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Xiii: The Series: Season 2
Stuart Townsend,
Aisha Tyler,
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XIII: The Series
After being betrayed by his country and those closest to him, Agent XIII (Stuart Townsend, Queen of the Damned) finds himself swept up in a resistance movement as he tries to figure out what his new place in the world will be. His life is thrown into further chaos when someone who looks exactly like him starts to carry out domestic terrorist attacks in his name. While XIII struggles to figure out who this mysterious double is, he uncovers an international arms race to develop a weapon capable of dealing catastrophic damage across the globe. As deceptions and conspiracies come to a head, the US government begins to crumble. In the end, the most wanted ex-agent in the world might be the one man capable of saving the country. The action never stops in this thrill-ride series from the producer of Lost Girl!


An area of 3 decares around the chapel is protected. The protected area was instituted to protect the area's special historical and natural qualities.
mittel - Gruppe. Minimum 15 stk, kr. 50,-.
geöffnet nach Vereinbarung - Omvising for grupper. Ring Åshild på 90 59 09 96.
englisch - Guided tour only for groups, min. 15 pers.


The Scottish Government has once again exceeded its IVF target, ahead of changes which will mean more people are eligible for treatment.
All but one eligible patient (99.7%) were screened for IVF treatment within 365 days across Scotland within 12 months of being referred, during the quarter ending June 2016.
From Thursday (1 September) couples where one partner does not have a biological child will be eligible for IVF access, even if there are children in the home. This is the first recommendation to be implemented from the National Infertility Group report published in June.
"Scotland is leading the way in the provision of NHS IVF treatment and I'm really pleased that we continue to meet and exceed our target.
"We want to make sure that access to treatment on the NHS is as fair as it possibly can be - giving more people the opportunity to conceive. Over the last four years we have invested around £18 million to reduce IVF waiting times and improve the outcomes for couples undergoing this treatment.
Reducing waiting times for IVF patients potentially improves the chance of a successful outcome.
The Scottish Government determined that the IVF waiting times target should be delivered for at least 90 per cent of patients from 1 April 2015, as for some it may not be clinically appropriate for treatment to begin within 12 months.


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FEU invites students to be part of the next generation of tech-ready innovators
e-Conomy SEA 2022: PH digital economy to reach USD20B by end of year
TechnologyFinTeche-Conomy SEA 2022: PH digital economy to reach USD20B by end of year
The Philippine digital economy could reach USD35B by 2025, and possibly USD100B- USD150B by 2030
This year's e-Conomy SEA report titled Through the waves, towards a sea of opportunity projects that the Philippine digital economy is on track to hit USD20B Gross Merchandise Value (GMV) in 2022, after growing at 22% over the past year. By 2025, the local digital economy is projected to reach USD35B GMV, growing at a 20% Compound Annual Growth Rate (CAGR), and possibly will hit USD100 to USD150B GMV by 2030.
The annual report, which combines Google Trends, Temasek insights, and Bain & Company's analysis in addition to industry sources and expert interviews, spotlights the digital economies of six countries in Southeast Asia (SEA): Indonesia, Malaysia, the Philippines, Singapore, Thailand, and Vietnam.
Growing at 17% from the previous year, e-commerce is expected to reach USD14B GMV by 2022 and USD22B GMV by 2025 as it continues to propel the local digital economy. Despite the partial resumption of in-store shopping, e-commerce accounted for 70% of the overall Philippine digital economy.
E-commerce, food delivery, and video-on-demand are the top three digital activities of Filipinos showing an adoption rate of 88%, 69%, and 58% respectively among digital urban users.
Here are the rest of the key insights into the PH digital economy:
Transport and food delivery are projected to reach USD1.9B GMV in 2022 and continue to grow at 29% CAGR to hit USD4B GMV in 2025. Demand growth is normalizing as more people eat out. The gradual return to the office, increase in brick-and-mortar shopping, and the resurgence of tourism are driving recovery from lockdown lows for Transport.
Travel is back, with 180% YoY growth to hit USD1B GMV in 2022. As recovery to pre-pandemic levels is likely to be gradual, travel is growing at 44% CAGR by 2025 where the sector is expected to reach USD4B GMV.
Online media which comprises music, video streaming, and online gaming is projected to grow at USD3.1B GMV in 2022, with YoY growth at 17% since the pandemic peak last year. Highest in Southeast Asia, 37% of paying users in the Philippines said that they play online games at least once a week.
Digital financial services show sustained growth, with lending and remittance hitting $6 billion this year. Remittance is expected to deliver USD4B in Gross Transaction Value (GTV) at 32% YoY growth. In 2022, digital payments will drive USD75B GTV and keep growing at 18% CAGR to reach USD123B GTV in 2025.
The Philippines will attract more investors over the long term
With the Philippines as the second fastest growing digital economy in SEA (next to Vietnam) in 2022 (at 22% YoY) and between 2022 to 2025 (at 20% CAGR), the country will be a hot investment hub across sectors including digital financial services.
The Philippines has the fastest-growing digital investments sector this year in SEA, at a 63% growth rate from 2021 to 2022. Seventy-three percent (73%) of investors (second highest in SEA after Vietnam) are expecting deal activity to increase in the country's local digital economy in the period of 2025 to 2030 as compared to the current situation.
Digital financial services are soaring high and the sector continues to attract investor interest, capturing 56% of total investor funding in 2022 and raising roughly USD450M in the first half of 2022 alone. Both Filipino unicorns Mynt and Voyager are in the fintech space.
Web 3.0 has attracted sizable investor funding raising USD90M in the first half of 2022 with investors bullish on cryptocurrency adoption in the Philippines which is considered one of the fastest growing in the world.
"The Philippine digital economy remains resilient despite headwinds and continues to provide boundless opportunities as it is projected to reach USD20B GMV by end of the year. This year's e-Conomy SEA report also suggests that the country will be a leading investment destination with over 70% of investors expecting deal activity to increase in the period of 2025 to 2030. Google is committed to helping the Philippines sustain the growth momentum of its digital economy through local partnerships, programs, and products that aim to improve the lives of Filipinos," said Bernadette Nacario, country director, Google Philippines.
"The Philippines' digital economy is one of the more attractive investment hubs in the region. Across internet sectors there remains tremendous whitespace for growth as the ecosystem drives greater digital inclusion in the country, particularly outside of metro areas. We expect growth to come from not only the physical goods and services internet sectors, but also digital financial services across payments, lending, and other Fintech products," said Willy Chang, associate partner at Bain & Company.
"The seventh edition of the e-Conomy SEA report shows that the digital future of the Philippines is bright as it has the fastest growing digital investments sector this year in the region. The Department of Trade and Industry is committed to digitalizing our MSMEs and working closely with our private sector partners like Google to help reach the full potential of the country's growing digital economy," said Alfredo Pascual, Secretary, Department of Trade and Industry.
BillEase partners with Alipay+ to allow Filipino consumers to shop global online
GCash, BSP, DILG join hands to launch PalengQR-PH in Bohol
GoTyme Bank wins Innovation Award from Visa

