text:
Boral Wollert Quarry
Boral's Wollert Quarry is one of five Boral hard rock sites positioned around Melbourne's outer metropolitan fringe.
It is primarily serves the needs of builders and developers in the city's rapidly expanding outer northern suburbs.
The site also hosts the Boral Wollert Concrete plant which makes use of the aggregates produced by the quarry for its products.
>About the Boral Wollert Quarry
>About Boral Wollert Concrete
>Planning & Approvals
>Resources & Products
>Our environment
>Our community (including latest news)
>Contact us
>About Boral - Community Information
Our Community Partnerships
Sustainability News and Submissions
- Gender Equality Report
- December 2013 - EEO Report
- Sustainability News Archive
Boral's 2017 Public report on Gender Equality was lodged with the Gender Equality Agency on 7 June 2017. Comment and feedback on Boral's Public report can be forwarded to the Group Human Resources Director, Boral Limited or the workplace Gender Equality Agency.
Boral's Energy Efficiencies Opportunities (EEO) public report
Click to see all Boral's Sustainability News Items.


text:
Workout mommy
Member since 6/24/2011
- 2 total posts
- 1 review
- 1 comment
Reviews & Stories
Dr. G Comes Highly Recommended - Seattle, WA
After breast feeding two children my breasts were sagging and lop-sided. I had an aereolar lift and implants and now have perfect breasts. I've actually had people ask if they are real because they look so natural. READ MORE
Recent comments from Workout mommy
I went from a 34C-D to a 34DD and maybe even a little larger. I was so lop-sided, that one implant was 300cc and the other is 375cc to even them out. I also did not go under the muscle with the implant because I had enough breast... READ COMMENT

subset: commoncrawl


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Big day for Zak Boggs; ex-USF Bulls star nominated for Sullivan Award
On the heels of signing with Major League Soccer's New England Revolution, former USF Bulls soccer star Zak Boggs has been nominated for the Sullivan Award. You can vote for him. The award is for the top amateur athlete of 2009. The Sullivan Award has been presented annually by the Amateur Athletic Union since 1930 as a salute to founder and past president of the AAU, and a pioneer in amateur sports, James E. Sullivan. Based on the qualities of leadership, character, sportsmanship and the ideals of amateurism, the AAU Sullivan Award goes far beyond athletic accomplishments and honors those who have shown strong moral character.


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[As prepared for delivery]
As you've probably had to explain to your grandparents, I'm Raj Shah, Administrator of the United States Agency for International Development.
Hopefully that explanation was easier than usual, because unlike many audiences I'm asked to speak to, most of you have actually heard of USAID.
How do I know?
Last week, I sent an e-mail to our Agency's staff, telling them that I'd be at SIS and asking alumni to share their experiences.
I assumed I'd get a handful of replies, with alumni telling me about their favorite classes or the name of a popular hangout that I could mention for an easy applause line.
But then e-mails started flooding in. From Angola and the Philippines and Lesotho and Bolivia.
From a gender advisor working to ensure pregnant mothers have access to HIV medication so their children are born AIDS-free.
And from a member of our cutting-edge mobile partnerships team, who's helping transform Haiti into one of the world's first mobile banking economies.
After reading these e-mails, two things became very clear to me:
First: as soon as this speech is over, I need to take my family to Guapo's for some Mexican food.
Second, and far more relevant: SIS grads don't sit on the sidelines. They get out in the field and learn about the world by actually seeing it.
The e-mails I got didn't talk about popular classes. They talked about peace-building internships in Northern Ireland and trips to the Thai-Burmese border.
Your choice to study at SIS has allowed you to learn from great professors and talented classmates, and you've already demonstrated an early and inspiring commitment to serve.
That's not just a throwaway line—I mean it when I say I find your choice inspiring.
When I was in your position, I didn't have the awareness or courage to make that choice.
I spent nearly all of my academic life pursuing a degree in medicine, even though I knew deep down that development work and public service felt more satisfying to me personally.
It wasn't until I finished medical school and took my board exams that I finally decided to take a leap, packing my car and driving to Nashville to volunteer on Al Gore's Presidential Campaign.
As a new volunteer, most of my peers were high school interns. We spent a lot of our time at the local public library, searching for articles about Al Gore's record on microfiche.
You may not know what microfiche is—but suffice it to say, it wasn't exactly the high stakes campaign work I was expecting.
I soon felt lost, thinking I had just wasted a quarter-million-dollar education without any real chance to make a meaningful contribution in public service.
And one night, just as I was ready to quit, I decided to call the two people who knew me best: my sister Ami and my fiance Shivam.
They told me exactly what I needed to hear: "Stick it out. You wanted this opportunity; you gave up a lot to get it. You need to see it through."
As you leave SIS, you will likely come to a similar crossroads. International service is not necessarily filled with tremendous financial rewards, nor is the work-life balance what most psychologists would call: "healthy."
The true rewards are the relationships you build, the experiences you share and seeing those you help along the way.
I had enough sense to listen—I stuck with the campaign and as a result, built some of my closest and most rewarding friendships.
And it was those friendships that led me to work at the Gates Foundation and begin my career in development.
When I started at the Gates Foundation, I spent a lot of time exploring why millions of children were still dying around the world from preventable diseases like measles pneumonia when simply vaccinations could save their lives.
The system for financing vaccines was broken and some colleagues and I thought we could help fix it. Our idea was to pool vaccine funding commitments from donors and sell them on capital markets, giving aid agencies more flexibility to invest in immunizing children.
I drafted a proposal for how this new scheme could work and sent it off to my new boss.
As I sat in my first one-on-one meeting with Bill Gates, I felt a bit nervous—and I had good reason.
As he fished my proposal out of his bag, I could see that it was covered in red ink.
In quick succession, he detailed its flaws with a tough and aggressive line of reasoning.
And he wasn't alone. As we sent the proposal around to experts in the field, we kept getting the same response: "This will never work. It's unrealistic."
And those experts were right. At the time, our proposal was unrealistic.
But that was the point.
When you choose to tackle the greatest challenges facing the world, you are by definition choosing an unrealistic goal. Your job is to turn that reality on its head; to make the impossible, possible.
So we kept at it. We got smart people to help us answer Bill's tough questions. And during three years of work, we earned his and other leaders' support.
Today, the International Finance Facility has used capital markets to raise nearly $6 billion to immunize children around the world, saving 4 million lives over the last six years. To me, the lesson was simple: if you want to tackle really big problems, you have to to endure the natural skepticism the world will throw at you.
I've been thinking a lot recently about that experience.
We're at a time in global history where our appetite to dream big and tackle our greatest challenges can seem limited by austerity and uncertainties about the world around us.
It would be easy in this environment to look at problems like global hunger, failed states, and extreme poverty and lower our sights.
But as President Obama and Secretary Clinton have made clear, America's global leadership cannot be taken for granted—it must be earned.
We have never been a country satisfied with taking the easy route.
We rid the world of smallpox, fed hundreds of millions during the Green Revolution and launched the largest humanitarian rescue mission in history in Haiti.
And under this Administration, we will continue to expand the reach of human dignity.
Because there are other trends at work today. More than ever before, people from all walks of life and all corners of our nation are inspired to tackle the challenges of development.
Students are oversubscribing to courses on global health and development.
Religious groups are raising money to buy bed nets and fight child trafficking.
Entrepreneurs and investors are working to develop businesses that can generate profits and yield strong social returns.
At the same time, economic growth in the developing world has taken off, making investment and trade with places like sub-Saharan Africa more profitable and likely.
Over the last few years, we've made difficult but necessary reforms at USAID to tap into these emerging trends.
We've developed new scientific partnerships with universities – allowing us to us satallite data to predict floods in Bangladesh and assess famine risk in Africa.
We're partnering with financial institutions to unlock capital for local entrepreneurs—allowing young leaders in Uganda and Egypt to start businesses and tap into international markets.
We've launched an innovation venture fund to invest in technological breakthroughs like mobile banking and off-grid solar energy.
And we've adopted a relentless focus on concrete, quantifiable results, which is helping us earn bipartisan support on Capitol Hill.
All these reforms mean we are playing a much more central role in our country's national and economic security—and hopfully—keeping the world focused on tackling big problems like global hunger and child survival.
Tonight, nearly one billion people will go to bed on empty stomachs, over 200 million of them children.
For those under two years old, we now know that chronic malnutrition is a lifelong curse, literally harming the way their brains grow and develop and irreversibly limiting their economic potential.
Last year, this brutal fact of life was drawn into stark relief as the world's worst drought in 60 years struck the Horn of Africa.
That drought put over 13 million people at dire risk, uprooting over 300,000 people who journeyed to refugee camps in search of aid. Coupled with poor and predatory governance in Somalia, it led to the death of nearly 30,000 children.
Along with Dr. Jill Biden and Dr. Bill Frist, I travelled to met with affected families in a refugee camp in Dadaab, Kenya.
While there, I talked to mothers who had suffered brutal, 100-mile plus treks on foot and I spoke to a little boy who's younger brother had perished earlier in the day in a camp medical facility.
The suffering wrought by last year's famine was tragic. But the larger tragedy is knowing that we can actually end this type of extraordinary human suffering.
We know how to beat severe hunger and malnutrition. By helping poor farmers get access to better seeds, fertilizer and irrigation, we can help them grow and sell more crops, boost their incomes and escape extreme poverty.
That same day, we met with agricultural scientists in Kenya that had developed stronger crop breeds that more than doubled food production in certain parts of the country – helping to keep millions of people from requiring food aid during the emergency.
President Obama launched Feed the Future – our new effort to move from food aid dependency to self-sufficiency –in 2009. In the 20 countries we're working in, we're already seeing agricultural productivity gains are eight times higher than the global average.
As a result of these efforts, we are on the verge of mobilizing dozens of companies—from multinationals like Unilever to small African firms like Ethiopia's Omega Farms—to invest billions of dollars in African agriculture ahead of next week's G8 meeting of world leaders.
With that kind of support, we can move tens of millions people out of a state of poverty and finally prove that hunger can be beaten.
We face a similar massive challenge when it comes to ensuring every child reaches their fifth birthday. We know that this year, over 7 million children will die before that special moment and that a child born in the developing world is as much as 15 times more likely to perish than one born in America.
These children may be born into different circumstances, but their families suffer exactly the same immense pain, insufferable anguish and tragic loss that we would.
Thankfully, what is true today that wasn't just a few years ago is that we now have the technology and know-how to change this brutal fact of life.
New vaccines against diarrhea and pneumonia, bed nets for malaria, nutrition supplements for pregnant women and young children, and a few other things – mostly low-cost technologies that only cost about $30 and fit neatly inside a backpack – could save nearly 6 million children a year.
We believe that if we can reach kids with these simple interventions, then we can achieve an incredible goal: eliminating preventable child death.
In June, we'll be co-convening a global summit on Child Survival with the Government of India—a country that has seen rapid economic growth but slow gains in child survival—to bring the world together behind that goal.
To raise awareness about this effort, we're asking people to visit our Web site and post pictures from their own fifth birthday.
Through that simple act, we want to remind people that no matter where we live around the world—we are united by the basic desire to give our children a bright, promising future.
When you confront persistant challenges like hunger and child death, or witness tragedies like the earthquake in Port-au-Prince or last year's crisis in the Horn of Africa, it's easy get discouraged.
It's easy to face a challenging economy or a job that's less inspiring than you'd hoped or feedback that's more critical than you care to hear—and lose your sense of enthusiasm.
But you made a very important choice to attend SIS.
You chose to forgo the easy rewards…to skip the clear paths…to throw the full weight of your intellect and creativity at the worlds toughest challenges.
Remember that choice.
Remember what drew you into service in the first place.
Remember what you accomplished today.
And draw from it the confidence you need to transform unrealistic goals…
…into inspiring realities.
Remember, the word commencement actually means…
…a beginning.
And though the journey you're beginning today will be long and challenging…
…though it will be, at times, dispiriting…
…the reward of helping a poor farmer sell her crops and feed her children…
…or giving a citizen the chance to vote for the first time…
…or meeting a young girl or boy who is alive because you didn't give up…
…makes this the most meaningful journey you can take.
To the Class of American University's School of International Service, I say congrualtions for what you've already accomplished…
…and thank you for the journer you're about to begin.
Related Speeches
- Remarks by Christina Lau, Deputy Director, Office of Public Health and Education, USAID/Cambodia, Opening Ceremony of the Kick-Off Workshop for "One Health Workforce"
- Remarks by Polly Dunford, Mission Director, USAID Cambodia, Inauguration of Center of Excellence for Sustainable Agricultural Intensification and Nutrition
- Remarks by Polly Dunford, Mission Director, USAID Cambodia, Scaling Up Nutrition (SUN) Annual Meeting
Last updated: September 26, 2017


