuel dilution...etc.
Not so great for monitoring what people like to call "wear metals", because they may not be from wear.
Here's a hypo to consider: you do two UOAs, same vehicle, different oils, where virtually all the other variables remain the same, or very similar. On the first UOA, the Iron is normal. On the second, it shoots up to a much higher level. Now, off the bat, obviously this does not necessarily mean that the second oil is at fault. It could very well be other things. But nevertheless, the UOA provides the initial information that the owner can use to track down whether there is an issue, and if so, how to fix it.
This involves trending though. And the size of the spike would have to be factored into how one categorized the severity. If it was only a few PPM, then that falls into the category of noise.
Since we (not you, as you do particle counts) in general are dealing with particles less than 10 microns, as the poster above noted, then exactly what ARE we looking at? Not much. Any sort of large mechanical failure is going to create particles LARGER than 10 microns, which, like in BuickGN's example, DO NOT show up in your typically Blackstone UOA.
So here, you can have somebody thinking things are hunky-dory, when they are NOT, or could have somebody thinking their engine is coming apart when a pocket of deposits became uprooted, dissolved, and some <10 micron iron particles were put in suspension in the oil.
This is why Doug, and OTR truckers use UOA's to monitor CONTAMINATION levels, check for coolant leaks, and watch TAN and TBN to gauge their OCI's. That is the PURPOSE of this TOOL.
Perhaps you should consider the foolishness of condemning another poster, without having bothered to understand what that poster is saying. Please show me where I've said, ever, that I advocate relying solely upon low cost UOA results in drawing "meaningful conclusions".
But MANY on here DO. And unless you explicitly preface your posts with "I do particle counts too, and not just basic $30.00 UOA's", and refrain from citing UOA's in posts that ARE just basic UOA's and using them as EXAMPLES, like was done earlier, than you ARE enforcing that mindset. Obviously not intentionally, but without that sort of disclaimer, nobody KNOWS that you are not just doing the same thing they are.
Beyond that, what's your alternative? Fly blind until one day, your engine fails with no warning? You attack those of us who try to make some use of UOAs, but on the other hand, you don't offer anything that's any better.
Lets flip this over:
He could live contently in ignorant bliss doing regular OCI's and his engine will likely outlive the rest of his car.
Isn't the reason for specifically shopping for a vehicle that is known to be RELIABLE done to eliminate the headaches?
I will repeat: I use UOA, blotter cards, filter inspection, and the subjective signs my engines send, in order to determine whether further examination is necessary. And even then, I very well understand that there will be many things I can not determine from readily available sources of information.
So, what alternative do you propose for finding those larger particles that MAY not be detected in a low cost UOA???
Without a tear-down, you really can't. And that's the point I think. I know it sort of goes in concert with the whole maintenance OCD of those of us on this site, but really, if you think about how anal retentive you are with the maintenance of your car, then think about how many people rack up (in blissful ignorance I might add) ridiculous mileage without doing ANY of those things, I think perhaps you may be able to see the other side of this.....
Remember, OTR transport guys DO perform periodic tear-downs. They do them for a reason......


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Immediate Manufacturers, the Canadian firm that makes the category-defining Immediate Pot multifunction stress cookers, introduced the launch of ten new small kitchen home equipment. The announcement contains two new Immediate Pot multicookers and quite a lot of meals preparation units, together with Immediate Vortex Air Fryers, Immediate Ace Cooking and Beverage Blenders, Immediate Zest Rice and Grain Cookers, and Immediate Omni Toaster Ovens.
A number of of the brand new kitchen home equipment had been obtainable in restricted distribution earlier, however now the total lineup is launched on the market at main retailers on-line and nationwide, together with Amazon, Walmart, Greatest Purchase, Goal, and extra. Multi-functionality and space-saving design are traits widespread to all the Immediate line.
Immediate Pot multicookers
The 7-in-1 Immediate Pot Duo Nova has 14 good packages for meals akin to stews, chili, poultry, rice, and yogurt. The Duo Nova is accessible in Three-quart, 6-quart, Eight-quart, and 10-quart variations.
The brand new Immediate Pot 6-quart Duo Evo Plus 10-in-1 multicooker can ferment and pasteurize along with the extra acquainted record of cooking features. The Duo Evo Plus, additionally obtainable in an Eight-quart dimension, has a brand new internal pot that can be utilized to saute meals on a range prime.
Immediate Vortex Air Fryers
You possibly can prepare dinner a Four-pound rooster or 4 kilos of French fries within the Immediate Vortx Air Fryer’s 6-quart container. This Four-in-1 cooker can bake, roast, reheat, and air fry.
The Immediate Vortex Plus Air Fryer Oven is a 10-quart, 7-in-1 air fryer with 14 pre-set cooking packages. This unit can air fry, roast, bake, broil, dehydrate, reheat, and rotisserie meals. Equipment embrace a rotisserie spit, a drip pan, and two cooking trays.
Immediate Ace Cooking and Beverage Blenders
Are you prepared for blenders that may prepare dinner meals whereas the chop, reduce, and mix it? The Immediate Ace Nova Cooking and Beverage Blender
The 10-speed Immediate Ace Nova Blender has a hid heating factor and eight hardened stainless-steel blades. Prepare dinner your meals after mixing and the Ace will preserve it heat for 2 hours.
The 54-ounce skilled glass pitcher with the Immediate Ace Plus Cooking and Cooling Blender has an 800-watt heating factor for making chunky soups, rice, and different sizzling dishes. 4 sizzling blender packages and 4 chilly blender packages are one-touch simple.
Immediate Zest Rice and Grain Cookers
The Eight-cup Immediate Zest Rice and Grain Cooker can prepare dinner rice the best way you prefer it in addition to steam veggies and make oatmeal.
The 20-cup Immediate Zest Plus Rice and Grain Cooker isn’t simply bigger than the Eight-cup mannequin above. The Zest Plus can steam, saute, and sluggish prepare dinner, plus it has pre-programmed settings for cooking a greater variety of rice and grains.
Immediate Omni Toaster Ovens
We weren’t capable of finding both of the Omni Toaster Ovens on the market after we revealed this submit, however we’ll swing again to replace with pricing information as quickly as the data is accessible.
Immediate Omni Toaster Oven
The Immediate Omni 26 L Toaster Oven is a 9-in-1 equipment with seven good packages and two cooking strategies. The Omni matches six slices of toast or a 12-inch pizza. With this toaster, you’ll be able to air fry, dehydrate, roast, toast, bake, and broil meals.