redpajama_set_name: RedPajamaCommonCrawl


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Milan's Alessandro Del Piero on the future of Gianluca Vialli
By Football Italian staffThis summer, Milan are set to lose one of their most successful players, Gianlucio Del Pieros.
The 26-year-old is expected to leave the Rossoneri this summer and his contract expires in the summer of 2019.
He joined the Rossos in 2011, making his debut in the Champions League, and was the first Italian to score for the club in the competition.
Del Pier, however, has been hampered by injuries throughout his career and was only part of the Rosso side that reached the Europa League final.
It is unclear how his future will play out, as he has not played for the Rossino since the final of the Europa Cup in June 2017.
While Milan have been in talks with the Juventus man over his future, they are yet to receive any response from Juventus.
Del Pieros was a regular fixture in the Milan defence in the years that he was at the club.
His contribution was always evident in his ability to keep a clean sheet, and he scored in all but four of Milan's Europa League wins.
In all, he scored 17 goals in the group stages of the competition, making him one of the most prolific strikers in the history of Italian football.
It is expected that Milan will continue their talks with Juventus over Del Pier's future, and Juventus may offer the Rossa an alternative.
Milan were also linked with Liverpool's Mamadou Sakho earlier this month.
The Merseysiders are also looking to sign Liverpool's James Milner.
The former Manchester United man is out of contract in the autumn, and the Merseyside outfit are yet a few months away from signing a replacement for the midfielder, which could be Liverpool's Milner or Liverpool's Sakho.
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Insurance receipt email missing "everything"!
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Town Bond Fraud
Town Bond Fraud	Jason Pickholz	2019-03-19T16:47:00+00:00
Types Of SEC Cases: Town Bond Fraud
SEC Charges Government Officials With Town Bond Fraud
The SEC accused Ramapo, N.Y. ("Ramapo"), its local development corporation, and four town officials with engaging in a municipal town bond fraud.
The SEC alleged that the four officials hid Ramapo's deteriorating financial situation from municipal bond investors.
In its court Complaint, the SEC charged Christopher P. St. Lawrence, Nachman Aaron Troodler, Michael Klein, and Nathan Oberman, with committing public statement fraud by making numerous false or misleading statements and omissions about the financial condition of the Town of Ramapo and the Ramapo Local Development Corporation ("RLDC") in connection with 16 municipal securities offerings between September 2010 and September 2015.
The SEC's court Complaint
Baseball Stadium Cover-Up Leads To Town Bond Fraud
According to the SEC, in 2010, the Town of Ramapo and the RLDC set out to build a minor league baseball stadium, later named Provident Bank Park. Politically, this turned out to be an unpopular municipal project. It cost at least $60 million to construct, and immediately began to put a strain on Ramapo's finances, which were already impaired by declining property and sales tax revenues.
In a press release, the Chief of the SEC Enforcement Division's Public Finance Abuse Unit summarized the SEC's case: "We allege that Ramapo's senior-most officials concealed the true condition of the town's declining finances to avoid further political fallout from the construction of the baseball stadium".
The financial strain and desire to avoid further political fallout supposedly led St. Lawrence and the other defendants to conceal the true condition of Ramapo's and RLDC's finances.
Specifically, the SEC alleged that the goal of the town bond fraud scheme was to mask the deterioration of Ramapo's primary operating fund (the "General Fund"), in order to facilitate various bond financings of more than $300 million, including $85 million of new capital.
The Complaint contended that the defendants' materially false representations to investors and potential investors concerning the financial health and wherewithal of the Town constituted municipal securities fraud.
Specific Acts In Furtherance Of The Town Bond Fraud
The SEC's court Complaint set forth numerous allegedly false statements and acts of misconduct that contributed to the municipal town bond fraud, including accounting fraud, false statements to auditors, and books and records violations. Some of the SEC's allegations included:
Lawrence masterminded and directed a scheme to artificially inflate the balance of the General Fund by recognizing fraudulent receivables, omitting unpaid liabilities, and improperly recording transfers from other funds with different tax bases.
Klein, the Town Attorney, aided in the town bond fraud by helping to conceal outstanding liabilities related to the baseball stadium and making repeated misleading statements to Ramapo's auditors.
Troodler, an assistant Ramapo attorney and former RLDC Executive Director, participated in the town bond fraud by certifying official statements that contained fraudulent receivables.
Oberman, Ramapo's Deputy Finance Director, joined in the scheme by, among other things, directing improper transfers from the Ambulance Fund to the General Fund.
According to the SEC, the alleged conspirators' false or misleading statements and misconduct made it appear as though Ramapo's General Fund had a positive balance, when in fact it had a negative cash balance throughout the alleged relevant period.
Source: SEC's court Complaint, page 3
The SEC Demands A Jury Trial
In its court Complaint, the SEC's demanded a jury trial against Ramapo. This made it one of the few cases in which the SEC has sought a jury trial against a municipality.
The first case ever in which the SEC pursued a federal jury trial against a municipality or one of its officers for violations of the federal securities laws involved the City of Miami (FL). That case was filed in court approximately three years before the Ramapo case, with the SEC winning a jury verdict in September 2016.
Whistleblowers Can Report Municipal Town Bond Fraud To The SEC
This case illustrates some types of misconduct that could give rise to SEC whistleblower cases if reported to the Commission through the SEC whistleblower program.
However, the SEC has not made any public statement as to whether this case was itself an actual SEC whistleblower case. The SEC Office of the Whistleblower posts Notices of Covered Action ("NoCA") for Commission actions where a final judgment or order results in monetary sanctions exceeding $1 million. The NoCA list does not disclose if a particular Enforcement action was brought as the result of an SEC whistleblower case, tip, complaint, or referral being filed with the Commission.
For more information about municipal town bond fraud, click on the links below:
The SEC's court Complaint & Jury Demand in SEC v. Ramapo. (External link to the SEC's website.)
The SEC's Press Release announcing the charges. (External link to the SEC's website.)


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What Second Language Will Be the Most Valuable for You?
Are you planning to learn a second language? The benefits are well-documented, ranging from a reduced risk of dementia in your old age to a more lucrative career. But which language should you study? There are almost 5,000 languages spoken throughout the world. How do you know what second language is the most valuable?
To help you out, we've chosen the six most valuable second languages for English speakers to learn.
Spanish (405 million native speakers)
Almost 6 percent of the world speaks Spanish, including millions of people in Mexico, Central and South America. Spanish is also an official language of numerous international organizations, including the United Nations, the European Union, and the World Trade Organization.
Without a doubt, Spanish is one of the most valuable second languages if you're in the United States-even if you never plan to travel outside the country. That's because it's the most common language in the US aside from English. It's also the most "in-demand" language among US employers. However, the number of US children who grow up bilingual in Spanish is also high. So, Americans who learn Spanish as a second language face more competition in the job market than those who acquire a less common language.
Mandarin Chinese (955 million native speakers)
Looking for a challenging language that could lead to a raise? Consider learning Mandarin, the most commonly spoken language in the world.
Many US-based companies depend on relationships with Chinese suppliers and manufacturers. Other businesses are expanding to court China's growing consumer class.
Consequently, demand for Mandarin speakers is high. In 2014, 21,770 job openings in the US required fluency in Mandarin. That's a drop in the bucket compared to the job openings for Spanish speakers. However, because Mandarin isn't a common language to study, there's not nearly as much competition.
German (95 million native speakers)
Demand for German speakers is high due to the country's bustling economy and close trading relationship with the United States. Jobs that require fluency in German often pay exceptionally well. For example, Kiplinger.com says "The median U.S. salary for jobs requiring German is higher than for any other language on our list." Meanwhile, The Economist found that learning German could translate into an extra $128,000 in earnings over the course of your career.
French (75 million native speakers)
The popularity of the French language may have declined in recent years, but it's still an important language for international business and diplomacy. According to Kiplinger, it's also the third most commonly requested language among US employers.
arabic (295 million native speakers)
Are you looking for a job in the oil and gas industry, diplomacy, or national security? Arabic might be the language for you. According to the National Security Education Program, Arabic is a "critical" language for the United States.
Unfortunately, like Mandarin, it's a difficult language for English speakers to learn.
portuguese (215 million native speakers)
The Brazilian economy is the eighth largest in the world. However, since most Brazilians are not fluent in English, organizations that wish to do business in Brazil need Portuguese-speaking employees.
Only 1% of Americans are fluent in a foreign language, so no matter which language you choose, you'll still be ahead of the crowd.
Of course, fluency in another language isn't always enough. Sometimes, businesses need a specific skill set combined with foreign language fluency. For example, just being bilingual doesn't give someone the ability to translate legal contracts or healthcare information. You also need expertise in the field.
If your business is looking for translation help from skilled linguists with expertise in specialized subjects, you've come to the right place. Take a look at the services we offer and the industries we serve, and then contact us for a quote.
Translation | Localization


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Citronlunden ( – Etz Limon; ) er en israelsk film fra 2008, der er instrueret af den israelske instruktør Eran Riklis.

Plot
Da den israelske forsvarsminister (Doron Tavory) flytter ind ved siden af den palæstinensiske kvinde Selma (Hiam Abbass), starter en kamp om Selmas citronlund, som hun har arvet efter sin far, idet denne udgør en sikkerhedstrussel og derfor skal ryddes. Selma allierer sig med advokaten Ziad Daud (Ali Suliman), og mellem de to opstår en gensidig sympati, der bunder i Selmas følelsesmæssige isolation: Hendes søn bor i USA, og hendes datter besøger hende sjældent, og Ziads savn efter sin datter, som han har efterladt i Rusland hos hendes mor. Filmen handler dels om Selmas kamp for sin ret og dels de menneskelige omkostninger ved den israelsk-palæstinensiske konflikt.

Priser

Eksterne henvisninger

Film fra Israel
Film fra 2008
Arabisksprogede film

redpajama_set_name: RedPajamaWikipedia


WordPress is actually a CMS (content management system) but the term cms often strikes fear into the hearts of users, because it implies a complicated and difficult to learn interface. Many people have experienced CMS that is full of bugs, slow to upload, requires constant updating by its creator or even all three. By contrast WordPress is a deeply intuitive system that allows you to add new images, blog posts or whole pages with the utmost simplicity. Because the system is so simple, and formatting time is so swift, you can dedicate more time to finding just the right words and images to convey your message.
A WordPress site is designed to be eminently scalable – whether you have a one page website or hundreds or even thousands of pages, site performance will remain completely clean, swift and efficient, unlike other sites which can bog down' as they become more complex and layered. Not only that, but by using your administrative function you can set up multiple users for the site, and give each one different access levels and capabilities – this means you can delegate areas of your site without worrying about giving people access to back office functions you'd prefer to keep private.
WordPress code is deliberately created to be clean and straightforward – this means that Google, and all other search engines, find it easy to read a WordPress site and index it. This means that a WordPress site is likely to rank higher than a less easy to read site. In addition, every single page, post and even image can be given its own meta tag and title, along with keywords so that every element of a WordPress site can be precisely and intensively optimized to ensure every search engine ranks each individual element of your site.
Unlike bespoke website designs WordPress allows you, the site owner, to have complete control of your website – no need to hire a web designer or even content manager, and therefore no hanging around waiting for somebody to update your website to your wishes. With your site's performance literally at your fingertips, you can update and alter it whenever you wish, making it highly responsive.
Because WordPress serves as the engine' of your website, the whole appearance and function of the site can be customized to let your brand, hobby or interests illuminate the entire website and make it uniquely yours. It's a very cost-effective process because there are literally thousands of WordPress themes that permit you to change your website design instantly. And unlike paying for web design, many themes are available for free. In addition to themes, there are over 50,000 plugins that allow you to add every kind of function to your website – from contact forms to spam catchers, from e-commerce add ons to analytics programs, there is everything you could need to run a business website or a personal web page.
Because WordPress is browser based it's possible to log into the system from any computer and manage your website. This is possible because all the technical functions such as html or ftp are built into the software you use to create and upload pages, no extra software is required, so no licenses, no 9-5 only software support and no tied to a single computer' packages – WordPress is ready to go whenever you want, wherever you are in the world, whatever you want to do.
Since 2003, WordPress has been powering websites across the internet – this means that not only are there hundreds of thousands of enthusiastic WordPress users out there, many of them are designers, developers, writers and SEO experts in their own right. They bring their expertise, as well as their enthusiasm, to the global WordPress community, which means that there are always people willing to help you get the best from your WordPress site. They create beautiful new elements to enhance their own WordPress websites and delight in making their creations available to others.