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Dear Soapdom,
(I am) desperately looking for song and artist from a song heard on One Live to Live. On Sept 27,2006 -- towards end of the show -- a beautiful song was heard. The episode is 9786. The song starts with David and Dorian saying goodbye, leads into several love scenes and then ends with Natalie crying by a window. This is the episode when they find out John is dead.
The song goes "It's okay. I'm going home now. You'll be okay tonight. I just want to be alone now with the dark of light. Let's not fade the color blue, and as a sun sets in the sky, there's just something in the hue there in the corner of my eye. In the dark and light. In the blue and white. All the things that I've forgotten. All the things that I am not holding back and never letting you see. I'll imagine the dark now. See any shadow on the wall. Doesn't seem too much like me, though its not like me at all."
There is more, but I hope you can help me. My daughter is a dancer and really wants to dance to it. Help. Please.
Thank you for your time and help,
Judie McClimans
via email
Dear Judie,
Thank you for contacting Soapdom. The song is called Blue and White. It is by Beth Waters, a singer/songwriter based in San Francisco and can be found on her self titled cd, "Beth Waters." It's available on Amazon.com
The Editors
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text:
Analyze some Data
Budget $250-750 USD
data base
11 freelancers are bidding on average $342 for this job
HIRE ME!! MICROSOFT CERTIFIED EXCEL EXPERT!! Excellent in Data Mining, Data collection and Data Analysis … Hourly Basis work I am an excel expert certified by Microsoft. I have hands on experience on creating dashb More
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Hello, I am so pleased to collaborate with each other. I am a professional in Data entry and collecting the data as I have mane access to websites and organization. I am a professional data entry operator. I have 5 y More
i can assure you i can finish it within time any guarantees you need i can give to you
I was a Financial Analyst for 5 years with a telco company dealing with thousands of data on a daily basis. I'm highly motivated, keen to details, and skilled for the project. Yes, I'm relatively new here building repu More


text:
Students & Mentors, I'd like to know how all of your projects came out. I know about a few of them, but not a lot of the others: Florian -- still working on HOT Standby Euler -- did not complete Michael -- pgUnitTest released Guodong Liu -- ??? German -- ??? Robert -- ??? Ivan -- Completed? Partial? -- Josh Berkus PostgreSQL @ Sun San Francisco
Next: From: Andrew Dunstan Date: 2007-08-29 17:51:39 Subject: Re: Final status of projects? Previous: From: Rober Mach Date: 2007-08-22 10:05:05 Subject: Re: Final Evaluations nearly due


text:
Hello, May I know can "case when " used by update clause. If yes, how? I use one small Example, table: test ============================= id == 5 6 8 try to update test.id update test case when id =5 then SET id = 6 end ; Thanks!
Next: From: Emi Lu Date: 2008-03-12 16:01:21 Subject: Re: case when... end in update clause? Previous: From: Rodrigo E. De León Plicet Date: 2008-03-11 20:31:48 Subject: Re: Composite UNIQUE across two tables?