Immediate Omni Plus Toaster Oven
The Immediate Omni Plus Toaster Oven is an 11-in-1 equipment. Along with toasting, you may also use the Omni Plus to air fry, sluggish prepare dinner, dehydrate, roast, bake broil, reheat, proof, convection prepare dinner, and rotisserie prepare dinner. Twin heating parts on the highest and backside of the oven allow convection cooking.
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Newbies from 1st place to 30th place on Instiz chart
67. HIGH4, IU – Not Spring, Love, or Cherry Blossoms
85. Jimin (AOA), MC META, Nuck – T4SA (Prod. by MC Meta)
91. GaIn – Free Will (feat. Dok2)
96. Yoon Hyun Sang – Time Forgets
98. Junhyung (2BiC) – Bye Bye Bye
91. GaIn – Guilty
95. GaIn – The First Temptation
98. Busker Busker – The Flowers
99. Naul – One’s Way Back
HIGH4 and IU song is old (if you didn’t know) but it follows the same path as Busker Busker, so we might see it pop up again next spring.
I think I would have prefer to get a MV for Guilty over Apple. Guilty sounds more fun altogether, with that little edge I like that GaIn usually go for.


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Research ArticleHedgehog Signaling and Bmi-1 Regulate Self-renewal ofNormal and Malignant Human Mammary Stem CellsSuling Liu, Gabriela Dontu, Ilia D. Mantle, Shivani Patel, Nam-shik Ahn,Kyle W. Jackson, Prerna Suri, and Max S. WichaComprehensive Cancer Center, Department of Internal Medicine, University of Michigan, Ann Arbor, MichiganAbstract the self-renewal of normal stem cells in the hematopoietic system, the skin, the nervous system, and the breast (1, 3, 4). In normalThe epithelial components of the mammary gland are thought breast development, the epithelial components of the mammaryto arise from stem cells with a capacity for self-renewal and gland are generated by a stem cell able to give rise to the lineagesmultilineage differentiation. Furthermore, these cells and/or found in the adult gland, including myoepithelial cells, ductaltheir immediate progeny may be targets for transformation. epithelial cells, and alveolar epithelial cells (5). In the past,We have used both in vitro cultivation and a xenograft mouse characterization of the pathways that regulate self-renewal ofmodel to examine the role of hedgehog signaling and Bmi-1 in mammary stem cells has been limited by the lack of systems thatregulating self-renewal of normal and malignant human support propagation of these cells in an undifferentiated statemammary stem cells. We show that hedgehog signaling in vitro. When primary cultures of mammary epithelium fromcomponents PTCH1, Gli1, and Gli2 are highly expressed in rodents or humans are cultured on solid substrata, they undergonormal human mammary stem/progenitor cells cultured as limited replication and terminally differentiate (6–8). Moreover, themammospheres and that these genes are down-regulated in vivo study of human mammary stem cells has been precluded bywhen cells are induced to differentiate. Activation of hedgehog the lack of xenotransplantation mouse models. We have recentlysignaling increases mammosphere-initiating cell number and described an in vitro system for the propagation of humanmammosphere size, whereas inhibition of the pathway results mammary stem and progenitor cells in suspension culture. Wein a reduction of these effects. These effects are mediated by showed that human mammary stem cells isolated from reductionthe polycomb gene Bmi-1. Overexpression of Gli2 in mammo- mammoplasties generate spherical colonies in suspension culture.sphere-initiating cells results in the production of ductal These colonies, which we have termed nonadherent mammo-hyperplasia, and modulation of Bmi-1 expression in mammo- spheres, are highly enriched in mammary stem and progenitor cellssphere-initiating cells alters mammary development in a capable of both self-renewal and multilineage differentiation (9).humanized nonobese diabetic-severe combined immuno- We have previously used this culture system to show that the Notchdeficient mouse model. Furthermore, we show that the hedge- pathway plays a role in cell fate determination of human mammaryhog signaling pathway is activated in human breast ‘‘cancer stem cells (10).stem cells’’ characterized as CD44+CD24À/lowLinÀ. Thesestudies support a cancer stem cell model in which the hedgehog The characterization of mouse mammary stem cells and study ofpathway and Bmi-1 play important roles in regulating self- mammary development has been greatly facilitated by the use ofrenewal of normal and tumorigenic human mammary stem transplantation models in which mammary cells can be trans-cells. (Cancer Res 2006; 66(12): 6063-71) planted into the cleared mammary fatpads of syngenic mice (5, 11). Recently, Kuperwasser et al. (12) described a system in which theIntroduction fatpads of nonobese diabetic-severe combined immunodeficient mouse (NOD-SCID) mice, ‘‘humanized’’ by implantation of Stem cells are characterized by their ability to self-renew as immortalized human mammary fibroblasts, were able to supportwell as generate differentiated cells within each organ. There is the growth of human mammary cells. The use of in vitro humanincreasing evidence that these cells or their immediate progeny mammosphere cultures and their transplantation into humanizedmay be targets for transformation. We have hypothesized that an NOD-SCID mouse fatpads has allowed us to further elucidate theearly event in carcinogenesis may involve dysregulation of stem pathways that regulate self-renewal of normal human mammarycell self-renewal leading to a clonal expansion of initiated stem stem cells.cells (1, 2). In addition to addressing the cell involved in tumor initiation, A number of developmental signaling pathways, such as Wnt, the ‘‘cancer stem cell hypothesis’’ postulates that tumors areNotch, and hedgehog, have been found to play a role in regulating driven by a cellular subpopulation retaining stem cell properties (2, 3, 13). Consistent with this model, we recently identified a Note: Supplementary data for this article are available at Cancer Research Online subpopulation of cells in human breast cancers with the phenotype(http://cancerres.aacrjournals.org/). CD44+CD24À/lowlineageÀ that display stem cell properties. As few as 200 cells that display this phenotype were capable of generating M.S. Wicha is a consultant for and has financial holdings in OncoMed tumors in NOD-SCID mice, whereas the bulk of the tumorPharmaceuticals. population was not tumorigenic. Furthermore, consistent with a stem cell model, these tumor-initiating cells produce tumors that Requests for reprints: Max S. Wicha, Comprehensive Cancer Center, Department recapitulate the phenotype of the initial tumor. Thus, these tumor-of Internal Medicine, University of Michigan, Room 7110, 1500 East Medical Center initiating cells display the stem cell characteristics of self-renewalDrive, 6303 CCGC, Ann Arbor, MI 48109-0946. Phone: 734-936-1831; Fax: 734-615-3947; and differentiation. Over the past several years, tumorigenic stemE-mail: [email protected] I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-0054www.aacrjournals.org 6063 Cancer Res 2006; 66: (12). June 15, 2006Downloaded from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.