A-frames are one of the oldest method of advertising that converts a passerby into a potential customer. Studies show that businesses that use signage placed outside on the premises increase their sales and profitability. A well designed A-frame is a perfect way to promote yourself or an event close to home.
Their effectiveness can be pushed the extra mile when you add a well worked design into the equation. A well designed sign that has a straightforward message makes a great first impression. Having a branded A-frame shows your market that you care about your appearance and will give the impression that you do business in a similar fashion!
They're portable too! You are not limited on where you can advertise as you can take them with you to a convention, trade shows, or local events. Their low cost and versatility makes an A-frame hard to beat when choosing products within your advertising budget.


For all of you who hope the U.S. government will lift the veil on its alleged stockpile of evidence that Earth is being visited by extraterrestrials -- take heart.
This week, former Clinton chief of staff and UFO advocate John Podesta was named as President Obama's newest advisor.
Podesta, 64, has more than once publicly urged the U.S. government to release any UFO files that could help scientists determine "the real nature of this phenomenon."
Podesta's stance on UFOs can be seen in the following video clip from the 2009 James Fox film, "I Know What I Saw." Podesta spoke at a 2002 news conference at the National Press Club in Washington, D.C., where he called upon the U.S. government "to declassify [UFO] records that are more than 25 years old."
I'm skeptical about many things, including the notion that government always knows best, and that the people can't be trusted with the truth. The time to pull the curtain back on this subject is long overdue. Presenting the facts, the book includes statements from only the most credible sources -- those in a position to know -- about a fascinating phenomenon, the nature of which is yet to be determined.
It's time to find out what the truth really is that's out there. The American people -- and people around the world -- want to know, and they can handle the truth.
Now that Podesta seemingly has Obama's ear, will the former White House chief of staff stay true to his stance on UFOs and use his new advisory position to sway the president on this topic?
Part of the answer might lie in whether or not Obama would consider releasing UFO information as a way to improve his popularity.
The other part of the answer, of course, is whether or not there truly are UFO secrets to even divulge.


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UN Rights Expert Urges Protection of Transgender People
Margaret Besheer
Victor Madrigal-Borloz, independent expert on protection against violence and discrimination based on sexual orientation and gender identity, briefs journalists at the United Nations in New York, Oct. 25, 2018. (M. Garten/U.N. photo)
The U.N. human rights expert on preventing violence and discrimination against people based on their sexual orientation and gender identity is urging countries to amend laws and policies to protect and include those from this part of society.
"These persons are suffering levels of violence and discrimination that are offensive to human conscience," U.N. Special Rapporteur Victor Madrigal-Borloz told reporters Thursday.
During the presentation of his report to the General Assembly committee that handles human rights, he urged countries that classify certain forms of gender as medical conditions to eliminate that practice, as it further marginalizes a vulnerable segment of the population.
Madrigal-Borloz focused his report on transgender people — those who identify with a different gender than the one assigned to them at birth — noting that most of them are not granted legal recognition of their gender identity.
Basic rights at stake
The special rapporteur warned that when a state does not recognize a person's self-identified gender, it can lead to the denial of many basic rights, including health care, education, employment and access to housing.
His comments came amid reports the Trump administration is looking to roll back recognition and civil rights protections of transgender persons in the United States.
Madrigal-Borloz noted that 71 countries still criminalize diversity in sexual orientation and gender identity. Of them, 20 countries criminalize certain activities or forms of gender identity.
"Clearly, criminalization is creating a situation where persons are not only not protected, but actively persecuted on the basis of their gender identity," he said.
Madrigal-Borloz said human rights advocates continue to receive reports of violence committed against these people, including murder, beatings, kidnappings, sexual assault and hate crimes. Often, when they seek help from police, they are harassed, abused or arrested.
The special rapporteur noted that U.N. conventions forbid discrimination against people based on their sexual orientation and gender identity, just as they prohibit discriminating against people on the basis of race or religion.
Paris Police, Activists Split Over Surge in LGBT+ Attacks
A GOP Favorite Faces a Democratic Groundbreaker in Vermont
Transgender Activists Rally Against Redefining Gender
Young Catholics Urge Vatican to Issue Inclusive LGBT Message
Bali Beauty Pageant Signals Renewed Anti-LGBT Crackdown


This year was typified by many small piles of rubbish dotted everywhere with a general spread along Gooreengi Way and Market Way / North Arm Cove Road. A large pile of household rubbish was found near Beauty Point near a 40ft container/squatter cottage there.
On the Saturday the Carter / Anderson team showed up and scoured the foreshore between Medina Bay and Baromee Point around to the end of the Esplanade. Tyres were the main culprits otherwise - just general flotsam. It wasn't an easy task so thank you all.
My admiration goes to Pat Brennan who took it upon herself to start on Gooreengi Way for the Saturday afternoon filling bags up with litter just from the west side of this road. Having exhausted herself that afternoon she came back for more on the Sunday morning. She is the hero of the weekend, and must be totally covered with Koala stamps. Sad to say her efforts were insulted the following Monday by a philistine leaving takeaway remnants in the same area.
Sunday morning saw vehicles and trailers in profusion, so everyone went out to their designated areas after signing in. The Hann/Brennan team heroically completing Gooreengi Way, the newly formed Easton Team really doing the miles with the Market Way/ North Arm Cove clean, which was a bit beyond the pale for first timers but they bounced back smiling.
With a convoy of 4WDs and a large tip trailer the 'keen clean team' comprising of John, Jennie, Karl, Tim, Michael, Maria, Adrienne and esteemed guest, Reinhilde, who came all the way from Germany......to pick up rubbish around Beauty Point environs...well not really, but her enthusiasm was appreciated by all. It took hours to clean this pile. I hear it was left under unfortunate circumstance, but this was too much of an eyesore to be ignored. That did mean other sites were unattended that day. Nearby there was a full porta loo left full by campers at the point, the onerous and odourous task of disposal, which Karl kindly attended to, can no further be elaborated on as this is a family article.
The Moller team cruised around the north east areas, and came back with many bags and larger items – another great effort. They reported of a site with many tyres further into the bush. Not sure what we can do about that – any suggestions? Tyres do fill with water and breed all sorts of nasties.
There wasn't anything particularly unusual found this year to comment about, just boring rubbish mainly. Three cars have been found and a 1800mm diameter reel of telstra cable is still there from last year. I will get onto Council about that. Speaking of which, at the end of the day this was the largest pile ever and I am sure we all agree Council and the machinery operators do a great job in removing the spoils promptly.


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Working Together to Nourish the Nation
USDA Makes it Easier to Feed Kids and Those Who Need Food During the COVID-19 National Emergency
Thursday, March 26, 2020 3:56 PM
Roger Szemraj (Administrator)
Contact: USDA Press
Email: press@usda.gov
Nationwide waivers grant states the flexibility to allow parent pick-up of meals for kids, ease burdens on local meal providers
(WASHINGTON, March 26, 2020) – U.S. Secretary of Agriculture Sonny Perdue announced additional flexibilities to make it easier for children, seniors, and individuals with disabilities to get food during the COVID-19 national emergency and remove administrative roadblocks for the dedicated local staff who serve them. These changes are in line with USDA's commitment to keep Americans safe, secure, and healthy during this national emergency and explore all options to keep kids fed during this unprecedented time.
"USDA is committed to maximizing our services and flexibilities to ensure children and others who need food can get it during this Coronavirus epidemic," said Secretary Perdue. "This is a challenging time for many Americans, but it is reassuring to see our Government and fellow Americans stepping up to the challenges facing us to make sure kids and those facing hunger are fed."
Under one of the newly-announced waivers, USDA is giving states the option to allow parents or guardians to take meals home to their children. Typically, children would need to be present to receive a meal through USDA's child nutrition programs.
However, USDA recognizes that this may not be practical during the current COVID-19 outbreak. This flexibility is also available for states to assist seniors and individuals with disabilities served through the Child and Adult Care Food Program. Additional nationwide flexibilities announced today include:
Allowing states to waive meal pattern requirements, so local operators can create meals with the foods they have available; and
Delaying administrative deadlines associated with the Community Eligibility Provision to ease burdens on schools that are currently closed due to COVID-19.
Since the outset of the COVID-19 outbreak, USDA has been working tirelessly with states and local authorities to ensure schools and other program operators are empowered to continue feeding children. These flexibilities complement previously-announced nationwide actions that temporarily waive:
Meal times requirements to make meal pick-up easier,
Requirements that meals be served in group settings to support social distancing, and
The requirement that afterschool meals and snacks served through certain programs be accompanied by educational activities to minimize exposure to the novel coronavirus.
USDA will continue to provide technical assistance to help state agencies swiftly implement these flexibilities.
Today's announcement is the latest in a series of actions that USDA's Food and Nutrition Service has taken to uphold the USDA's commitment to "Do Right and Feed Everyone" during this national emergency. Other actions include:
Launching a new coronavirus webpage to proactively inform the public about USDA's efforts to keep children and families fed;
Investing in a public-private partnership to feed rural children impacted by school closures due to COVID-19;
Allowing states to issue Pandemic EBT (electronic benefits transfer, similar to food stamps) to families of children eligible for free or reduced-price meals dealing with school closures;
Providing administrative flexibilities in the Supplemental Nutrition Assistance Program (SNAP) and the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) to allow for social distancing; and
Allowing states to issue emergency supplemental SNAP benefits to increase recipients' purchasing power during the national emergency.
These actions and more are part of USDA's focus on service during the COVID-19 outbreak. To learn more about FNS's response to COVID-19, visit www.fns.usda.gov/coronavirus
USDA's Food and Nutrition Service (FNS) administers 15 nutrition assistance programs that leverage American's agricultural abundance to ensure children and low-income individuals and families have nutritious food to eat. FNS also co-develops the Dietary Guidelines for Americans,which provide science-based nutrition recommendations and serve as the cornerstone of federal nutrition policy.