text: The Elk River junior varsity softball team coached by Amy Kahl finished 13-0 and outscored opponents 83-13. They had three shutouts and six other games allowing only one run. Top batting averages were Sydney Person .385, Olivia Wolters .375, Melea Bruns .360 and Ema Pieper .349. Wolters was the main pitcher with a 0.40 ERA, 119 strikeouts and 16 walks in 70 innings. Bruns pitched 8 innings with a 0.88 ERA and six strikeouts.


text:
Deep-in-the-money calls are the perfect investing tool for a volatile stock market. For those of you who have been using my deep-in-the-money calls strategy, you know I prefer to go out four to six months with our strike price. Because of the time we give our picks to make a move up, our chances of executing a winning trade are improved tremendously, thereby improving the risk/reward profile.This week, we focus on General Dynamics' ( GD) November $55 deep-in-the-money calls, which I'm looking to buy for $11.50 or better. For the simple reason that this particular option hasn't traded in a while, I am only going to add a small "premium" to the stock price. General Dynamics closed Friday at $66.12; let's see if it comes in. There is no reason to chase anything in this roller-coaster of a market. Until things calm down, we need to find companies that will alleviate the daily pain we have been experiencing with these massive market swings. This market is so volatile right now, it's downright scary! That is why we are going to buy the General Dynamics deep-in-the-money calls. When market conditions are this unpredictable, a company needs to have a weapon to keep the "operators" from taking their stock down. Otherwise, it will get pummeled in an environment like this. In other words, we need to find companies that are buying back their stock. General Dynamics plans to buy back up to 10 million shares, or 2.5% of its outstanding stock. Also, let me add that General Dynamics is arguably the premier defense contractor in the world. Bank of America recently boosted its profit estimates and raised its price target to $73. This is a company that has about 72,000 employees; it produced about $22 billion in revenue in the past 12 months. General Dynamics has a forward P/E of 14.31, return on equity of 18.87%, and to top it off, it has $1.70 billion in free cash flow. Remember, that's after it pays all its bills. This is the definition of a "major league" company!
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text:
Details & Care
A subtly sheer knit of speckled marl fashions a soft and slouchy sweater with fine cabling down both sleeves.
- Approx. length from shoulder: 24 1/2".
- Measurements taken from size M and may vary slightly by size.
- Acrylic/cotton; hand wash.
- By BP.; imported.
- Item #574747


text:
On August 31, the UW Law School class of 2014 put aside their textbooks for a day of community service.
More than 200 students partnered with Project Linus, a nonprofit organization dedicated to "providing security through blankets" for ill or traumatized children and teens. Working together, the students made handmade blankets and cards to be given as gifts.
"Community Outreach Day is a reminder that law is a service profession," says Kimberly Frank, student services coordinator at the Law School, who organized the day of service. "The blankets are a way for our students to engage with their new community."
To view a slideshow of Community Outreach Day, click here.
Submitted by UW Law News on September 20, 2011
This article appears in the categories: Articles


text:
Dollfie Fate T Harlaown
Taken at the Dolls Party - the gorgeous Fate T Harlaown.
In her other gear below.
Full body shot. Gorgeous accessories.
Not sure if the cloak has wires inside to hold the position.
And a few of the lovely Nanoha too.
Nanoha in her officer gear.
Fate adoption mission complete?!
BTW, I've just been approved by Volks for my current and future press coverage - as you can see by the blurb below ^^;
(C)1998-2009 VOLKS INC. All rights are reserved.
(C)2003-2009 VOLKS INC. All rights are reserved.
"Super Dollfie(R)""Dollfie Dream(R)" is the registered trademark of VOLKS INC. All rights are reserved.


text:
The Marilyn Monroe Slot Machine
From 1947 to 1962, Marilyn Monroe graced the silver screen with a halo of unprecedented fame. Tough men like Marlon Brando bragged about flings with the blonde from California. She spent quality time with President John F. Kennedy and his brother Robert.
50 years after her tragic death, Marilyn Monroe is an online slot machine. The new Marilyn Monroe slot machine is a 5 reel, 20 payline beauty. The game takes you back to the classic era of the late 50s to early 60's.
Marilyn's slot game has a very revealing stacked wild, a Backstage Bonus with free spins and a x10 multiplier. She had style like no woman of her time. No one could blush on the silver screen like Norma Jean. Relive Marilyn Monroe's greatest moments in this 5 star slot game!
Norma Jean's Symbols
Marilyn stars as all of the symbols of her slot machine. The star-studded Marilyn Monroe is worth 750 coins. Marilyn's Hollywood Walk of Fame Symbol is worth 400 coins when matched 5 times.
Marilyn's TV Magazine is worth 250 coins when matched twice. There are two different Marilyn TV Magazine Symbols. The Backstage Bonus Symbol is worth 500 coins when matched 5 times.
The Glittered Marilyn is the Wild Symbol in this slot machine. It substitutes all symbols of the game except the Backstage Bonus Symbol. The other symbols include the letters A, J, K, Q and the numbers 9 and 10.
The Wild Marilyn Monroe!
Marilyn stars as ALL of the symbols!
Match Marilyn 5 times for thousands of instant payouts
Marilyn on the Magazine is worth 250 coins
Match Marilyn's Hollywood Walk of Fame Symbol for 400 coins!
Marilyn's Backstage Bonus
Imagine a backstage ticket to see Marilyn Monroe live and in person? That's what you get when you match 5 Backstage Bonus Scatter Symbols anywhere on the reels.
Ms. Monroe starts you off with 10 free spins and a x2 multiplier. Immediately after, you get to choose 2 of 5 pictures of the lovely lady for even more free spins and a multiplier.
If 3 or more Backstage Bonus Symbols show up anywhere during your free spins, you get another 35 free spins with the same multiplier!! All in all, you can win up to 45 free spins with a x10 multiplier.
Backstage with Marilyn Monroe!
Start off with 10 free spins and a x2 multiplier
Win another 35 free spins and the same multiplier
For a total of 45 free spins and a x10 multiplier
Imagine the payouts after all of these bonuses?!
Your Jackpot With Marilyn
The Marilyn Monroe Slot Machine has one huge jackpot. The Glittered Marilyn Wild Symbol is worth a stunning 10,000 coins when matched 5 times. Match it 4 times for a sweet 2,500 coins. Even 3 matches is worth 250 and 2 are worth 10 coins.
One big screen sized jackpot is enough, isn't it? No, its not! Marilyn's slot machine has a very cool extra way to win payouts. Every winning that happens thanks to a Wild Symbol is doubled!
The 750 coin Star-Studded Marilyn is worth 1,500 coins when one Wild Symbol is a part of the action. The Walk of Fame Symbol is worth 800 coins - and not 400 - when the Wild Symbol joins in. When the Wild Marilyn join you, you have twice the fun and twice the winnings!
Marilyn's 5 Star Jackpot!
Win 10,000 coins in one spin!
Wild symbols double your winnings
750 coin payout is worth 1,500 when a wild joins in!
Even the 400 coin payout turns to 800 coins with a wild
Take Marilyn For a Spin!
We've all seen Marilyn Monroe on TV and magazine ads. Her inheritance must be worth a lot. Here's your chance to cash in on Marilyn's fame and fortune! A few payouts is all it takes.
After a few spins, you're likely to see Marilyn's Stacked Wild. I bet you've seen that pose before! Its windy and her dress is rising. When's the last time a pose like that paid you so well?!
The graphics and sound of the Marilyn Monroe Slot Machine are immersive and the game play is fun. Look at that skyline! This is one slot game you'll want to show your friends - it exudes "cool" from the era that defined the word. Why not take Marilyn for a spin - she's waiting for you!