Cancer Researchcells have been detected in myeloma, brain cancers, sarcoma, and was used to generate Bmi-1-expressing (hBmi-1-GFP) and green fluorescentprostate cancers (14–16), lending support to the cancer stem cell protein (GFP)–expressing (GFP alone) lentiviruses in University of Michiganhypothesis. However, it remains unclear how pathways such as Hh Vector Core Facility.regulate the self-renewal of normal stem cells and the role thatderegulation of these pathways plays in carcinogenesis. Small interfering RNA constructions. Three human Bmi-1 (hBmi-1) siRNA oligos were purchased from Ambion, Inc. (Austin, TX; Silencer In the present studies, we have used both in vitro and mouse Predesigned siRNAs) and were used to confirm the knockdown of Bmi-1model systems to elucidate the role of hedgehog signaling and the expression in primary human mammary epithelial cells. All the siRNApolycomb gene Bmi-1 in regulating the self-renewal of normal sequences were converted to small hairpins (shRNA) and inserted intohuman mammary stem cells. Furthermore, we have examined the lentivirus vector LentiLox 3.7. The GFP is expressed in lentivirus-infectedactivation of these pathways in breast cancer stem cells. These cells as the marker to indicate that the cells express the shRNA for hBmi-1.studies provide support for the cancer stem cell hypothesis in which In our experiments, >90% of cells were infected with the control (GFP alone)dysregulation of normal stem cell self-renewal pathways generates or siRNA lentiviruses (hBmi-1-siRNA1-GFP, hBmi-1-siRNA2-GFP, and hBmi-tumors driven by cells that maintain stem cell characteristics. 1-siRNA3-GFP).Materials and Methods Mammosphere implantation into the cleared fatpads of NOD-SCID mice. Three-week-old female NOD-SCID mice were anesthetized by an i.p. Dissociation of mammary tissue and mammosphere culture. One injection (21). The no. 4 inguinal mammary glands were cleared andhundred to 200 g normal breast tissue from reduction mammoplasties were humanized with 2.5 Â 105 nonirradiated telomerase immortalized humanminced and dissociated, and single cells were cultured in suspension as mammary fibroblasts (a generous gift from John Stingl and Connie Eaves,described previously (9). Primary mammospheres were dissociated Terry Fox Laboratory, Vancouver, British Columbia, Canada) and 2.5 Â 105enzymatically and mechanically, and then cultured in suspension to irradiated (4 Gy) fibroblasts as previously described (12), followingproduce mammospheres or on a collagen substratum, as described a previously established protocol (22). A 60-day release estrogen pelletpreviously (9). After mammospheres were formed in suspension culture (0.72 mg/pellet, Innovative Research of America, Sarasota, FL) was placedor cells reached 85% confluency on the collagen plate (f7 days), total RNA s.c. on the back of the neck of the mouse by using a trocar, and 400was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) and used for mammospheres were mixed with 2.5 Â 105 normal human mammaryreal-time quantitative reverse transcription-PCR (qRT-PCR) assays in a ABI fibroblasts and resuspended in 10 AL of 1:1 Matrigel: 5% serum Ham’s F-12PRISM 7900HT sequence detection system with 384-well block module and and injected into each of the cleared fatpads. All of the implantationautomation accessory (Applied Biosystems, Foster City, CA) as described in experiments were repeated five times using mammospheres from differentSupplementary Data. patients with three mice implanted per patient sample. Treatments of mammospheres with hedgehog agonists and antag- Preparation of mammary fatpad sections. Approximately 8 weeksonist. Single cells from epithelial organoids were plated in six-well ultra-low after the implantation, the fatpads were removed and fixed in Carnoy’sattachment plates (Corning, Acton, MA) as described previously (9). solution for 1 hour and subsequently stained with carmine alum overnight.Biologically active, unmodified amino-terminal recombinant human Sonic The tissue was then defatted through graded ethanol and cleared in 5 mL ofhedgehog (Shh) and mouse Indian hedgehog (Ihh; R&D Systems, xylene for 1 hour. The tissue was then embedded in the paraffin andMinneapolis, MN), cyclopamine (TRC, Inc., North York, Ontario, Canada) sectioned for H&E staining.were used. We tested different concentrations of Shh and determined theoptimum stimulation or inhibition was obtained with 3 Ag/mL Shh (17) or Preparation of single-cell suspensions of tumor cells, xenografts,300 nmol/L cyclopamine (18) in our studies. Tomatidine was used as a and flow cytometry. Human mammary tumors were passaged in NOD-negative control for cyclopamine. Mammospheres were then collected at SCID mice as previously described (21). Following tumor growth, which tookdays 1, 3, 5, or 7. All of these collected mammospheres were used for RNA 1 to 2 months, tumors were removed and single cells were obtained byextraction and qRT-PCR and the mammospheres treated for 7 days were also collagenase digestion as described previously (21). One part of the single cellsused for in vitro self-renewal assays as described in Supplementary Data. was used for flow cytometry to sort out the H2Kd-CD44+CD24À/lowlineageÀ population and H2KdÀCD44+CD24+lineageÀ population as described previ- Immunostaining. To assess lineage composition of the colonies, single- ously (21). RNA was extracted from these two populations and real-timecell suspensions were plated on collagen-coated dishes and cultured as RT-PCR was used to quantitate gene expression.described previously (9) for 7 days. Cells were fixed on plates in À20jCmethanol for 20 minutes and stained using Peroxidase Histostain-Plus and Statistical analysis. Results are presented as the mean F SD for at leastAlkaline-Phosphatase Histostain-Plus kits (Zymed, South San Francisco, three repeated individual experiments for each group. Analysis was doneCA), according to the protocol of the manufacturer. The primary antibodies, using Minitab statistical software for Windows (Minitab, Inc., State College,cytokeratin 18 for epithelial cells and cytokeratin 14 (Novocastra, Norwell, PA). Statistical differences were determined by using one-way ANOVA forMA) for myoepithelial cells, were used at the dilutions indicated by the independent samples. P < 0.05 was considered statistically significant.manufacturer. AEC and 3,3¶-diaminobenzidine (Zymed) were used assubstrates for peroxidase and nitroblue tetrazolium/5-bromo-4-chloro-3- Resultsindolyl phosphate (Life Technologies, Gaithersburg, MD) for alkalinephosphatase. Components of the hedgehog pathway are highly expressed in mammary stem/progenitor cells. We have previously de- Virus production, infection, and cell culture. The retroviral plasmid scribed the development of an in vitro culture system and aDNAs for Vector only (SIN-IP-EGFP), Gli1 (SIN-GLI1-EGFP; ref. 19), and Gli2 xenograft mouse model for the propagation of mammary stem/(SIN-GLI2-EGFP; ref. 20) were generous gifts from Dr. Graham W. Neil. progenitor cells. This system is outlined in Fig. 1A. When primaryRetroviruses for SIN-IP-EGFP, SIN-GLI1-EGFP, and SIN-GLI2-EGFP were human mammary epithelium isolated from reduction mammo-produced by stable transfection of 293 cells and were used to infect the plasties are cultured in nonadhering conditions, the vast majority ofsingle cells isolated from primary mammosphere (see Supplementary Data cells undergo anoikis. However, a small number (mammosphere-for details). A highly efficient lentiviral expression system (pLentiLox 3.7)1 initiating cells; f4 per 1,000 cells) are able to form floating spherical colonies (mammospheres). Utilizing retroviral marking studies, we 1 http://www.med.umich.edu/vcore/. showed that these mammospheres could be dissociated and serially passaged at clonal density, with secondary and subsequent generation of mammospheres generated from single cells (9), maintaining a relatively constant number of mammospheres over aCancer Res 2006; 66: (12). June 15, 2006 6064 www.aacrjournals.orgDownloaded from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.