Christine Wartman, an associate civil engineer with Los Angeles County Flood Control, at a soccer field at Sun Valley Park. The soccer field sits atop underground caverns used to drain stormwater into the earth.
Concrete caverns are being set into place in Sun Valley Park. They would be covered over with about five feet of fill dirt. Baseball and soccer fields were placed atop the caverns.
Armando Mendoza enjoys a day in Sun Valley Park with family and friends. Under nearby baseball and soccer fields, a series of buried caverns percolate stormwater into the earth.
Concrete storm water percolation caverns as they appeared in 2007 before they were buried underneath the baseball and soccer fields at Sun Valley Park. Water that has been filtered of metals and pollutants flows into the caverns and it percolates into groundwater aquifers.
These canisters in a vault under Sun Valley Park filter pollutants and metals from stormwater runoff before it is filtered into the ground.
The engineered Dominguez Gap Wetlands in Long Beach filters stormwater and runoff from the Los Angeles River. Then the water is siphoned under the river to a spreading ground to the west.
Earth movers claw at piles of inert construction debris at Strathern Pit near Strathern Street and Tujunga Ave. It is a materials recycling dump that will be transformed into an eningeered wetlands park, funneling stormwater into underwater caverns at adjacent Sun Valley Park. The future Rory Shaw Wetlands Park will be named for a Los Angeles city civil engineer who was killed when he was swept into a giant sinkhole nearby during heavy rain and flooding in Sun Valley.
In its first century, Los Angeles County Flood Control District built mostly dams and storm channels. The idea was to funnel water to the ocean as fast as possible. But as it enters its second century, the challenge is to store much of that water underground.
The Flood Control District isn't about to change its name to StormCatchers, but it is laser-focused on new ways to direct runoff underground to increase our water supply, rather than lose it to the Pacific Ocean. The district is also making many of its projects do double-duty as recreation places.
Projects like these represent not just the future of the county but also California as a whole. Storm water capture and reuse is one of the cornerstones of efforts at the local level to be more water independent.
One such project is hidden under Sun Valley Park. As kids play on a baseball field and a wandering vendor selling frozen paletas from a cart rings his bell, a grid of underground caverns beneath their feet awaits the next big rain.
Armando Mendoza, 16, studies physics at a local high school. He's been coming to this park for five years, ever since moving to Sun Valley. But he wasn't around to see the caverns constructed. "That's so cool, I didn't know that," he said.
In 2007, earth movers tore out the baseball and soccer fields, dug down about 30 feet and loaded in a series of open-sided concrete boxes, each about six feet tall. Then the boxes were covered over with dirt and the ball fields replaced.
The boxes function as huge drains. Rain that falls on flood-prone Sun Valley percolates underground here.
In a fenced corner of the park, Flood Control civil engineer Christine Wartman opens up a heavy steel door covering a 10-foot deep pit. It's full of cylinders the size of trash barrels. This is where the storm water comes in and gets filtered.
"Inside these canisters that you see, there are different media types, like soils, different types of soils or metals that take out other metals as the water flows through them," she said.
From there the water flows to the caverns under the ball fields.
"They look like tombs or chambers of some sort," she said.
A drop of water filtering into the earth at Sun Valley Park will be pumped, treated and emerge in home faucets as drinking water in a few years.
"The point of the project was to see how these systems worked. This was the first of its kind in L.A. and we wanted to see how it would function before we do something similar in different parks around the county," Wartman said.
The caves were part of an experiment, one of 16 pilot projects designed for the 5-square mile watershed in Sun Valley. They will direct storm water underground. These projects have been successful enough that they now represent the Flood Control District's new strategies for managing storm water that falls elsewhere in the county. The agency plans to duplicate the Sun Valley Park approach in many other locations — not just parks, but other large and small parcels, including parking lots.
It's a big change from the agency's first century, during which it built 14 dams, 500 miles of open channels and 2,800 miles of storm drains, all to move storm water into the ocean.
"Over the first hundred years, the focus was largely on providing flood protection to the public," said Gary Hildebrand, the district's deputy director. "Our local groundwater supplies, which provide about one-third of the supply to L.A. County, also needed to be replenished."
The Flood Control District has been storing groundwater for several decades, but these types of multiple-use projects — pairing water storage with recreation or habitat — will become an even larger portion of its project to-do list, Hildebrand said.
Hanging onto storm water for future use takes on more importance as California deals with a fourth year of drought. It's also a policy response to predictions that changing climate patterns will bring rain in similar amounts as in the past but in swifter and greater concentrations.
Sun Valley has a history of flooding. In the early 2000s, the Flood Control District had proposed building another channel to carry the runoff from Sun Valley's industrial and residential streets into the Los Angeles River. But heavy community pushback to a concrete channel led to this unusual dual-purposing of park and groundwater replenishment.
The water caves at Sun Valley Park are percolating only enough water for about 30 households, but a companion project next door will increase that amount to about 620 households.
Strathern Pit is a loud and dusty recycling dump for inert construction materials. It's 46 acres of dirt fill, ceramic tiles, broken toilets, concrete slabs and bricks. Earthmoving machines claw at the piles not far from homes on Strathern Avenue.
The dump started out as flat land, but in the 1940s, it was excavated for gravel that was used in construction at the Port of Los Angeles. Once the gravel was exhausted, the pit was refilled with inert construction materials, said Wartman. It's now closed, and the materials that are left on the site will be crushed and used as fill for a new park or sold.
In just another year, this dump will be remade into an engineered wetlands park. The wetlands portion will collect and filter water that is headed to the Sun Valley Park underground caverns next door.
The new Rory Shaw Wetlands Park will have a tot lot, tennis and basketball courts, amphitheater and synthetic soccer field. It will also have a 50-foot-deep pond for holding storm water – that part will be fenced off from the public.
The park is named for Rory Shaw, 47, a city of Los Angeles civil engineer who was killed when he was swept by floodwaters and heavy rain into a giant sinkhole that opened up on Tujunga Avenue in February 2005. At that time, the community of Sun Valley still had no flood control or storm drains.
To get an idea of what the engineered wetlands would be like, visit the Dominguez Gap Wetlands in Long Beach.
"Back in the early 2000s we started re-envisioning this facility to not only improve its ability to capture stormwater for groundwater recharge, but also fold in water quality improvements, habitat by including wetlands features, and also recreational amenities for the surrounding community," Hildebrand said.
"If you were to have come here 10 years ago — it was closed off to the public — you would have seen a very barren facility with very little vegetation, not much water in it, at times," Hildebrand said.
Now it's a half-mile-long engineered wetlands. It pulls in water from the Los Angeles River. The water circulates around a half-mile loop, feeding plants and providing habitat for birds and animals. Then it's siphoned underneath the L.A. River to a big spreading ground to the west.
The prototypes have led to other projects across Los Angeles county. The Santa Monica Library, for example, has an underground cistern that collects some 200,000 gallons of urban runoff each year. Harbor City has a half-mile long greenway that collects water. The Mar Vista Recreation Center in Los Angeles is getting some underground caverns. Other projects are planned for foothill communities in Pacoima and Tujunga.
The projects are paid for out of grants from the state, contributions from local water agencies and money from property owners throughout L.A. County who pay into the flood control system each year – there's a flood control district assessment on annual county property tax bills.
Where does LA's rainfall end up?