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This is a daily SPX chart of closing prices. Thursday's rally is starting to look like a bear flag (red lines on chart). If that proves to be the correct interpretation, a 100% completion of the pattern (blue line) implies a test of the October 2 intra-day low at 1020 at some point within the next few days.
49 thoughts on "SPX Bear Flag"
I started to put a jar of peanuts in the picture but I didn't want to make everyone hungry.
How do you use that tinypic thing, but I can't seem to come up with a url like the one you posted. I have a long string of html code. Didn't want to post it without knowing what it would do.
Happy Halloween!
Help me understand the rule of thumb you posted. Dow ended 9712 so it is 971. SPX is 1036. So is the rule of thumb that the SPX has more to fall?
Matt if we don't break 1018-1020 do we rally back up to 1070?
1060 is the new resistance now
The company plans to file for bankruptcy in New York as soon as Sunday night or early Monday, said people familiar with the matter. CIT is poised to enter bankruptcy with enough creditor support to approve its reorganization plan and shorten its stay in Chapter 11 …
I put those pictures on a CD and went to tinypic, it had a "browse" button then I picked the picture I wanted and it uploaded it. Use the last URL they give so there is a direct link.
Tinypic gives I belive 4 different ways to access the pic that's uploaded. Use the bottom one.
That was scary!
I'm the oracle eh?
Someone wants to dump his broker and have me manage his 50k+ lol
I gotta get to writing basic contract saying I want 20% of profits and i will charge nothing if i lose your money.
his current broker charges 1% of the account value.
something funny resisted to post during trading hours
how google came out with their chrome logo
The K from a year ago would be buying Apple now. But the new K has been killing it on the short side, going with the flow instead of against it. What do you think is the factor that helped you make that evolution?
I'm leaving Sunday for the Outer Banks (OBX), N.C. for a week-long fishing trip. A bunch of guys, mostly relatives, staying in a nice house near Kitty Hawk.
The problem is, I don't like to fish. I used to but it just isn't fun for me now. So I'm taking two laptops and you know what I'll be doing! Yep,,, and if I get hog-tied to go on the pier to watch them pull a couple in, I have my iPhone to keep tabs of the market and trade.
Besides, I don't eat dead fish.
K can you give me a little feed back on the S&P's
I'll make a stab at this to show how close we are to being twins. First should be full view with window unit and space heater and putters. Second is close up of monitors.
A Goldman Insider. 😉
I've been trading since sept 2007. In the beginning it was all buy and hold. Heck I bought ORCL, and GOOG and made cash (beginner's luck).
Right as market topped I went into college. for a year I traded like a lost sheep trading on WSJ news and discussion board trash tips.
I then got sick of being a loser, (greed and fear). Joined school fund 2nd semester of freshman year. Unknowingly chose the role of an economic reporter and thanks to my previous geek skills was able to start digging a goldmine and creating macro economic presentations. They helped me a lot along with our fund.
From then till about december of 2008 I learned a lot on technical analysis. december i opened my forex account and started exploring new worlds. Months later i was wiped out (1k). Took forex break and started back focusing on equities. Watched all steve nison candlestick seminars (google it) and since then candlestick patterns began to burn into my brain.
Going into options while on vacation this summer was another milestone…
Final straw was selling short an AIG Call and losing $1k same day (~30-40% of my total portfolio lol). that desensitized me somewhat to looking at large negative numbers and not running into a wall to escape.
Anyways after running into the steaming train a few times I became "super"(man)K so at that point I gained the ability to crush the trains and stop them where i wanted them. (remember oct 1 1035 call, and today's 1037 call for recent ones)
Bottom line,
I started using a winner's strategy by skimming through blogs for news, participating here, using long lost indicators (those forgotten by the news after 2008 crash such as TED spread), trendlines, moving averages, pivots, and currency correlations. Of course I think I can psychologically handle more pain now when i lost $100-200 a day I don't freak out.
I have gained respect from coworkers, other people, chair of finance, my investing oriented friends and such (not being cocky). I think I will start posting more on my blog about things such as daily recaps of the grains of salt, my own take, the weekly presentations I make for our school etc.
Thanks to all Here for their help I don't want to name names because there are so many but I will name what I can remember anyways.
Matt, George, Stringm, Phil, Charlie, JG, Julie, Paula, Randall, After, Scotty, Jim, 2thfixr and the other sporiadic and frequent posters (although I'm sure I also get award for the most comments lol!)
Is that any help? Further questions require 0.25% of your total account value!
Here's my setup, crappy quality but bb sucks in low light lol
Learning to trade really is a trial-by-fire – even for very smart, accomplished people.
Not losing real money will get you nowhere. The 2 of my friends I introduced to trading have done better than me since march but now when tide turns and there is no longer a rally like march/october they will lose because they took easy way out of buy and hold.
Smart people lose the most money in the market. Common sense and brains get you nowhere in wall st but take your money!
This tinypic thing is fun, and is it OK Matt? Since it is the weekend.
Out my backdoor and the Pavillion I built 3 years ago when my back worked.
Heh String! lucky you got pool but must be pain to maintain.
Where are these bank failures? !!
The wife wanted it for the baby girl, so she maintains it, I only help at times. It is not that bad though, however leaves are a killer.
No problem, String.
U.S. set to seize FBOP, congressmen say
Federal regulators are expected to seize Friday night the banks owned by Oak Park-based FBOP Corp., the troubled owner of Park National Bank of Chicago and eight other U.S. banks, people familiar with the situation say.
Pacific National Bank Going to FDIC
San Francisco-based Pacific National Bank is being eyeballed for takeover by federal authorities, according to several sources in the Silicon Valley and San Francisco commercial real estate industry. … The Federal Deposit Insurance Corp. was expected to take control as early as this afternoon. … The bank … reported assets of $2.1 billion
Ted Up 1.72% on days it would usually go down. not good!
Grain of Salt alert:
Stringm, K, nice setups.
I broke my piggy bank does that count?
No George,
Wanna give me some money? 😛
I hate begging people for things 🙂
did you notice in my setup the monitors have no stands? 😉 I went all out and got a dual monitor stand so i can raise and lower them as desired. 😛 $250 it cost me the bloody thing!
Yes, at first I thought they were wall mounted. Nice.
My office is rather cluttered due to the home improvements here and I'll soon get it back into order and have my monitors closer together.
I have one more monitor I'm going to add. I'll hook it to my other laptop that has Scottrade. I have a scheme to doing this: I will be day-trading but on a longer timeframe scale like the 5min on up. That way it will be easier to trade multiple stocks due to their slower movement.
Matt, Stringm and K, along with any others that automate, I have been working on the problem of ranging. One thing I've noticed is that if price is under the 15min 36MA, then the stochastic (kiss, cross or 80) should be used to exit on the 1min; if price is over the 15min 36MA, then the MACD cross can be used. This solves the problem of using MACD crosses where it crosses at a lower price from entry as well as which exit to use overall.
Going up a notch, the 15min and 60min work well together doing the same thing. The time frames must be far apart because it doesn't work as well with like the 1min and 5min or other closely spaced time frames.
Check it out and see what you think.
Getting there.
I no longer automate 😛
SuperK has been turned off for over a week 🙂
Sorry, Paula is a better writer than me. Take
the daily DOW gain or loss and divide it by
10. If the SPX gain or loss exceeds that
number, look for the trend to continue.
If it does not, look for a trend change.
Old traders use this to decide if they
should start thinking about closing their
swing trade positions.
hmm nicee!! 9 failed banks. awesome
Fewer bank failures each week K. You're going to have withdrawal symptoms when it reaches zeroes.
That pavillion is awesome. You have some talent there dude. I couldn't build a dog house.
Thanks, It was about 2 months of labor, but it has served the family well. The school has faculty, and sports team parties here, the church youth and family night parties, as well as familiy reunions. It has been enjoyable. I can smoke a mean chicken on that pit you see in the pic. I need that missteak recipe you talked about too.
What I mean about "trending vs. ranging" is how do you predict when a period of ranging will begin? In the chart that you posted back here:
…SDS ranged for two hours. If you were looking at your SDS chart at 1:33pm, how could you tell that the uptrend was done and SDS would commence ranging? I don't know if there is an answer to that question, but if there is, it would be hugely profitable.
Fewer bank failures George?
2-3 weeks ago it was 2-3 then 1 then 6 now 9 LOL
The 115 bank failures this year compare with 25 last year and three in 2007.
The number of banks on the FDIC's confidential "problem list" jumped to 416 at the end of June from 305 in the first quarter. That's the most since June 1994. About 13 percent of banks on the list generally end up failing, according to the FDIC.
Last week the SPX weekly chart had a key reversal…
…made a higher high than the week before and
closed below the close of the week before. And
now the SPX monthly chart has a key reversal…
SPX made a higher high above the prior month
and closed below the close of the prior month.
The last SPX weekly correction took the SPX to
the 1019 area. If this correction does the same, keep
your eye on the test results of that prior low. If
successful, you have a double bottom. If it fails, the
door to 980 opens…and one could build a case for
You are a master builder (number 1). Hope you have
a great pain-free weekend. Best wishes from all of
Based on the number of blogs you read and
great links that you post, Paula wants to know
how you ever have time to study…and attend
class…plus homework…quite a workload…
you've constructed a mini rendition of the Babylonian Gardens right there in your Texas back yard. I'm inspired!
I've learned to skim blogs, 3 years expertise of reading stuff has caused me to know worthy info at first sight.
School I make time somehow. Only get 5-6hrs of sleep.
Getting rid of facebook has been the biggest help and instead of degrading my relationship with others it has enhanced it.
this site is my facebook now but instead of wasting time it makes me smarter and to have meet amazing people (so far virtually) all over the world who respect eachother makes things great.
Hope Paula, grandfather and you are doing well.
Comments are closed.