Regulation of Mammary Stem Cell Self-renewalFigure 1. A, experimental strategies for testing self-renewal and differentiation of human mammary stem cells in vitro and in vivo. I, self-renewal is assessed byevaluating the ability of mammosphere-derived cells to form mammospheres containing multipotent cells. II, lineage-specific differentiation potential is assessed byculturing cells on a collagen-coated substratum in the presence of serum. III, differentiation and self-renewal in vivo are tested by implanting human mammary epithelialcells into the humanized cleared mammary fatpads of NOD/SCID mice. EGF, epidermal growth factor. B to D, components of the hedgehog pathway are highlyexpressed in mammary stem/progenitor cells. Mammary epithelial cells were cultured as mammospheres in suspension or as differentiated mammary cells on collagensubstrata, and mammary fibroblasts from the same patient were cultured on collagen substrata. Total RNA was isolated and mRNA was quantitated by real-timeRT-PCR. Columns, means; bars, SD. *, P < 0.05, statistically significant differences from the differentiated cells. B, mRNA expression of hedgehog ligands Shh, Ihh,and Desert hedgehog (Dhh ). C, mRNA expression of hedgehog receptors PTCH1, PTCH2, and SMO. D, mRNA expression of transcription factors Gli1 and Gli2.number of generations. The lineage-specific differentiation poten- expression of transcription factors Gli1 and Gli2, which aretial was assessed by plating these cells at clonal density on collagen downstream components of the hedgehog pathway, and foundsubstrata (9). These studies suggest that mammospheres are that mammary stem/progenitor cells have almost 25-fold highercomposed of a small number of stem cells capable of mammo- levels of Gli1 mRNA and 6-fold higher levels of Gli2 mRNA thansphere formation and progenitors capable of multilineage differen- differentiated mammary cells (Fig. 1D). Taken together, thesetiation, but not sphere formation. Similar findings have been results indicate that hedgehog signaling pathway is activated inreported for neural stem cells in neurospheres (1, 3, 4, 6). Attach- mammary stem/progenitor cells and is down-regulated duringment of cells to collagen substrata induces irreversible differenti- differentiation.ation of these cells (9). Hedgehog signaling agonists and antagonist regulate We compared expression of the genes in the hedgehog pathway mammary stem cell self-renewal and multilineage differenti-in mammary stem/progenitor cells to that of differentiated ation. We have previously shown that mammosphere numbermammary cells, using mammosphere-derived cells grown in upon multiple passages reflects stem cell self-renewal, whereassuspension culture versus mammosphere-derived cells cultured mammosphere size reflects progenitor cell proliferation (9, 10).on a collagen substratum. As shown in Fig. 1B, Ihh is the major Hh We examined the effects of the hedgehog ligand Shh andligands expressed in mammary epithelial cells and its expression hedgehog signaling inhibitor cyclopamine on primary andlevel is f9-fold higher in stem/progenitor cells in mammospheres secondary mammosphere formation. We found that 3 Ag/mLthan in differentiated cells cultured on a collagen substratum. Shh increased primary mammosphere formation by 57% and theDuring normal mammary development, hedgehog signaling is average cell number in these mammospheres by 62% (Fig. 2A). Inpresent in the stroma as well as the epithelium (23). We found that contrast, cyclopamine decreased primary mammosphere forma-mammary fibroblasts produce Hh ligands although at lower level tion by 45% and the average cell number in the primarythan in mammospheres. Figure 1C shows that hedgehog receptors mammospheres by 51% (Fig. 2A). The specificity of ShhPTCHs and SMO are expressed in both cell populations. However, stimulation was shown by reduction of this effect by cyclopaminemammosphere-derived mammary stem/progenitor cells express (Fig. 2A), but not by tomatidine, an inactive cyclopaminef4-fold higher levels of PTCH mRNA, and 3-fold higher levels of analogue (Supplementary Fig. S1). The degree of reversal wasSMO mRNA than differentiated mammary cells. We measured the dependent on the concentrations of cyclopamine and Shh. Thiswww.aacrjournals.org 6065 Cancer Res 2006; 66: (12). June 15, 2006Downloaded from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.