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Protesters Detained Outside Moscow FSB Headquarters
MOSCOW -- Russian police have arrested seven people protesting outside the headquarters of the Federal Security Service (FSB) in Moscow, according to OVD-Info, an independent group that monitors crackdowns on demonstrations.
Several dozen protesters had gathered outside the Lublyanka building on December 16 for an unauthorized demonstration against what they said were abuses committed by the security forces and the conflict in eastern Ukraine.
Some of the demonstrators denounced the jailing of Lev Ponomaryov, a 77-year-old human rights activist who is serving a 16-day term in detention -- in part for a Facebook post about a protest.
There was no immediate comment from Moscow police.
Russia seized Ukraine's Crimea region in March 2014 and supports separatists battling Ukrainian forces in eastern Ukraine in a conflict that has killed more than 10,300 people since April 2014.
Russia denies interfering in Ukraine's internal affairs, despite compelling evidence that Moscow has provided military, economic, and political support to the separatists fighting against Kyiv.
Copyright (c) 2010-2018. RFE/RL, Inc. Reprinted with the permission of Radio Free Europe/Radio Liberty, 1201 Connecticut Ave., N.W. Washington DC 20036.
ecoi.net summary:
Moscow: Police arrest 7 participants in unauthorised demonstration outside headquarters of the Federal Security Service (FSB) protesting against abuses committed by the security forces and the conflict in eastern Ukraine
Original link:
https://www.rferl.org/a/protesters-detained-outside-moscow-fsb-headquarters/29659305.html
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In honor of Daisy Dirt Dog. It's her birthday this week. Daisy hates her booties, even though they keep her paws from getting raw from long days in sharp granite. Super DirtBomb. I know I dug a giant hole in the lawn! You guys just don't understand, I was protecting the lawn from marauding voles!
Daisy Snow Dog- ripping it near Barker Pass. She gets more face shots than any two-legger.
A twofer this week. There's something about tent pics that represents a bit of home in the outdoors. Home is where you pitch it. Daisy Trail dog- sacked out on the "trail couch" in the camp living room/kitchen.
Daisy Snow Dog- prancing with the simple joy of fresh tracks.
Daisy in her float coat sailing off the West Shore on her International 420 (Named the DaisyDela of course). TahoePix will appear periodically as we share some of the thousands of photos we've taken on our Tahoe adventures.


When creating the world of Jardins D'Écrivains, Anaïs Beguine combined two of her passions, gardens and literature.
Symbolizing an era, a character or a place, a trail of scent evokes the stories of famous writers who live on through their words and with a perfume that arouses the soul, each telling its own story.
Anaïs Beguine has begun a new chapter, focusing on the nineteenth-century French courtesans known as the Queens of Paris, The Cocottes.
They entertained, seduced and made headlines to the point where they imposed their style and taste on Europe and the Americas. Les Cocottes de Paris brings you new perfumes that tell a story centered on three scandalous icons.
As fascinating, sensational and beguiling as a fragrance can be.


SHERMAN, Texas - Austin College Athletics is excited to announce the Modern Woodmen Fraternal Financial Student-Athlete of the Week, an award highlighing our great student-athletes on and off the field of play each week. This week's honoree is 'Roo women's soccer player Hallie Dickerson.
Dickerson, a sophomore from McKinney, was recently honored by the Southern Collegiate Athletic Conference with the Character and Community Award for her vast community service and volunteer work. A two-year starter for the Kangaroo women's soccer team, Dickerson has consistently shown high character and leadership on the field and in the community. She's committed 100 hours of community service in each of the last three years, including working with Habitat for Humanity, the Sherman Women's Crisis Shelter, and Sneed Restoration Prairie.
"Hallie is a pleasure to coach and to mentor," said assistant women's soccer coach Grace Sokolow. "She is bright, inquisitive, and brings dignity and leadership to her work both on and off the field. She is a good student, a good soccer player, a good teammate, and most importantly, a good friend."
In two seasons playing primarily as a defender for the 'Roos, Dickerson has appeared in 31 games, starting 29.
Modern Woodmen Fraternal Financial is proud to recognize and support the ongoing achievements of Austin College student-athletes. Their North Texas Region understands that the pursuit of excellence on and off the field creates a foundation for continued excellence after graduation. For more information about opportunities for careers and internships, go to MWAcareers.org.