text:
Giant Killer.
Those two words said it all about Brian Westbrook. That he was cut yesterday by the Eagles says more about being a RB in the NFL than about the player. Two concussions on top of the myriad injuries he suffered over the years simply took their cumulative toll not only on him but the Eagles. It is hard to gameplan and plan period when this great player who can do so many things is not around consistently enough to do the things you map out for him. How do you maintain your playbook when so many things can only be done by him? The answer was to let Westbrook go and move on. If someone else in the NFL wants to take a shot, they know exactly what they are getting.
A great player who can make a great impact every time he touches the ball.
A player who will be injured at least twice during the season and who will miss extended time.
Westbrook the Giant killer did some of his earliest damage to Jim Fassel. Fassel's Prevent Offense was set ablaze by Brian, who was in his second year. The year was 2003. The Eagles had 0.0 offense. They were down 10-7 and likely would not be able to score if you gave them 5 quarters. Good teams put bad ones away. Instead, Fassel played clock instead of football. A punt returner named Westbrook took one back 84 yards for the win, leaving the faithful at the Meadowlands howling in disgust. What we preach here is NOT that the special teams unit made an error. The ill-informed fan can blame the special teams coach or the special teams player for not making his tackle. They can yell at the ref for not calling clipping. Where the fault truly lies is with a head coach who stops playing football midway through the second half. Bad things happen when you leave things to chance.
If Fassel had understood how good Westbrook would be, maybe he would have been more respectful of him. Maybe he would have been more respectful of the Eagles too. The better players and better teams find ways to burn you when you play Prevent. Westbrook killed the Giants that day. He would go on to kill the gmen many more times. You never wish a player harm, but it is a good thing Westbrook was not healthier. He could have easily cost us a Super Bowl in 2007 if he was not injured. These were the ups and downs of going against Westbrook. In Game 1 (Week 4) of the 2007 series, the game where Umenyiora sacked McNabb 6 times: "without Brian Westbrook in the lineup the Eagles were not able to exploit the fierce rush with effective screens or explosive edge runs." Playing against the Eagles with and without him in the lineup was night and day.
In 2008, the lost championship season, it was Westbrook who unraveled the Giants. He put just enough pressure on Gilbride's offense to do something, and Gilgarbage came up short. Adversity is supposed to make you stronger. Westbrook always gave the Giants fits, but he prepared the Giants for whatever else they'd see in the NFL from that checkdown RB. Maybe seeing Westbrook in Game 2 of 2007 got them ready for Kevin Faulk of the Patriots.
Giants fans can pay tribute to Brian Westbrook and be grateful he did not do a lot more damage to the gmen than he did. When he was healthy, he was one of the best.


text:
found a new home!
Plenty of my friends are looking
for one too. Find a pet to adopt.
I am already neutered, up to date with shots, and good with cats.
Buddy is 4 years old and fine with other cats, but he would be fine as a single cat too. He is also super gentle, so he would be fine being adopted to a home with small children. Buddy's previous caretaker has become terminally ill and now Buddy and the other cats from her home are in need of new homes.
Buddy is neutered, has tested negative for FIV and Feline Leukemia, and is up to date on vaccinations.
Buddy, a Merrifield Domestic Shorthair cat was adopted! Here's how you can benefit by looking at adoption as an option to buying a Domestic Shorthair for sale in Merrifield, or seeking a Domestic Shorthair cat from a breeder in Merrifield.


text:
'Inside Man' Synopsis & Production InfoBy Christian
August 22, 2000 - 12:57 PM
The official Star Trek site has been updated with both plot and background info for 'Inside Man', the episode featuring the return of Dwight Schultz (Reginald Barclay) and Marina Sirtis (Deanna Troi).
Shooting on the episode began this week on Stages 8 and 9, in both the permanent 'Voyager' sets as well as the Pathfinder Project research lab. Next week filming for the episode will conclude at Stage 16, where the Astrometics set is located.
In addition to this, StarTrek.com also posted the following official synopsis of the episode:
The crew of the U.S.S. Voyager gets an unexpected bonus in their regular "mail" beamed from Starfleet in the Alpha Quadrant. When a hologram of Reg Barclay appears telling the crew of a new plan that can get them home within days, everyone is elated. The plan seems dangerous in theory, but the Barclay hologram persuades the crew the risk is minimal. Meanwhile, back at Starfleet, the real Reg Barclay is completely unaware of his doppelganger's actions.
For the original report on this, please visit the official site's Production News section. Thanks go out to Doug Wilson for pointing me to this!
Discuss this news item at Trek BBS!
Add TrekToday RSS feed to your news reader or My Yahoo!
Also a CSI: Crime Scene Investigation fan? Then visit CSIFiles.com!