Cancer ResearchFigure 2. Hedgehog signaling regulates mammosphere number and size. Columns, mean; bars, SD. *, P < 0.05, statistically significant differences from the controlgroup. Bar, 100 Am. A, effects of hedgehog agonist and antagonist on primary and secondary mammosphere formation. Primary mammospheres were grown insuspension for 7 to 10 days in the presence or absence of 3 Ag/mL Shh, 300 nmol/L cyclopamine (CP ), or both. Single cells dissociated from each group were grown assecondary mammospheres in suspension for 7 to 10 days without treatment. The number of mammospheres represents total mammospheres formed from 10,000single cells; the number of cells represents the total cells per mammosphere. B, effect of hedgehog activation and inhibition on lineage specification of human mammaryprogenitor cells. C, effect of hedgehog agonist and antagonist treatment on differentiated mammary epithelial cells cultured on a collagen substratum. Primarymammary epithelial cells cultured on collagen-coated six-well plates were treated with 3 Ag/mL Shh or 300 nmol/L of cyclopamine for 7 days, after which the cells werecollected and counted. D, effects of Gli1 and Gli2 overexpression on mammosphere number and size. Secondary mammospheres were infected with SIN-IP-EGFPvirus, SIN-GLI1-EGFP virus, SIN-GLI2-EGFP virus, or none as the control.suggests that at low concentration of cyclopamine, inhibition of primitive mammary cells capable of multilineage differentiation.Smoothened was incomplete. To assess this, we plated mammosphere-derived cells at clonal density on collagen plates in the presence of FCS, conditions that Modulation of hedgehog signaling had an even greater effect on we have previously determined promote cell differentiation (9). Wesecondary mammosphere formation. Shh-treated primary mam- used cytokeratin 14 as a marker of myoepithelial cells andmospheres formed 100% more secondary mammospheres and the cytokeratin 18 as marker of epithelial cells. Similar results wereaverage cell numbers per secondary mammosphere were increased obtained with the markers ESA and CD10, respectively (data not67% (Fig. 2A). This stimulation could be reversed by addition of 300 shown). Addition of Shh resulted in a 3.5-fold increase in thenmol/L cyclopamine (Fig. 2A). Single cells from primary mammo- number of multipotent cells, whereas cyclopamine decreased thespheres treated with cyclopamine generated 54% less secondary number of these cells by 1.8-fold (Fig. 2B), demonstrating that Hhmammospheres and the average cell numbers per secondary activation increased the generation of undifferentiated cells.mammosphere were decreased 56% (Fig. 2A) compared withcontrols. Because Ihh was the main hedgehog ligand expressed in the mammospheres as assayed by real-time quantitative RT-PCR, we To show that Hh stimulated self-renewal of undifferentiated also determined the effects of recombinant Ihh on the system.cells, we examined the differentiation potential of cells treated with Consistent with the previously reported interchangeability ofHh ligand. If the hedgehog pathway acts on primitive cells, then hedgehog ligands, Ihh had same effects as Shh on mammospherestimulation of this pathway should increase the number ofCancer Res 2006; 66: (12). June 15, 2006 6066 www.aacrjournals.orgDownloaded from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.
Regulation of Mammary Stem Cell Self-renewalformation and production of multilineage progenitors (data not and 70% (Fig. 3D), respectively. Furthermore, the effects of Hhshown). activation on both primary and secondary mammosphere forma- tion were significantly reduced by Bmi-1 down-regulation (Fig. 3D, In contrast to the effect on mammospheres, addition of 3 Ag/mL Supplementary Fig. S3). Taken together, these studies suggest thatShh or 300 nmol/L cyclopamine to mammary epithelial cells Hh effects on mammary stem/progenitor cells are mediated by thecultured on a collagen substratum had no effect on the polycomb gene Bmi-1.proliferation of these cells (Fig. 2C). This suggests that the Hhpathway primarily affects undifferentiated cell proliferation. Effects of Gli2 and Bmi-1 expression on mammary develop- ment in humanized NOD-SCID xenotransplants. Because Hh Mammary stem cell self-renewal is regulated by Gli signaling modulates both Glis and Bmi-1, we determined thetranscription factors. To determine whether the effects of Hh effects of Gli and Bmi-1 expression on mammary development.signaling on self renewal of stem cells and proliferation of This was accomplished using a modification of the modelprogenitor cells was mediated by the Gli transcription factors, we described recently by Kuperwasser et al. (12) in which irradiatedinfected mammosphere-initiating cells with retroviral vectors and nonirradiated human mammary fibroblasts are implanted intocontaining Gli1 or Gli2 and determined the effect of constitutive the cleared fatpads of NOD-SCID mice to support the growth ofexpression of these transcription factors on mammosphere normal human mammary epithelial cells. The cleared fatpads offormation. 3-week-old NOD-SCID mice were humanized with telomerase immortalized human mammary fibroblasts. Subsequently, they A highly efficient retroviral expression system (19, 24) was used were implanted with control mammospheres, mammospheresto generate Gli1-, Gli2-, and EGFP-expressing human mammo- overexpressing Gli2, or mammospheres with Bmi-1 overexpressionspheres. We found that compared with uninfected controls or the or Bmi-1 down-regulation. After 8 weeks, the mammary glandsEGFP-expressing cells, overexpression of Gli1 and Gli2 in were removed and examined by histologic analysis. Dense humanmammary epithelial cells in primary suspension culture stimulated mammary stroma was apparent in the humanized NOD-SCIDmammosphere formation by 49% and 66%, respectively (Fig. 2D). mouse fatpad that expressed GFP (data not shown). ControlFurthermore, overexpression of Gli1 and Gli2 increased the number mammospheres (SIN-IP-EGFP, GFP alone) produced limited ductalof cells per mammosphere by 77% and 100%, respectively (Fig. 2D). growth in these areas (Fig. 4A and C). In contrast, both Gli2-Thus, Gli1 or Gli2 overexpression recapitulates the effects of overxpressing mammospheres (SIN-GLI2-EGFP) and Bmi-1-over-hedgehog activation in this system. xpressing mammospheres (hBmi-1-GFP) developed substantially more outgrowths (Fig. 4B and D) than control mammospheres. Hedgehog effects on mammary stem cell self-renewal are Furthermore, down-regulation of Bmi-1 expression in mammo-mediated by the polycomb gene Bmi-1. Bmi-1 is a polycomb spheres by siRNAs inhibited the mammary development (Fig. 4E).gene, which has recently been shown to play a role in the Microscopic examination indicated that Gli2-transfected mammo-regulation of hematopoietic (25) and neural stem cell self-renewal spheres but not control mammospheres produced ductal hyper-(26). Interestingly, we found that Bmi-1 mRNA levels are increased plasia (Fig. 4B). Gli2-transfected mammospheres produced ductalf3.5-fold in mammospheres compared with differentiated mam- hyperplasia in f90% of ductal structures, and these were notmary cells (Fig. 3A). We hypothesized that Bmi-1 might function as detected with the implantation of control mammospheres. Thea downstream target of the hedgehog pathway. To test this human origin of these cells was confirmed by immunostaining withhypothesis we investigated the effect of hedgehog activation on human specific antibodies, such as ESA and cytokeratins (data notBmi-1 expression. We found that activation of the hedgehog shown). These results show that mammospheres can generatepathway by addition of Shh resulted in a 6-fold increase in human ductal/alveolar structures when implanted into theexpression of Bmi-1 in