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Corporate Score Card
Wednesday, July 17, 2019.
New Mail Nigeria
Ex-Pope Benedict denies covering up sexual abuse
Semiu Salami
Former Pope Benedict
Former Pope Benedict has denied that he tried to cover up sexual abuse of children by Roman Catholic priests, in his first direct published comments since he stepped down.
The comments came in an 11-page letter to Italian author and mathematician Piergiorgio Odifreddi, who had written a book about the problems facing the Roman Catholic Church before the pope resigned in February.
"As far as you mentioning the moral abuse of minors by priests, I can only, as you know, acknowledge it with profound consternation. But I never tried to cover up these things," Benedict, who now has the title Emeritus Pope, said.
Excerpts of Benedict's letter were published in the Rome Newspaper La Republica, on Tuesday with the former pope's permission.
It was believed to be the first time Benedict has responded to the sexual abuse accusations in the first person, although the Vatican has always said he did much to put an end to sexual abuse of minors by priests and never tried to cover it up.
It was also the first time since Benedict resigned on Feb. 28 that anything precise that he has written or said was published, although some people who have visited him in the Vatican house where he is living out retirement have indirectly reported to outsiders some of his comments to them.
Victims groups have accused Benedict of not doing enough to stop the abuse of children by priests while he was pope and before when he was head of the Vatican's doctrinal office.
They say there is much still to be discovered about how the Church behaved in the past and want more bishops who were aware of abuse to be held responsible.
The Catholic Church's crisis came to light in Boston in 2002 when media began reporting how cases of abuse were systematically covered up and abusive priests were shuttled from parish to parish instead of being defrocked and handed over to civil authorities.
Since then, the Catholic Church in many countries has set up new guidelines to deal with cases of past abuse, prevent new cases, report abuse to police, and stop potential abusers from entering the priesthood in the first place.
The rest of the letter from Benedict to Odifreddi referred to other aspects of the author's book, called "Dear Pope, I Am Writing You", such as the conflict between good and evil.
Piergiorgio Odifreddi
Roman Catholic priests
Rome Newspaper La Republica
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Bone is constantly being remodeled in a process where osteoclasts resorb bone, osteoblasts deposit type I collagen and other proteins in the resorbed lacunae, and, lastly, the collagen mineralizes to form bone (1, 2). The first partner in this "pas de deux" is the osteoclast, which arises from hematopoietic monocytic precursors and resorbs bone. The formation of osteoclasts requires the cytokine receptor activator of nuclear factor kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) (3). RANKL enhances the expression of nuclear factor of activated T cells, cytoplasmic 1 protein (NFATc1), a transcriptional factor that up-regulates the expression of osteoclast differentiation–associated genes, such as tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CALCR), and cathepsin K (CTSK), whereas the transcriptional factor interferon regulatory factor 8 (IRF8) can suppress RANKL-induced NFATc1 expression (4). The second player in the remodeling cycle is the osteoblast, which is differentiated from mesenchymal stem cells (MSCs). This process requires the activation of core-binding factor α-1/runt-related transcription factor 2 (RUNX2) and osterix, which stimulate the expression of osteoblast differentiation–associated genes, such as bone γ-carboxyglutamic acid–containing protein (BGLAP), alkaline phosphatase (ALP), and collagen type I α1 (COL1A1).
This delicate balance between osteoclast-mediated bone resorption and osteoblast deposition of matrix is disrupted in certain types of malignancies, including multiple myeloma and solid tumors, such as breast and lung cancer, among others (5, 6). In multiple myeloma, tumor cells secrete RANKL or stimulate the release of RANKL by surrounding stromal cells, leading to enhanced osteoclast differentiation. Myeloma cells can also secrete dickkopf-related protein 1 (DKK1), which inhibits the Wnt/β-catenin signaling pathway and suppresses osteoblast differentiation. Attempts to target RANKL and DKK1 in myeloma therapeutically have achieved only modest success. For example, the anti-resorptive agent denosumab (a monoclonal antibody against RANKL) was examined in a phase 3 trial, but only moderately affected myeloma-induced lytic lesions were shown (7). BHQ880 (a monoclonal antibody against DKK1) is now in a phase 1/2 study, but its application in myeloma fails to restore new bone formation (8). Thus, bisphosphonates, which suppress osteoclast function, remain the mainstay in treatment of myeloma-induced bone disease. Unfortunately, bisphosphonates are less than fully effective and cause osteonecrosis of the jaw in 2 to 5% of treated patients (7). The goal of the current studies is to identify other factors produced by myeloma cells, which regulate both resorption and formation, and to determine whether this information can be used to prevent myeloma-induced bone disease.
Thymidine phosphorylase (TP), also called platelet-derived endothelial cell growth factor, is an enzyme that can reversibly catalyze the conversion of thymidine to thymine and 2-deoxy-d-ribose-1-phosphate (2DDR1P), which is further dephosphorylated into a smaller, more stable molecule, 2-deoxy-d-ribose (2DDR) (9). TP has been found in a wide range of normal tissues (10) and participates in wound healing and a variety of chronic inflammatory diseases (11). TP is highly expressed in many types of cancers, including lung and breast cancer, and plays an important role in angiogenesis and anti-apoptosis (12, 13). Clinically, elevated levels of TP are associated with cancer aggressiveness and poor prognosis (11), but TP has never been implicated in the regulation of bone resorption or formation. In previous studies, we demonstrated that p38 MAPK (mitogen-activated protein kinase) activity in myeloma cells induces osteolytic bone lesions (14, 15), and others demonstrated that this signaling regulates TP expression (16, 17). We therefore hypothesized that the TP gene plays a role in the pathogenesis of cancer-induced bone destruction in myeloma.
Here, we report an association of cancer-expressed TP with osteolytic bone lesions, as well as the ability of TP to orchestrate osteoclast-mediated resorption and decreased bone formation. Specifically, TP down-regulated the expression of IRF8 and thereby activated RANKL-induced NFATc1 expression, leading to an increase in osteoclastogenesis and bone resorption. We further observed that myeloma-expressed TP suppressed osteoblastogenesis and bone formation by down-regulating the expression of RUNX2 and osterix in human MSCs. Our findings not only elucidate a mechanism of cancer-induced suppression of osteoblast differentiation and activation of osteoclast differentiation and activity, but also implicate a potential therapeutic approach for cancer patients with osteolytic bone lesions by targeting TP.
We confirmed the presence of myeloma (CD138+) cells in tissue array biopsies and found that TP+ cells in the bone marrow from 14 myeloma patients were significantly greater than those from 14 healthy donors (Fig. 1, A and B). TP was expressed in most of the bone marrow aspirates of primary myeloma cells (4 of 6 patients) and in most of the established human myeloma cell lines (4 of 6), but not in aspirates of plasma cells from normal subjects (Fig. 1C).
To determine whether TP expression correlated with the frequency of bone lesions in myeloma patients, we performed two experiments using samples from tissue banks. In the first experiment, there was a strong positive correlation between TP gene expression in myeloma cells and bone lesion numbers in 52 patients (Fig. 2A); in the second experiment, there was a robust positive correlation between TP immunohistochemistry and bone lesion numbers (Fig. 2B). TP expression was higher in myeloma cells from patients with high bone lesion scores than in those from patients with low lesion numbers (Fig. 2C).
We then isolated primary myeloma cells from the bone marrow aspirates of 10 newly diagnosed patients. On the basis of the TP expression in myeloma cells as detected by Western blot, the aspirates were separated into high and low TP expression groups: TPhigh and TPlow (n = 5 patients' bone marrow aspirates per group) (fig. S1A). To determine the functional role of myeloma-expressed TP in lytic bone lesions, we injected TPhigh or TPlow cells into human bone chips that had been implanted into severe combined immunodeficient (SCID)–hu mice. Mice with no tumor cells (but with bone chips implanted) were controls. More lytic lesions and a lower percentage of bone volume versus total volume (BV/TV) were observed in the bone chips of mice injected with TPhigh cells than in those injected with TPlow cells (Fig. 2, D and H). Moreover, injecting wild-type ARP-1 cells, which have high TP expression, into mouse femurs caused more lytic lesions than wild-type MM.1S cells, which have low TP expression (Fig. 2, E and H, and fig. S1B). ARP-1 cells with TP knocked down by small hairpin RNAs (shRNAs; shTP ARP-1) were injected into mouse femurs and caused fewer lytic lesions than did ARP-1 cells with a nontargeted shRNA (shCtrl ARP-1) (Fig. 2, F and H, and fig. S1C). Conversely, MM.1S cells expressing TP complementary DNA (cDNA; TP MM.1S) caused more femur lesions than did wild-type MM.1S cells expressing a control vector (Vec MM.1S) (Fig. 2, G and H, and fig. S1D). In summary, cells with high TP expression induce the formation of lytic lesions in mouse and human bone.
To determine whether lytic lesions result from a change in the tumor burden within the marrow milieu, we measured the levels of myeloma-secreted M protein, a reflection of tumor burden, in the mouse sera. The modulation of TP expression in myeloma cells did not change the serum levels of M protein (fig. S1E). In addition, there were no differences in viability or apoptosis in control and modified ARP-1 or MM.1S cell lines (fig. S1, F and G), indicating that changes in TP expression did not affect myeloma cell growth or survival. Together, these results reveal that myeloma cells express TP and enhance lytic bone lesions in patients and mice with myeloma.
We studied the ability of myeloma-expressed TP to regulate osteoclast differentiation in vitro using a standard osteoclast differentiation protocol (18), in which TRAP+ cell number and TRAP5b secretion were assessed in the presence of RANKL (10 ng/ml) and M-CSF (25 ng/ml), both of which are needed for osteoclast formation. The coculture of preosteoclasts (preOCs) with TPhigh patient myeloma cells or wild-type ARP-1 cells induced higher numbers of multinuclear TRAP+ cells (fig. S2A), more TRAP5b secretion (fig. S2B), and higher expression of the osteoclast genes TRAP, CALCR, and CTSK (fig. S2, C and D) than coculture with cells expressing low levels of TP (TPlow or wild-type MM.1S). Knockdown of TP expression in wild-type ARP-1 cells reduced osteoclast differentiation and activity (fig. S2, A, B, and E). In contrast, overexpression of TP in wild-type MM.1S cells enhanced the osteoclast differentiation and activity (fig. S2, A, B, and F).
To assess the role of myeloma-expressed TP in osteoclast-mediated bone resorption in vivo, we stained myeloma-bearing human bone chips or mouse femurs for TRAP and analyzed the percentage of bone surface eroded by osteoclasts (ES/BS) and the percentage of bone surface covered with osteoclasts (Oc. S/BS). The percentage of ES/BS (Fig. 3A) and Oc. S/BS was higher in the mice injected with myeloma cells that had high TP expression (TPhigh, wild-type ARP-1, shCtrl ARP-1, or TP MM.1S) than in those that had low TP expression (TPlow, shTP ARP-1, wild-type MM.1S, or Vec MM.1S) (Fig. 3, A and B). These results indicate that myeloma-expressed TP enhances osteoclastogenesis.