text:
bay(redirected from bays)
Also found in: Dictionary, Thesaurus, Medical, Legal, Financial, Acronyms, Encyclopedia.
Related to bays: Bayes, Headlands and bays
flourish like a green bay tree
To thrive; to grow very successfully. The green bay tree is known to grow several new branches every year. Wow, you have just been flourishing like a green bay tree ever since you changed majors.
at bay
At a safe distance; held back by some action. That preventative course of antibiotics really kept the illness at bay—I was hardly sick at all! We have a security system to keep burglars at bay.
bay at
To howl at. A: "What's that noise?" B: "Just some coyotes baying at the moon."
bay for blood
To seek punishment for a person or group. My co-workers have been baying for blood ever since they found out that I lied about my credentials on my resume.
*at bay
Fig. at a distance. (*Typically: be ~; keep someone or something ~; remain ~.) I have to keep the bill collectors at bay until I get my paycheck. The mosquitoes will not remain at bay for very long.
bay at something
to howl at something. (Usually said of a dog, wolf, or coyote.) The dogs were baying at the moon. We heard a coyote in the distance, baying at the moon.
hold someone or something at bay
Fig. to make someone, a group, or an animal stay at a safe distance. (Originally referred only to animals.) I held the attacker at bay while Mary got away and called the police. The dogs held the bear at bay while I got my gun loaded.
keep somebody at bayalso hold somebody at bay
to prevent someone from moving closer He held the police at bay with a gun for several hours.
keep something at bayalso hold something at bay
to control something and prevent it from causing you problems She fought to keep her unhappiness at bay. In the garden, there's no security system to keep the rabbits at bay. Experts hope the economy will slow enough to keep inflation at bay.
be baying for blood(British)
if a group of people are baying for blood, they want someone to be hurt or punished Families of the victims were baying for blood during the trial.
keep something/somebody at bay
to prevent something or someone unpleasant from coming too near you or harming you If we can keep the rabbits at bay, we should have a good crop of vegetables in the garden. For me, overeating is a way of keeping my feelings at bay.
at bay
Cornered, in distress, as in Angry bystanders chased the thief into an alley and held him at bay until the police arrived . This idiom originally came from hunting, where it describes an animal that has been driven back and now faces pursuing hounds. Its use for other situations dates from the late 1500s.
bay window
n. a belly; an abdomen. You are going to have to do something about that bay window.
at bay
To keep someone or something at a safe distance. The phrase derives from stag hunting, from a French word that also is the source of the English word for the baying howl that hounds make during a chance. A tired and cornered stag that turns to face the pursuing hounds is, for the moment, at a safe distance from its attackers.


text:
Car shows are different in Tijuana
"You do not have to babysit the people from leaning or touching the cars."
Mike Madriaga 2:30 p.m., Sept. 19
Valentine's day is always somewhat of a spectacle in Golden Hill. Nearly every corner from B St. to the 94 becomes a makeshift flower/teddy bear store. Mexican families wave at cars passing by in hopes that one stops to buy a bouquet of flowers or perhaps a 3 ½-foot-tall plush bear. The purpose of the stands is undoubtedly entrepreneurial, but the end result seems to be two-fold. Hapless saps in the hopes of love are pressured by a sense of guilt to buy tokens of their sappiness. Or, hapless saps without the hope of love turn bitter by their hopeless. What a wonderful holiday! This year I fell into category B.
Valentine's day marked the end of my lease in Golden Hill. For the last two years I lived in the area with my girlfriend but recently things between us atrophied and blah blah blah it was time to move on. She had moved out several weeks prior and I had slowly begun the process myself, but today was the official finale. I knew there was going to be an ironic, maybe even cynical tone to the day and I prepared myself for it. I wanted to make the transition as smooth as possible and not turn into the bitter, drunken curmudgeon that always seems to be looming in the bottom of my soul.
8am. I woke with a formidable hangover that the sun preyed upon like a bloodthirsty shark, taking full advantage of the fact I had already packed my curtains. No big deal, I thought. Carpe Diem! I borrowed my friend's truck and packed the last of my stuff and my bulldog and headed to my temporary residence east, aka my parent's house. I unloaded my things and headed back for some last minute tidying up.
11am. Driving back I passed the throng of flower stands and what seemed like an unending multitude of lovers holding hands, enjoying the day's warmth and each other. Good for them, I thought. No need to be a hater. It seemed as if my hangover was passing. My mood brightened; I focused on the task at hand. The house was empty save for a few miscellaneous items of junk and a couple bottles of near-empty cleaning supplies. I discarded the junk, cleaned what needed to be cleaned, and wandered through the hollow house taking in the bareness. The transformation was definitely underway.
1:30pm. After a brief moment of rumination I realized I neglected an important task. I had to pick up the dog crap in the yard. Menial tasks turn into chores in the absence of adequate preparation and this was certainly the case. I sifted through the garbage looking for something to protect my hand from canine fecal matter but found nothing. I walked around the corner to the green dog-post thing that once in a blue moon has poop bags, which was obviously empty. I was finally able to borrow grocery bags from a neighbor and get down to business. This system worked well until I ripped through a bag and covered my hand in turd. Not to worry, the Starbucks on the corner is good for something!
2:30pm. I sauntered around the corner under the hot sun and made my way to the bathroom. I washed up and decided to order a coffee. A caffeine buzz would treat me nicely.
Me: Let me get a venti iced-coffee with soy.
Overzealous Starbucks Guy: Hey what's up!?! Haven't seen you in a while!?! Is this for (ex-girlfriend's name)!?!
Me: Nope. We broke up. Moving out today actually.
Overzealous Starbucks Guy: Aww that sux!! On Valentine's Day!?! Do you want your coffee sweetened man!?!
Me (with a sh*t-eating grin): No thanks, I'll take it bitter.
3pm. It was time for the walk-through and my landlady was promptly on time. She was always good about that. It was a little strange, though, that she arrived with her girlfriend. We exchanged pleasantries and took care of our mundane business. The ladies walked about the house and inspected. I could hear them quietly reminisce about happy times they had spent together in each of the rooms, in the sort of saccharine prattle happy people are known to make. By now the irony of the day was at its zenith and I could do nothing but admire it. It's power was immense. This was real; this was happening. This was pretty funny if you looked at it right. As I closed the door for the last time the Indian-summer sun shone in my face and a gentle breeze caressed my skin. I knew this wasn't the last time I'd feel these golden rays in Golden Hill. I'll be back. It's the only place in San Diego I want to live!
This made me laugh out loud!! I hope you keep posting, I love your style!
Great thread, look forward to reading more. :)
Way to capture the essence of the archetypal Overzealous Starbucks Guy - that dude is everywhere. Something to be said about In-N-Out Resurrected Teen From the '50s as well...
Looking forward to your future blogs.


text:
West One Student Accommodation is an established family run business providing top quality student accommodation for more than 800 students in Sheffield. We are property owners and not letting agents, so we really care about our properties and make sure they are well maintained and cared for.
In our experience location is the key factor for convenience during your student years, and therefore you will find all our student properties are in "The Places to be" such as Broomhill, Crookesmoor, Broomhall, Ecclesall Road, the increasingly popular Sharrow/London Road area, and of course Sheffield City Centre.
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text:
New slideshow view for sharing launches today
Photobucket today launched two new "views" for sharing your photos on Photobucket. The two views are called: "Grid" and "Slideshow." They are pretty straightforward.
Click on "Share Album" and choose whichever view you want to share with friends. The "Links" view is the regular, usual Photobucket view with links for copying HTML. THe "Grid" and "Slideshow" views just offer nice, friendly ways to share your albums of photos with your friends and family.
To switch your album to the new views look in the top right in your album:
This is what the "Slideshow" view of your album looks like:
This is what the "Grid" view of your album looks like:
4 Comments
Alice LankesterJanuary 10, 2008
Adding music and text to the slideshows would certainly be fun. We're bear that in mind. Thanks for the suggestion!
EllyseJanuary 5, 2008
can we add text to the slideshow? or music?
edwin ceronDecember 26, 2007
muy chevere y eficaz
gracias por su servicio
SMDecember 19, 2007
Very nice! Love the look of the slideshows! I love how Photobucket is always developing and always getting better.