mammospheres, an effect that was blocked humanized cleared fatpad of NOD-SCID mice. Furthermore,by the hedgehog pathway specific inhibitor cyclopamine (Fig. 3B). generation of these mammary outgrowths is modulated by theFurthermore, both Gli1-overexpressing and Gli2-overexpressing expression of Gli2 and Bmi-1.mammospheres displayed a 6-fold higher Bmi-1 expressioncompared with control cultures (Fig. 3B). Together, these results The hedgehog pathway and Bmi-1 are activated in breastsuggest that Bmi-1 expression can be up-regulated by Hh signaling tumor-initiating cells. We have recently reported that humanin human mammary stem/progenitor cells. breast cancers are driven by a small subset of cancer stem cells that are characterized by the cell surface phenotype CD44+ To test whether Bmi-1 plays a role in regulating mammary stem CD24À/lowlinÀ. These cells functionally resemble normal stemcell self-renewal, we infected mammosphere-initiating cells with cells in that they are able to self-renew generating tumors in NOD-lentiviral vectors containing Bmi-1. We found that compared with SCID mice, as well as to differentiate into nontumorigenic cells thatuninfected controls or GFP-expressing cells, overexpression of Bmi- form the bulk of tumors (21). To determine whether the Hh pathway1 stimulated mammosphere formation by 80% and increased the is activated in these cells, we used flow cytometry to isolatenumber of cells per mammosphere by 67% (Fig. 3C). CD44+CD24À/lowlineageÀ cells from a metastatic human breast carcinoma xenografted in NOD-SCID mice. CD44 is an adhesion To provide further evidence that Hh effects on stem cell self- receptor for extracellular matrix ligands, such as hyaluronic acid,renewal are mediated by Bmi-1, we used siRNA delivered in a whose expression has been linked to aggressive behavior and tumorlentiviral vector tagged with GFP to down regulate Bmi-1 metastasis (27). CD24 may regulate cell adhesion by down-expression in mammospheres. Two different siRNA lentiviruses regulation of CXCR4 an important receptor in stem cell homingsignificantly reduced the Bmi-1 expression at both the mRNA (over and tumor metastasis (28). Mouse cells are eliminated in these80% reduction) and protein levels (over 70% reduction; Supple- studies by eliminating H2K-positive cells. LinÀ cells were depleted ofmentary Fig. S2). We used these vectors to examine the effect of cells displaying mammary differentiation antigens using a cocktaildown-regulation of Bmi-1 on mammosphere formation in the of monoclonal antibodies as previously described (21). The levels ofpresence or absence of Hh activation. Down-regulation of Bmi-1expression reduced primary and secondary mammosphere forma-tion by 80% (Fig. 3D) and 70% (Fig. 3D), respectively; and reducedthe primary and secondary mammosphere size by 60% (Fig. 3D)www.aacrjournals.org 6067 Cancer Res 2006; 66: (12). 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Cancer ResearchmRNAs for Hh pathway components and Bmi-1 were measured by DiscussionqPCR. As indicated in Fig. 5A, CD44+CD24À/lowlinÀ cells displayed There is increasing evidence that stem cells or their immediateincreased expression of Hh pathway components PTCH1, Gli1, and progeny may be the targets of transformation during carcinogen-Gli2 by f1.7-fold, 30-fold, and 6-fold, respectively, as well as 5-fold esis. Carcinomas are believed to arise through a series of mutations that occur over many years. Adult stem cells are slowly dividingincrease in Bmi-1 compared with the cells isolated from the same long-lived cells, which by their very nature are exposed totumor, which lacked these cancer stem cell markers (Fig. 5). Figure 3. Hh effects on mammospheres are mediated by hBmi-1. Columns, mean; bars, SD. *, P < 0.05; &, P < 0.05, statistically significant differences from the control group or untreated group, respectively. A, hBmi-1 mRNA level in mammospheres compared with that in differentiated mammary cells. B, effects of hedgehog signaling on hBmi-1 mRNA level. C, effects of Bmi-1 overexpression on mammosphere number and size. D, effects of Bmi-1 knockdown on mammosphere number and size. Primary mammospheres were infected with the control virus (GFP alone) or siRNA lentiviruses (hBmi-1-siRNA1-GFP, hBmi-1-siRNA2-GFP, and hBmi-1-siRNA3- GFP), or uninfected (Non ) as the control, and cultured in suspension in the absence (untreated) or presence of 3 Ag/mL Shh for 7 days. The total number of mammospheres formed from 10,000 single cells and the total cells per mammosphere were counted. The single cells dissociated from each group of primary mammospheres were grown as secondary mammospheres in suspension for 7 to 10 days without treatment.Cancer Res 2006; 66: (12). June 15, 2006 6068 www.aacrjournals.orgDownloaded from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.
Regulation of Mammary Stem Cell Self-renewalFigure 4. Gli2 and Bmi-1 moderates mammary gland development in humanized NOD-SCID mice. H&E staining of sections through the mouse fatpads forSIN-IP-EGFP control virus–infected mammosphere xenograft outgrowth (A ), SIN-GLI2-EGFP virus–infected mammosphere xenograft outgrowth (B ), GFP alonecontrol virus–infected mammosphere xenograft outgrowth (C ), hBmi-1-GFP virus–infected mammosphere xenograft outgrowth (D ), and hBmi-1-siRNA-GFPvirus–infected mammosphere xenograft outgrowth (E). Arrow, ductal hyperplasia. Bar, 100 Am.damaging agents over long periods of time. Therefore, they may show that activation of this pathway with Hh ligands promotes theaccumulate mutations that result in transformation (1). Stem cells self-renewal of mammary stem cells, as evidenced by increasedare characterized by their ability to undergo self-renewal divisions, number of mammosphere-initiating cells. This effect was blockedas well as to differentiate into cell lineages that form adult organs. by cyclopamine, a specific inhibitor of this pathway. Hh activationThe property of self-renewal in which a stem cell can produce one also increases the proliferation of mammary progenitor cells asor two exact copies of itself is a property that is unique to stem reflected by increased mammosphere size.cells. The development of in vitro culture system that maintainhuman mammary stem and progenitor cells in an undifferentiated We have used this system to investigate the downstream targetsstate as well as NOD-SCID mouse models has permitted a more of Hh signaling responsible for mediating these effects. Addition ofdirect analysis of these pathways in normal and tumorigenic Hh ligands increases the expression of the transcription factorsmammary stem and progenitor cells. We have shown that Gli1 and Gli2, which was inhibited by cyclopamine. Because Gli1components of Hh signaling (PTCH1, Gli1, and Gli2) are highly and Gli2 are positive mediators of Hh signaling, whereas Gli3expressed in normal mammary stem/progenitor cells compared functions as a negative regulator of this pathway (29, 30), wewith differentiated cells on a collagen substratum. Furthermore, we focused on Gli1 and Gli2 in the current studies. Forced over- expression of Gli1 or Gli2 in mammosphere-initiating cells byFigure 5. Activation of hedgehog signaling in breast tumor-initiating cells. Hh signaling components and Bmi-1 expression in breast tumor stem cells. Tumor cells wereisolated from the mouse xenografts, both CD44+CD24À/lowlinÀ population (tumorigenic stem cells) and CD44+CD24+linÀ (nontumorigenic cells) were sorted by flowcytometry. Total RNA was isolated and mRNA for Hh components and Bmi-1 were quantitated by real-time RT-PCR. Columns, mean; bars, SD. *, P < 0.05, statisticallysignificant differences from the control group.www.aacrjournals.org 6069 Cancer Res 2006; 66: (12). June 15, 2006Downloaded from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.