In preOCs, myeloma cells have been shown to enhance the expression of NFATc1, a transcriptional factor that plays a pivotal role in osteoclast gene expression and can be up-regulated by RANKL (19). We confirmed this effect of myeloma cells on NFATc1 expression in preOCs (fig. S3A) and further demonstrated that myeloma cells with high but not low TP down-regulated expression of the transcription factor IRF8 in preOCs (fig. S3A). To determine whether IRF8 mediates TP-induced osteoclast differentiation, its expression in preOCs was knocked down using IRF8 shRNA (shIRF8). When cocultured with wild-type ARP-1 cells, shIRF8-preOCs expressed higher levels of NFATc1 protein (fig. S3B) and secreted more TRAP5b (fig. S3C) than did control preOCs. Thus, myeloma-expressed TP abrogated the inhibitory effect of IRF8 on NFATc1 shown previously (3), leading to the promotion of osteoclast gene expression and osteoclastogenesis.
To determine whether myeloma-expressed TP regulates osteoblast differentiation in vitro, we cocultured the precursors of osteoblasts, MSCs, with patient myeloma cells and myeloma cell lines in osteoblast medium for 2 weeks. MSCs cultured alone in this medium served as a positive control. Mature osteoblasts that produced soluble ALP were positive for Alizarin red S staining (which is indicative of osteoblast-mediated bone formation activity) and expressed osteoblast differentiation–associated genes (fig. S4). In line with previous studies (20), the coculture of MSCs with myeloma cells inhibited osteoblast activity, but coculture with TPlow myeloma cells and low TP–expressing myeloma cell lines (wild-type MM.1S, Vec MM.1S, or shTP ARP-1) had comparatively more mature osteoblasts than those with high levels of TP (fig. S4).
To determine the role of myeloma-expressed TP in osteoblast-mediated bone formation in vivo, we collected myeloma-bearing human bone chips or mouse femurs, enumerated the osteoblasts localized on trabecular bone, and counted the percentage of osteoid surface (OS/BS) and of bone surface lined with osteoblasts (Ob. S/BS). The percentages of OS/BS (Fig. 3C) and Ob. S/BS (Fig. 3D) were lower in the mice injected with myeloma cells expressing high levels of TP (TPhigh, wild-type ARP-1, shCtrl ARP-1, or TP MM.1S) than in those injected with low-TP myeloma cells (TPlow, shTP ARP-1, wild-type MM.1S, or Vec MM.1S). In agreement with these data, bone formation rate was increased in mice injected with shTP ARP-1 cells (Fig. 3E) and decreased in mice with TP MM.1S cells (Fig. 3F).
Transcription factors such as RUNX2 and osterix promote osteoblast differentiation by up-regulating the expression of osteoblast differentiation–associated genes (21). In line with previous studies (20), we observed that the coculture of MSCs with myeloma cells reduced the expression of RUNX2 and osterix (fig. S5). Moreover, the expression of these transcription factors was significantly lower when they were cocultured with myeloma cells with high levels of TP than when they were cocultured with lower-TP myeloma cells (fig. S5A). Moreover, myeloma-expressed TP modulated the binding activity of RUNX2 and osterix to the promoter of osteoblast genes BGLAP and COL1A1 (fig. S5, B and C), a finding consistent with the effect of TP on bone formation.
DNA methylation of CpG dinucleotides is a key epigenetic modification that influences tissue- and context-specific gene expression (22) and is generally associated with gene silencing (23). To determine whether myeloma-expressed TP regulates the methylation of CpG islands (CGI) in RUNX2, osterix, and IRF8, we designed MSP [methylation-specific polymerase chain reaction (PCR)] and BSP (bisulfite sequencing PCR) primers targeting their CpG-rich regions (Fig. 4A and table S2). Methylation of RUNX2 and osterix in MSCs and IRF8 in preOCs was higher in the coculture of myeloma cells with high TP expression (wild-type ARP-1, shCtrl ARP-1, and TP MM.1S) than in the coculture of those with low TP expression (shTP ARP-1, wild-type MM.1S, and Vec MM.1S) (Fig. 4B). BSP analysis confirmed these results (Fig. 4, C to E), and similar methylation data were obtained from healthy individual and myeloma patient samples expressing different levels of TP (Fig. 4F).
DNA methyltransferases (DNMTs) are important for the methylation of gene promoters. We observed higher gene expression and enzyme activity of DNMT3A, but not DNMT1 or DNMT3B, in MSCs and preOCs cocultured with high-TP myeloma cells (wild-type ARP-1, shCtrl ARP-1, and TP MM.1S) than in those cocultured with low-TP cells (shTP ARP-1, wild-type MM.1S, and Vec MM.1S) (fig. S6, A and B). The clinical relevance was further studied by correlating TP expression in myeloma cells from patients or number of bone lesions in these patients with DNMT3A expression in MSCs and preOCs from the same patients. We found positive correlations between TP expression and number of bone lesions with DNMT3A in MSCs or preOCs (fig. S6C). Collectively, these data demonstrate that myeloma-expressed TP up-regulates DNMT3A expression and activity in MSCs and preOCs, consequently enhancing methylation of CGIs in the promoters of RUNX2, osterix, and IRF8 genes, suppressing osteoblast differentiation, and increasing osteoclast activity. These signaling changes shift the balance of bone remodeling toward a net loss of bone tissue.
TP degrades thymidine into thymine and 2DDR1P; the unstable 2DDR1P is further dephosphorylated to 2DDR (11). As expected, in vitro myeloma cells with high levels of TP secreted more 2DDR than did low-TP cells (fig. S7A). Similarly, higher levels of 2DDR were observed in the serum of mice bearing high-TP myeloma cells (TP MM.1S and wild-type ARP-1) than that of mice bearing low-TP cells (wild-type MM.1S and shTP ARP-1) (fig. S7B).
To test whether 2DDR mediates TP-induced suppression of osteoblastogenesis, we cultured MSCs in osteoblast medium with incremental doses of 2DDR for 2 weeks. The addition of 2DDR down-regulated ALP production, Alizarin red S staining, and the expression of osteoblast genes in a dose-dependent manner (fig. S7C). Furthermore, the addition of 2DDR decreased the expression of RUNX2 and osterix (Fig. 5A) and up-regulated the expression and activity of DNMT3A in MSCs (Fig. 5B). The culture of MSCs in medium with 2DDR induced the hypermethylation of CGIs on the RUNX2 and osterix genes (Fig. 5C) and inhibited ALP production and Alizarin red S staining (Fig. 5D). Knockdown of DNMT3A in MSCs partially abrogated the effects of 2DDR (Fig. 5, C and D).
To examine the role of 2DDR in the activation of osteoclastogenesis, we cultured preOCs with RANKL (10 ng/ml) and M-CSF (25 ng/ml) without or with escalating doses of 2DDR for 1 week. 2DDR enhanced multinuclear TRAP+ cell numbers, TRAP5b secretion, and the expression of the osteoclast genes TRAP, CALCR, and CTSK in a dose-dependent manner (fig. S7D). 2DDR also reduced IRF8 expression (Fig. 5E), up-regulated DNMT3A expression (Fig. 5F), and induced hypermethylation of CGIs in IRF8 (Fig. 5G). Knocking down DNMT3A in preOCs reversed 2DDR-mediated IRF8 hypermethylation (Fig. 5G) and increased osteoclast formation (Fig. 5H).
We next investigated the signaling pathways by which 2DDR may regulate DNMT3A expression. 2DDR is known to bind integrins αVβ3 and α5β1 (11). We confirmed in the literature that all MSCs in this study express both integrins (24) and that preOCs express αVβ3 (25). The addition of an antibody against either the α5 subunit of α5β1 or the αV subunit of αVβ3, but not control immunoglobulin G, to the coculture of MSCs with 2DDR enhanced osteoblast formation; the addition of both anti-α5 and anti-αV antibodies had a synergistic effect (fig. S8A). Moreover, application of both antibodies inhibited 2DDR-stimulated DNMT3A expression in MSCs (fig. S8B), indicating that 2DDR regulates osteoblast differentiation and DNMT3A expression via αVβ3 and/or α5β1.
Furthermore, we examined the extracellular signal–regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways downstream of α5β1 and αVβ3. The addition of 2DDR to the culture of MSCs up-regulated the phosphorylated levels of Akt and the integrin-mediated downstream molecules focal adhesion kinase (FAK), paxillin, and p130Cas but did not change phosphorylated levels of ERK or nonphosphorylated levels of these molecules (fig. S8C). Akt phosphorylation induced by 2DDR was abrogated with antibodies against both α5 and αV (fig. S8D). An Akt inhibitor, LY294002, blocked 2DDR-induced DNMT3A expression in MSCs (fig. S8E).
Addition of anti-αV antibodies to the culture of preOCs reduced 2DDR-induced TRAP5b secretion and DNMT3A expression (fig. S8, F and G). Addition of 2DDR up-regulated the levels of phosphorylated Akt, FAK, paxillin, and p130Cas in cultured preOCs (fig. S8H), and blocking αV with the antibodies (fig. S8I) reduced the phosphorylation of Akt. Similar to the results in MSCs, administration of LY294002 significantly reduced 2DDR-induced DNMT3A expression in preOCs (fig. S8J).
To validate the antibody blocking study, we knocked down αV and/or α5 expression in MSCs using small interfering RNAs (siRNAs) (fig. S9A). Adding 2DDR to cultures of siαV or siα5 MSCs reduced osteoblast formation, DNMT3A expression, and Akt phosphorylation in MSCs (fig. S9, B to D). The siRNAs against Akt1/2 blocked 2DDR-induced DNMT3A expression in MSCs (fig. S9, E and F). Moreover, knockdown of αV expression in preOCs reduced 2DDR-induced TRAP5b secretion and DNMT3A expression (fig. S9, G to I), and knockdown of Akt1/2 (fig. S9J) significantly reduced 2DDR-induced DNMT3A mRNA (fig. S9, K and L). These results suggest that TP regulates 2DDR secretion from myeloma cells, and 2DDR enhances DNA methylation of RUNX2 and osterix in MSCs and IRF8 in preOCs through the αVβ3/α5β1-PI3K/Akt-DNMT3A signaling pathway.
Toward a therapeutic approach, we asked whether inhibiting TP can prevent myeloma-induced osteolytic bone lesions. For this purpose, ARP-1 cells, which express high levels of TP, were directly injected into the femurs of SCID mice. Mouse serum was collected to measure circulating M protein levels for monitoring tumor burden. When myeloma was established, mice were treated with vehicle control or TP inhibitors 7-deazaxanthine (7DX) or tipiracil hydrochloride (TPI). ARP-1 cells caused osteolytic bone lesions, increased osteoclastogenesis, and reduced bone volume and osteoblastogenesis (Fig. 6, A to D). TPI or 7DX treatment significantly reduced ARP-1–induced bone lesions (Fig. 6, A to D). Treatment with 7DX or TPI significantly reduced Dnmt3a expression in mouse MSCs or preOCs (Fig. 6E) and 2DDR levels in the serum of ARP-1 tumor-bearing mice (Fig. 6F).
Using two additional human myeloma cell lines, RPMI 8226 (high TP expression) and U266 (low TP expression), we observed similar effects of TP inhibitors on myeloma-induced bone lesions both in vitro and in vivo (Fig. 7). The coculture of RPMI 8226 cells secreting more 2DDR (Fig. 7A) increased osteoclast (Fig. 7B) and suppressed osteoblast (Fig. 7C) differentiation and activity, compared to those in the coculture of U266 cells (secreting less 2DDR). Treatment of RPMI 8226–bearing mice with TPI or 7DX enhanced bone volume and osteoblastogenesis and reduced osteoclastogenesis (Fig. 7, D to F).
Our study reveals an important biological function of TP in the pathogenesis of myeloma-associated osteolytic bone lesions (Fig. 6G) and indicates that counteracting TP activity may be effective for prevention or treatment of osteolytic bone lesions in myeloma patients. We found that TP reversibly catalyzes conversion of thymidine into thymine and 2DDR. Myeloma-secreted 2DDR binds to integrins αVβ3 and α5β1 in osteoblast progenitors, activates PI3K/Akt signaling, and increases DNMT3A expression and methylation of RUNX2 and osterix, leading to decreased osteoblastogenesis. The secreted 2DDR also binds to integrin αVβ3 in osteoclast progenitors, activates PI3K/Akt signaling, and increases DNMT3A expression and methylation of IRF8, leading to increased NFATc1 expression and osteoclastogenesis. The net effect of TP is to suppress osteoblast-mediated bone formation and activate osteoclast-mediated bone resorption, the hallmarks of myeloma-induced bone disease.