text:
About half way done...
Finished product....
Those are some great looking ribs!
I'm getting away from the 321. Overdone. I want the perfect bite. Also striving for the TBS. Trying turbinado sugar this time.
Any more pointers. They look great.
I only wrap BBs because I find them more temperamental than spares. Spares, I quit wrapping. I like the bark, smoke ring, tenderness and flavor better unwrapped with the spares.
How much time on the spares and what temp. I'm using a modified semi ecb, chunk charcoal with apple/cherry wood.
I wet smoke spares at 225-235F so you're right in the ballpark with your 230-245F temp. My wife and I prefer hickory or mesquite, the stronger woods. Apple was too mild. I only spritz 2-3 times during the smoke with apple cider. Smoke time is usually 5.5-6.5 hours, depending on the size of the racks. I always try to get racks close to the same weight. I don't trim, personal preference. I determine when they are done by the drawup on the bone (about half an inch) and the bend test.
Ribs are one of my easiest smokes. I can set and forget, spritz occasionally, and check when they are done. Since I started smoking unwrapped I have not had even a single over done rack of ribs.
When you say wet smoke you filling a water pan?
Yes. A wet smoke uses a water pan. Water helps the meat absorb more smoke flavor.
I am going to be chasing the TBS. Food turns out almost black.
anything on that?