Cancer Researchretroviral transduction recapulated the effects of Hh ligands. These shown to be activated in a subset of human cancers based oneffects were unaffected by cyclopamine, an observation consistent immunohistochemical staining of a set of 52 invasive breast cancerswith previous reports that Gli1 and Gli2 act downstream of (35). We have found that overexpression of the Hh target Gli2 insmoothened, the target of cyclopamine (23). mammospheres produces ductal hyperplasias when these cells are implanted into the humanized fatpads of NOD-SCID mice. These It has recently been reported that the polycomb gene Bmi-1 findings are consistent with a stem cell model of carcinogenesis inplays an important role in the regulation of self-renewal of which early events involve deregulation of Hh signaling resulting inhematopoitic (25) and neuronal stem cells (26). Bmi-1 is a clonal expansion of stem or progenitor cells. These cells in turn maytranscriptional repressor that may regulate stem cell self-renewal undergo further mutation to acquire a fully malignant phenotype.through the repression of important cell cycle regulatory genes inthe INK-4A/ADP ribosylation factor (ARF) complex, p16 INK-4A, We have recently described the existence of a cancer stem celland p19 ARF (25, 26). These studies have recently been confirmed population in human breast cancers (21). In the present study, weand extended using mouse knockout models of INK-4A and p19 show that these cancer stem cells display activation of Hh signalingARF (31). We have shown that Bmi-1 is expressed at increased components as well as increased expression of Bmi-1.levels in undifferentiated compared with differentiated mammarycells. Activation of Hh signaling increases Bmi-1 expression, and Taken together, these studies lend support to the cancer stemBmi-1 overexpression promotes mammary stem cell self-renewal cell hypothesis in which carcinogenesis results from deregulationand proliferation as indicated by an increase in mammosphere of self-renewal pathways in normal stem cells generating a cancernumber and size in vitro and increases ductal/alveolar develop- stem cell population that drives tumorigenesis. In normalment in humanized NOD-SCID mammary fatpads. In contrast, mammary development, Hh and the downstream transcriptiondown-regulation of Bmi-1 using siRNA abrogates the effects of Hh factor Bmi-1 play an important role in regulating stem cell self-signaling on mammosphere formation in vitro and inhibited renewal. These processes are tightly regulated by factors in theductal/alveolar development in NOD-SCID mice. These studies stem cell niche. Deregulation of these processes during carcino-suggest that the effects of Hh signaling on mammary stem cell self- genesis may result in stem cell expansion, a key event inrenewal may be mediated by Bmi-1. carcinogenesis. A hypothetical model depicting the role of Hh and Bmi-1 in the regulation of mammary stem cell self-renewal and We have previously proposed that deregulation of self-renewal deregulation of this pathway in cancer stem cells is shown in Fig. 6.may be one of the key events involved in the initial stages of The clinical importance of this is highlighted by a recent reportcarcinogenesis (1). Activation of the Hh signaling pathway as well as demonstrating a strong correlation between the expression of anBmi-1 has been shown to result in the generation of mammary 11-gene Bmi-1 stem cell signature and poor prognosis in patientscarcinomas in vitro or in transgenic models (32, 33). PTCH with a wide variety of malignancies (36). Recently, inhibitors of Hhmutations have been found in a subset of human breast cancers signaling, such as cyclopamine and related compounds, have been(33). A specific mutation in PTCH1 was linked to increased risk of shown to have antitumor activity with minimal systemic toxicity inbreast cancers with oral contraceptives (34). Hh signaling was also mouse tumor models (37, 38). Our studies highlight the importance Figure 6. Hypothetical model depicting mammary stem cell self-renewal pathways in normal and cancer stem cells. Arrows, activation of the pathways by ligands; cyclopamine is an inhibitor of hedgehog signaling.Cancer Res 2006; 66: (12). June 15, 2006 6070 www.aacrjournals.orgDownloaded from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.
Regulation of Mammary Stem Cell Self-renewalof the Hh signaling pathway and Bmi-1 in the regulation of normal 5 P 30 CA46592 (M.S. Wicha), and Susan G. Komen Postdoctoral Fellowship grantand malignant stem cell self-renewal and suggest that strategies PDF0503599 (S. Liu).aimed at inhibiting these pathways represent a rationale thera-peutic approach to target cancer stem cells. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordanceAcknowledgments with 18 U.S.C. Section 1734 solely to indicate this fact.Received 1/6/2006; revised 4/12/2006; accepted 4/17/2006. We thank Dr. Thomas Giordano for tissue procurement, Dr. Michael Clarke for Grant support: NIH grant CA101860 (M.S. Wicha), Department of Defense grant technical advice, Dr. Celina G. Kleer for examining the H&E staining slides, Zhifen Wu for helping with the Western blotting, the University of Michigan CancerBC030214 (M.S. Wicha), University of Michigan Cancer Center NIH Support grant Center Flow Cytometry and Vector Core Facilities, and Dr. Graham W. Neill (Centre for Cutaneous Research, Barts and the London, Queen Mary’s School of Medicine and Dentistry, London, United Kingdom) for generously providing the retroviral plasmid DNAs.References 14. Pellat-Deceunynck C, Bataille R. Normal and malig- 26. Molofsky AV, Pardal R, Iwashita T, et al. Bmi-1 nant human plasma cells: proliferation, differentiation, dependence distinguishes neural stem cell self-renewal1. Dontu G, Al-Hajj M, Abdallah WM, Clarke MF, Wicha and expansions in relation to CD45 expression. Blood from progenitor proliferation. Nature 2003;425:962–7. MS. Stem cells in normal breast development and breast Cells Mol Dis 2004;32:293–301. cancer. Cell Prolif 2003;36:59–72. 27. Avigdor A, Goichberg P, Shivtiel S, et al. CD44 and 15. Singh SK, Clarke ID, Terasaki M, et al. Identification hyaluronic acid cooperate with SDF-1 in the trafficking2. Wicha MS, Liu S, Dontu G. Cancer stem cells: an old of a cancer stem cell in human brain tumors. Cancer Res of human CD34+ stem/progenitor cells to bone marrow. idea—a paradigm shift. Cancer Res 2006;66:1883–90. 