DNA methylation regulation has been shown to be one of the most important regulatory mechanisms of gene expression (26). For instance, CpG methylation is catalyzed by a family of DNMTs (27), and DNMT3A and DNMT3B specifically are able to modify unmethylated DNAs. However, the involvement of DNA methylation in osteoblast and osteoclast differentiation is unclear. Our results demonstrated that myeloma-expressed TP up-regulated DNMT3A, but not DNMT1 or DNMT3B, expression in MSCs via 2DDR, the metabolic product of thymidine, leading to highly methylated CGIs in RUNX2 and osterix gene promoters. Hypermethylation caused down-regulation of these two genes, resulting in decreased osteoblast-mediated bone formation, a hallmark of myeloma. In addition, myeloma-expressed TP enhanced osteoclast differentiation. TP/2DDR down-regulated IRF8 expression in preOCs through DNA hypermethylation by DNMT3A, thereby enhancing RANKL-induced NFATc1 expression, demonstrating a critical role of DNA methylation in myeloma-induced activation of osteoclastogenesis. These results point to a new epigenetic mechanism underlying myeloma-induced bone disease.
Integrins are important for bone remodeling (28). For example, integrins α5β1 and αVβ3, which are expressed in MSCs and mature osteoblasts, have been shown to promote osteoblast differentiation through FAK-ERK1/2 signaling and maintain osteoblast survival through activation of PI3K/Akt signaling (24). In addition, αVβ3 expressed in preOCs and mature osteoclasts has been implicated in the regulation of osteoclast attachment (28). The αVβ3 is also essential for osteoclast-mediated matrix degradation, an effect that occurs through collaboration of c-Fms with the ERK/c-Fos signaling pathway. Hotchkiss et al. suggested that activation of α5β1 and αVβ3 and downstream signaling pathways by TP and 2DDR affects endothelial cell migration (29), leading us to examine the role of integrins in TP-mediated bone remodeling. We found that TP/2DDR enhances osteoclast differentiation by interaction with preOC-expressed αVβ3 and inhibits osteoblast differentiation by interacting with MSC-expressed α5β1 and αVβ3. The interaction of 2DDR with these integrins activated the PI3K/Akt signaling pathway, but not the ERK1/2 signaling pathway, leading to DNMT3A expression. Collectively, these results demonstrate that αVβ3/α5β1-mediated signaling plays an important role in bone remodeling through the effects of TP on regulation of osteoclastogenesis and osteoblastogenesis.
There are limitations to this study. Bone remodeling is a complex event in which multiple factors, regulating both formation and resorption, collaborate to maintain bone stability. In myeloma, several other factors, including RANKL, DKK1, macrophage inflammatory protein–1α, and stromal cell–derived factor–1, are known to be dysregulated, and it is not clear how TP may interact with these factors. In addition, although our results with TP inhibitors in animal models show promising results, questions remain about their efficacy in human disease. Because these models operate in a murine bone marrow microenvironment, which could be somewhat different from human marrow, it is not clear whether TP inhibitors will be effective in the human marrow environment. Despite these concerns, we think that it is reasonable to extend these studies to humans because TP inhibitors are now U.S. Food and Drug Administration (FDA)–approved for other purposes. In 2015, a TP inhibitor used in this study, TPI, combined with a nucleoside metabolic inhibitor (trifluridine) was approved by the FDA for treatment of patients with metastatic colorectal cancer (30). In addition, several DNMT inhibitors are now in clinical trials for cancer treatment: azacitidine is in phase 3 trials for myelodysplastic syndromes and acute myeloid leukemia (AML), and decitabine is in phase 3 trials for AML and chronic myelogenous leukemia (31).
The therapeutic effect of these drugs in myeloma or myeloma-induced bone disease remains unknown, but our data in cell lines, patient cells, and rodent models make a compelling case for a role of TP in the genesis of myeloma-induced bone disease and, thus, encourage repurposing of these inhibitors. An in-depth analysis of the therapeutic efficacy of TP inhibitors in the SCID-hu mouse model, an excellent model for myeloma translational research because it provides a human-like marrow microenvironment, would be a logical next step. This model could facilitate the translation of these inhibitors into human studies of myeloma bone disease. Because TP is often expressed by other malignancies including breast, prostate, and lung cancer, these findings may also have broader implications for the genesis of bone metastasis caused by these and other tumors.
We hypothesized that TP induces bone destruction based on an observation of high TP expression in tumor cells (12, 13). In a myeloma setting, this study was designed to evaluate the relationship between TP expression and cancer-associated bone lesions. It encompassed three main objectives: to determine the role of TP in myeloma-induced bone lesions, to elucidate the mechanism of TP-induced bone lesions, and to validate the mechanisms in vivo and in vitro using mouse models and patient samples, respectively. In the first objective, we separated all tested primary myeloma cells and human myeloma cell lines into high-TP– and low-TP–expressing cells, injected these cells into mice, and assessed osteoclast-mediated bone resorption and osteoblast-mediated bone formation by radiography and bone histomorphometry. We also knocked down or overexpressed TP in myeloma cells to assess whether modulating TP expression affects bone formation/resorption. TRAP and Alizarin red S staining in the coculture of myeloma cells with MSCs or preOCs determined the importance of TP to bone cell differentiation and activity. In the second aim, MSP and BSP were performed to analyze DNA methylation in the promoters of RUNX2 and osterix in MSCs or in the promoter of IRF8 in preOCs. We also examined DNMT3A and 2DDR expression and the integrin-PI3K/Akt signaling pathway in MSCs or preOCs. In the third objective, we evaluated the correlations among TP expression, DNMT3A expression, and bone lesions using samples from randomly selected myeloma patients. In addition, we confirmed the mechanism using the mouse models. Myeloma-bearing mice were randomly selected for the treatment with two TP inhibitors.
All patient samples were obtained from the Myeloma Tissue Bank of the University of Texas MD Anderson Cancer Center. Bone lesions in humans were characterized by a radiologist who was blinded to the severity of clinical bone disease. The number of samples required to achieve a correlation coefficient (r) ≥ 0.7, a power of 80%, and the level of significance at 5% was determined to be at least seven samples. In mouse studies, the sample size, the composition of replicates, and the intermediate end point were based on previous knowledge of the mouse models (14). The sample size was initially estimated using power analysis with our previous knowledge on the bone histomorphometric analysis in myeloma-bearing mice. We needed five mice per group to ensure a power of 80% to detect the changes in bone volume between the different TP expression groups with two-sided type I error rate controlled at the 0.05 level.
The final end point before sacrifice was in accordance with the Institutional Animal Care and Use Committee policies and was predefined. All data were included in the analysis, and the criteria for interpretation were established prospectively. Experiments were performed three to five times (as indicated in the figure legends). Animal results were verified by repetition over a 3-year period.
Primary myeloma cells were isolated from the bone marrow aspirates of newly diagnosed myeloma patients using anti-CD138 antibody–coated magnetic beads (Miltenyi Biotec Inc.). The cells were maintained in RPMI 1640 medium with 10% fetal bovine serum. Normal plasma cells were isolated from the peripheral blood of healthy donors, as previously described (18). MSCs and monocytes from myeloma patients were isolated from bone marrow aspirates and cultured as previously described (14). This study was approved by the Institutional Review Board of the University of Texas MD Anderson Cancer Center.
Nuclear extracts were isolated using the EpiQuik Nuclear Extraction Kit (EpiGentek), and 3 μl of nuclear extracts from cells was added to each reaction well according to the manufacturer's protocol. DNMT3A activity was measured using the EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (EpiGentek) as described previously (32).
The relative levels of 2DDR in culture medium and mouse serum were measured as described previously (33). Briefly, the samples were degraded in 1.0 M HCl at 80°C, and the absorbance at 261 and 277 nm was taken. The concentration was determined on the basis of the calibration curve.
C.B-17 SCID mice purchased from Harlan Laboratories were maintained in American Association of Laboratory Animal Care–accredited facilities. The mouse studies were approved by the Institutional Animal Care and Use Committee of the University of Texas MD Anderson Cancer Center. Cultured myeloma cells (5 × 105 cells per mouse) were injected into the femurs of 6- to 8-week-old SCID mice (34). SCID-hu hosts were established as reported previously (14). Human fetal bone chips were implanted subcutaneously into the right flanks of the mice. Primary myeloma cells (1 × 106 cell per mouse) were injected into the implanted human bone chips to establish myeloma. In some experiments, PBS or 7DX (200 μg/kg) or TPI (300 μg/kg) was injected into the peritoneum of mice three times a week for 2 weeks, beginning 3 weeks after myeloma cell injection.
Sera were collected from the mice weekly and were tested for myeloma-secreted M proteins or light chains by enzyme-linked immunosorbent assay or for 2DDR levels by spectrophotometric analysis. To measure the size of lytic bone lesions, radiographs were scanned with a Faxitron cabinet X-ray system.
SCID mouse femurs or human bone chips of SCID-hu mice were fixed in 10% neutral-buffered formalin and were decalcified, and the bone sections were stained with toluidine blue or TRAP following standard protocols. To assess dynamic histomorphometric indices, mice were given two injections of calcein (20 mg/kg; Sigma-Aldrich) at 3 and 6 days before dissection. The paraffin-fixed femurs were embedded and sectioned. Both analyses were carried out using BIOQUANT OSTEO software (BIOQUANT Image Analysis Co.). Mouse MSCs and monocytes were isolated and cultured as previously described (35, 36).
In directly comparing two sets of quantitative data, statistical significance was determined with SPSS software (version 10.0) using unpaired Student's t tests. P < 0.05 was considered statistically significant. All results were reproduced in at least three independent experiments.
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Acknowledgments: We thank the staff at MD Anderson's Small Animal Imaging Facility for their assistance with x-ray imaging. Funding: This work was supported by the National Cancer Institute R01s (CA190863 and CA193362), the National Cancer Institute UTMDACC SPORE in Multiple Myeloma Career Development Award (CDP-060315) and Developmental Research Program (DRP-00013585), the American Cancer Society Research scholar grant (127337-RSG-15-069-01-TBG), the University of Texas MD Anderson Cancer Center Institutional Research Grants Basic Research, the Leukemia Research Foundation, the American Society of Hematology, and the National Natural Science Foundation of China (grant no. 81470356). Author contributions: H.L. and J.Y. designed all experiments and wrote the manuscript; H.L., Z.L., J.D., and J. He performed all experiments and statistical analyses; P.L. and J.J.S. provided the myeloma patients' samples; R.E.D., R.F.G., J. Hou, and B.A. provided critical suggestions; and M.W.S. and N.N. provided the injection technology and osteodata analysis. All authors reviewed the final manuscript. Competing interests: The authors declare that they have no competing financial interests. Data and materials availability: Myeloma cell lines ARP-1, CAG, and ARK were provided by the University of Arkansas for Medical Sciences. Modified cell lines can be obtained from the corresponding authors through a material transfer agreement.