2003;63:5821–8. Blood 2004;103:2981–9.3. Kopper L, Hajdu M. Tumor stem cells. Pathol Oncol 16. Xin L, Lawson DA, Witte ON. The Sca-1 cell surface 28. Zhang M, Rosen JM. Stem cells in the etiology and Res 2004;10:69–73. marker enriches for a prostate-regenerating cell sub- treatment of cancer. Curr Opin Genet Dev 2006;16:60–4. population that can initiate prostate tumorigenesis.4. Taipale J, Beachy PA. The hedgehog and Wnt signalling Proc Natl Acad Sci U S A 2005;102:6942–7. 29. Stecca B, Mas C, Ruiz i Altaba A. Interference with pathways in cancer. Nature 2001;411:349–54. HH-GLI signaling inhibits prostate cancer. Trends Mol 17. Kenney AM, Rowitch DH. Sonic hedgehog promotes Med 2005;11:199–203.5. Smith GH, Chepko G. Mammary epithelial stem cells. G(1) cyclin expression and sustained cell cycle progres- Microsc Res Tech 2001;52:190–203. sion in mammalian neuronal precursors. Mol Cell Biol 30. Ingham PW, McMahon AP. Hedgehog signaling in 2000;20:9055–67. animal development: paradigms and principles. Genes6. Reynolds BA, Weiss S. Clonal and population analyses Dev 2001;15:3059–87. demonstrate that an EGF-responsive mammalian em- 18. Chen JK, Taipale J, Cooper MK, Beachy PA. Inhibition bryonic CNS precursor is a stem cell. Dev Biol 1996;175: of Hedgehog signaling by direct binding of cyclopamine 31. Molofsky AV, He S, Bydon M, Morrison SJ, Pardal R. 1–13. to Smoothened. Genes Dev 2002;16:2743–8. Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by7. Muschler J, Lochter A, Roskelley CD, Yurchenco P, 19. Regl G, Neill GW, Eichberger T, et al. Human GLI2 repressing the p16Ink4a and p19Arf senescence path- Bissell MJ. Division of labor among the a6h4 integrin, h1 and GLI1 are part of a positive feedback mechanism in ways. Genes Dev 2005;19:1432–7. integrins, and an E3 laminin receptor to signal basal cell carcinoma. Oncogene 2002;21:5529–39. morphogenesis and h-casein expression in mammary 32. Dimri GP, Martinez JL, Jacobs JJ, et al. The Bmi-1 epithelial cells. Mol Biol Cell 1999;10:2817–28. 20. Ikram MS, Neill GW, Regl G, et al. GLI2 is expressed oncogene induces telomerase activity and immortalizes in normal human epidermis and BCC and induces GLI1 human mammary epithelial cells. Cancer Res 2002;62:8. Romanov SR, Kozakiewicz BK, Holst CR, Stampfer expression by binding to its promoter. J Invest Dermatol 4736–45. MR, Haupt LM, Tisty TD. Normal human mammary 2004;122:1503–9. epithelial cells spontaneously escape senescence and 33. Xie J, Johnson RL, Zhang X, et al. Mutations of the acquire genomic changes. Nature 2001;409:633–7. 21. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison PATCHED gene in several types of sporadic extracuta- SJ, Clarke MF. Prospective identification of tumorigenic neous tumors. Cancer Res 1997;57:2369–72.9. Dontu G, Abdallah W, Foley J, et al. In vitro breast cancer cells. Proc Natl Acad Sci U S A 2003;100: propagation and transcriptional profiling of human 3983–8. 34. Strange RC, El-Genidy N, Ramachandran S, et al. mammary stem/progenitor cells. Genes Dev 2003;17: Susceptibility to basal cell carcinoma: associations with 1253–70. 22. Ip MM, Asch BB. Introduction: an histology atlas of PTCH polymorphisms. Ann Hum Genet 2004;68:536–45. the rodent mammary gland and human breast during10. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, normal postnatal development and in cancer. J Mam- 35. Kubo M, Nakamura M, Tasaki A, et al. Hedgehog Abdallah WM, Wicha MS. Role of Notch signaling in mary Gland Biol Neoplasia 2000;5:117–8. signaling pathway is a new therapeutic target for cell-fate determination of human mammary stem/ patients with breast cancer. Cancer Res 2004;64:6071–4. progenitor cells. Breast Cancer Res 2004;6:605–15. 23. Lewis MT, Veltmaat JM. Next stop, the twilight zone: hedgehog network regulation of mammary gland 36. Glinsky GV, Berezovska O, Glinskii AB. Microarray11. Stingl J, Eirew P, Ricketson I, et al. Purification and development. J Mammary Gland Biol Neoplasia 2004;9: analysis identifies a death-from-cancer signature pre- unique properties of mammary epithelial stem cells. 165–81. dicting therapy failure in patients with multiple types of Nature 2006;439:993–7. cancer. J Clin Invest 2005;115:1503–21. 24. Eichberger T, Regl G, Ikram MS, et al. FOXE1, a new12. Kuperwasser C, Chavarria T, Wu M, et al. Recon- transcriptional target of GLI2 is expressed in human 37. Karhadkar SS, Bova GS, Abdallah N, et al. Hedgehog struction of functionally normal and malignant human epidermis and basal cell carcinoma. J Invest Dermatol signalling in prostate regeneration, neoplasia and breast tissues in mice. Proc Natl Acad Sci U S A 2004; 2004;122:1180–7. metastasis. Nature 2004;431:707–12. 101:4966–71. 25. Park IK, Qian D, Kiel M, et al. Bmi-1 is required for 38. Romer JT, Kimura H, Magdaleno S, et al. Suppression13. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem maintenance of adult self-renewing haematopoietic of the Shh pathway using a small molecule inhibitor cells, cancer, and cancer stem cells. Nature 2001;414: stem cells. Nature 2003;423:302–5. eliminates medulloblastoma in Ptc1(+/À)p53(À/À) 105–11. mice. Cancer Cell 2004;6:229–40.www.aacrjournals.org 6071 Cancer Res 2006; 66: (12). June 15, 2006Downloaded from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.
Hedgehog Signaling and Bmi-1 Regulate Self-renewal ofNormal and Malignant Human Mammary Stem CellsSuling Liu, Gabriela Dontu, Ilia D. Mantle, et al.Cancer Res 2006;66:6063-6071.Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/66/12/6063 Supplementary Material Access the most recent supplemental material at: http://cancerres.aacrjournals.org/content/suppl/2006/06/19/66.12.6063.DC1.htmlCited Articles This article cites by 36 articles, 15 of which you can access for free at:Citing articles http://cancerres.aacrjournals.org/content/66/12/6063.full.html#ref-list-1 This article has been cited by 100 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/66/12/6063.full.html#related-urls E-mail alerts Sign up to receive free email-alerts related to this article or journal. Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR PublicationsSubscriptions Department at [email protected] Permissions To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected] from cancerres.aacrjournals.org on May 22, 2015. © 2006 American Association for Cancer Research.

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