text:
Patent application title: CELLULAR RESPONSE ASSAY FOR BIOFLUID BIOMARKER DISCOVERY AND DETECTION
Jennifer Joy Smith (Seattle, WA, US)
John David Aitchison (Seattle, WA, US)
Leslie Rae Miller (Seattle, WA, US)
IPC8 Class: AC40B3004FI
USPC Class:
506 9
Class name: Combinatorial chemistry technology: method, library, apparatus method of screening a library by measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
Publication date: 2012-09-27
Patent application number: 20120245048
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The invention provides a method to detect an abnormal condition or
disease state by assessing a characteristic response pattern of responder
cells to indicators contained in a biological fluid of a subject. A
characteristic disease-associated pattern can be obtained by determining
the response pattern of responder cells to that of bodily fluids or
fractions thereof from subjects known to exhibit a particular abnormal
condition optionally comparing said pattern to such pattern elicited by
fluids or fractions from normal subjects and identifying elements that
differ. The identified elements constitute a characteristic or
differential pattern which can then be used to identify the presence or
stage of a disease in a test subject.
1. A method to determine a response pattern characteristic of an abnormal
condition or a disease stage in a subject which method comprises
contacting a first sample of a culture of responder cells with a
biological fluid or fraction thereof of at least one subject known to
have said abnormal condition or disease stage and determining a first
response pattern of said cells to said fluid.
2. The method of claim 1 which further comprises comparing the first response pattern with a second normalizing response pattern which has been obtained by contacting a second sample of said culture of responder cells with biological fluid or fraction thereof of one or more normal subjects or culturing said responder cells in the absence of extraneous biological fluid and determining said second response pattern of said responder cells and identifying elements of the first response pattern that differ from corresponding elements in the second as representing a third, differential, response pattern characteristic of said abnormal condition or disease stage.
3. The method of claim 1 wherein the response patterns are transcriptome and/or proteome and/or metabolome and/or secretion profiles of the responder cells.
4. The method of claim 1 wherein the method employs at least two types of responder cells.
5. The method of claim 1 wherein the response pattern characteristic of an abnormal condition or disease stage contains at least five elements.
6. The method of claim 2 wherein the response patterns are transcriptome and/or proteome and/or metabolome and/or secretion profiles of the responder cells.
7. The method of claim 2 wherein the method employs at least two types of responder cells.
8. The method of claim 2 wherein the differential response pattern characteristic of an abnormal condition or disease stage contains at least five elements.
9. The method of claim 1 wherein the subject is human.
10. The method of claim 1 wherein the subject is a laboratory model of said condition.
11. A method to detect an abnormal condition or disease stage in a test subject, which method comprises determining whether said test subject is associated with a response pattern characteristic of said condition or stage by a method comprising contacting a biological fluid or fraction thereof of said test subject with a culture of responder cells and determining a response pattern of said cells to the fluid or fraction, and comparing the response pattern of said test subject to the response pattern determined by the method of claim 1 as characteristic of the abnormal condition or disease stage whereby similarity of the pattern obtained from the test subject to the pattern characteristic of the abnormal condition or disease stage detects the abnormal condition or disease stage in said subject.
12. The method of claim 11 wherein the pattern is a transcriptome and/or proteome and/or metabolome and/or secretion profile of said cells.
13. The method of claim 11 wherein the method employs at least two types of responder cells.
14. The method of claim 11 wherein the response pattern characteristic of an abnormal condition or disease stage contains at least five elements.
15. The method of claim 11 wherein the subject is human.
16. The method of claim 11 wherein the subject is a laboratory model of said condition.
17. A method to detect an abnormal condition or disease stage in a test subject, which method comprises determining whether said subject is associated with a differential response pattern characteristic of said condition or stage by a method comprising a) contacting a biological fluid or fraction thereof of said subject with a culture of responder cells and determining a response pattern of said cells to the fluid, b) comparing the response pattern of said subject in a) to a response pattern of control cells that have been contacted with biological fluid or fraction thereof of a normal subject or culturing said responder cells in the absence of extraneous biological fluid to obtain a differential response pattern associated with the subject, c) comparing the differential pattern in b) with a characteristic, differential, pattern determined by the method of claim 2 whereby similarity of the differential pattern associated with the subject to the differential pattern characteristic of the abnormal condition or disease stage detects the same in said subject.
18. The method of claim 17 wherein the pattern is a transcriptome and/or proteome and/or metabolome and/or secretion profile of said cells.
19. The method of claim 17 wherein the method employs at least two types of responder cells.
20. The method of claim 17 wherein the differential response pattern characteristic of an abnormal condition or disease stage contains at least five elements.
21. The method of claim 17 wherein the subject is human.
22. The method of claim 17 wherein the subject is a laboratory model of a disease or condition.
23. The method of claim 11 which is performed at least two different times on a fluid or fraction from a test subject in order to determine disease progression.
24. The method of claim 11 which is performed at least two times on a fluid or fraction from a subject treated with a protocol, wherein said method is performed before and after treatment with said protocol to determine effectiveness of said protocol.
25. The method of claim 17 which is performed at least two different times on a fluid or fraction from a test subject in order to determine disease progression.
26. The method of claim 17 which is performed at least two times on a fluid or fraction from a subject treated with a protocol, wherein said method is performed before and after treatment with said protocol to determine effectiveness of said protocol.
27. The method of claim 2 wherein the subject is human.
28. The method of claim 2 wherein the subject is a laboratory model of said condition.
[0001] This application claims priority from U.S. provisional application 61/467,898 filed 25 Mar. 2011. The contents of this document are incorporated herein by reference.
[0002] The invention is in the field of diagnosis and measurement of abnormal conditions in a vertebrate subject. More precisely, it concerns using responder cells to detect indicators of the condition of a subject which indicators are present in the biological fluids of said subject.
[0003] Many approaches have been used to identify markers, in particular for disease conditions. For example, expression profiles of cancer cells, as opposed to normal cells, have been used as indicators both of the presence of cancer and the stage to which it has advanced. Molecular markers such as creatine kinase MB (CK-MB) and troponin in serum have been used as indicators of myocardial infarction. Prostate-specific antigen (PSA) has been widely used as a marker for the presence of prostate cancer. Using combinations of such individual markers has also been suggested.
[0004] Levels of previously known markers of Alzheimer's disease have been measured in neuronal cells exposed to serum from elderly Alzheimer's patients, or serum from patients not afflicted with the disease. Although the markers were observed in response to disease serum, but not serum from young adults, they were not specific to Alzheimer's as they were also observed in response to serum from elderly spouses not afflicted by the disease. Neuronal viability and somal area were also assessed. Brewer, G. J., et al., J. Neurosci. Res. (1992) 33:355-359. In other studies, exposure of cultured motor neurons to serum or cerebral spinal fluid (CSF) from patients with ALS has been shown to result in increased neuronal death (Roisen, F. J., et al., Muscle Nerve (1984) 5:48-53; Couratier, P., et al., Lancet (1993) 134:265-268) and reduce abundance of ion channels (Gunasekaran, R., et al., Brain Res. (2009) 1255:170-179). In addition, conditioned medium from hepatocellular carcinoma cells was able to activate hepatic stellate cells eliciting morphological changes as well as elevation of actin expression, beta platelet derived growth factor receptor mRNA and matrix metalloprotease-2 expression. Faouzi, S., et al., Lab. Invest. (1999) 79:485-493.
[0005] The present inventors recognize that it is unlikely that any particular condition to be assessed will be associated only with a single marker that can conveniently be assayed in the biological fluid of a subject. Patterns of such markers can also be employed to enhance the accuracy of any diagnostic assessment. The complex expression patterns obtained in cellular assays are generally associated with determining the condition of the cell itself, for example, whether it is malignant or not. To applicants' knowledge, however, the complex responses of cells to multiple external factors carried, for example, in the circulation of a subject to be tested has not been employed with respect to assessing conditions associated with these unknown markers. The present invention takes advantage of the complex responses measurable in cells and thus uses responder cells as indicators of the presence or absence of any pattern of markers associated with an abnormal condition in the subject.
[0006] The invention is grounded in the ability of cells to translate a complex signal or pattern, even when the nature of this signal or pattern is not known, into a measurable complex response. This is achieved by using responder cells as detectors such that a complex pattern associated with the responder cell provides the readout of the assay. Since responder cells are themselves complex systems, they provide a multiplicity of parameters that can be measured substantially simultaneously to provide a detectable pattern.
[0007] One application of this concept is to employ responder cells to assess complex changes in biological fluid of a subject which occur when the condition of the subject deviates from normal. The presence of cancer, for example, or Alzheimer's disease, or heart disease will result in a change in the contents of bodily fluids, for example, blood or cerebrospinal fluid (CSF). When these fluids are contacted with responder cells, the cells themselves exhibit a response that constitutes a complex pattern.
[0008] Thus, in one aspect, the invention is directed to a method to determine a differential response pattern characteristic of an abnormal condition or a disease stage in a subject which method comprises
[0009] a) contacting a first sample of a culture of responder cells with a biological fluid or fraction thereof of a subject known to have said abnormal condition or disease stage and determining a first response pattern of said cells to said fluid;
[0010] b) contacting a second sample of said culture of responder cells with the bodily fluid or fraction thereof of a normal subject or cells that have not been contacted with bodily fluid and determining a second response pattern of said responder cells to said fluid;
[0011] c) comparing the response pattern in a) with the response pattern in b); and
[0012] d) identifying elements or parameters of the response pattern in a) that differ from corresponding elements or parameters in b) as representing a third, differential, response pattern characteristic of said abnormal condition.
[0013] In another aspect, the invention is directed to a method to detect an abnormal condition or disease stage in a subject, by determining whether the subject has a differential response pattern characteristic of the abnormal condition or disease stage. This is done by contacting a biological fluid or fraction thereof of said subject with responder cells and determining a response pattern of said cells. The response pattern is then compared to those of control cells which have not been contacted with said biological fluid, or that are contacted with the corresponding biological fluid of a normal control subject, or to a standard normal response pattern compiled from other subjects which may be accessed in a database. This "normalized" differential response pattern can be compared to the differential response pattern determined as described above.
[0014] It is also possible directly to compare the profile obtained when a culture is exposed to a fluid of one or more subjects with a known condition or with a particular stage of said condition with the profile obtained by subjecting a second culture of cells to fluids or fractions from a subject with an unknown condition or stage. Similar profiles indicate the correspondence of the condition or stage of the test subject with that obtained from the subject who is afflicted with the known condition or stage.
[0015] In some embodiments, the response patterns are the transcriptome, and/or proteome and/or the secretion profile and/or metabolome of said cells.
[0016] The invention employs responder cells that will exhibit complex measurable alterations as a result of exposure to stimuli associated with a condition to be assessed. In effect, the invention method translates complex variations in the composition of fluid samples into a complex response that is reproducible and indicative of the composition pattern in the fluid.
[0017] The results of culturing the responder cells will be a pattern of a number or parameters or elements that constitutes the fingerprint of the response. Thus, the collection of individual elements or parameters measured in the responder cells or their secretions will be referred to as a "pattern" or "fingerprint" or "profile." In this context, "parameters" and "elements" are used interchangeably.
[0018] The effectors of the response pattern will be those components of the biological fluid or fraction thereof containing them that corresponds to a particular disease, condition, or stage of progression of a disease or condition. Because these components are indicators of the disease, condition or stage, they are referred to as "indicators." Their identity is not, and need not be, apparent from the resultant pattern.
[0019] In general, the number of parameters in the profile should be at least 3, preferably more. The number of parameters may be 3-50 or more including all integer numbers between 3 and 50.
[0020] More than one type of responder cell can be used to create the pattern where these are grown either in the same culture or in different cultures. Thus, 2, 3 or even 4 or more, such as 5-10, types of responder cells could be employed. Some may be of the type related to the condition and others unrelated to obtain patterns of complexity. Further, as the number of responder cells used in the assay is increased, it may be possible to decrease the number of parameters in the pattern for each cell. For example, four responder cells measuring a single parameter in each, such as the expression level of a single gene, could substitute for measuring the expression levels of four or five different genes in the same responder cell.
[0021] When a range such as those above is set forth in the present application, whose components are inherently integers, each of these integers is included in the specification as if specifically set forth. Further, the articles "a" and "an" are intended to mean one or more than one unless otherwise specifically indicated otherwise. "Bodily" and "biological" fluid are used interchangeably.
[0022] Means to measure multiplicities of elements or parameters--i.e., components in the resulting pattern from the response cells either internally to cells or components that are secreted by them or both are known in the art. For example, commercially available platforms can measure expression levels of hundreds or thousands of genes typically as a transcriptome--i.e., levels of mRNA present in the cell. Less developed, but nevertheless useful, are means to measure the proteome--i.e., levels of multiple proteins that are produced in the cell. Methods are also available to measure secretion profiles wherein cells may secrete multiplicities of materials into the environment. Other parameters that may be measured are the levels of various small molecules in the cells--i.e., the metabolome. Other criteria are behaviors of the cells themselves such as proliferation, changes in morphology and the like.
[0023] More complex measurements can also be obtained by measuring components of cellular regulation such as microRNA and variations in RNA splicing. Also, the compositions of molecular complexes containing protein and/or DNA can be monitored on a large scale through analyzing components of protein or chromatin immunoprecipitations.
[0024] Elements of each of these types of measurements can also be combined to obtain a complex and meaningful readout for the assays.
[0025] Further, it is not necessary that the parameters that are members of the profile be those which are known to characterize the disease; indeed, it is typically the case that the measured parameters will be different from the disease indicators. Thus, the utility of the methods of the present invention does not depend on any prior knowledge of what parameters should be measured, or what the indicators are.
[0026] In general, the test substrates are biological fluids. As used herein, "biological fluid" includes not only the fluid itself, but if desired a fraction thereof may be tested. For example, if the biological fluid is plasma or serum, it may be useful to remove albumin in order to provide a cleaner test substrate. Thus, "biological fluid" should be understood to include fractions of the fluid as well as pretreated forms thereof. Any biological fluid is appropriate as a test substrate. In addition to serum, plasma, or cerebrospinal fluid or fractions thereof, other fluids that might be tested are semen, urine, saliva and bile.
[0027] The subjects from which the biological fluids are obtained may be mammals, including primates, such as humans and laboratory models such as rabbits, rats and mice, as well as livestock such as sheep, goats, horses, cattle, and pigs and companion animals such as dogs and cats. The subjects may also be birds or fish. The responder cells may also be derived from these species and need not be of the same species as the subject. The cells need not be vertebrate cells but can derive from eukaryotes, prokaryotes or archaea. The methods of the invention may be particularly useful in combination with model systems for disease, and in testing the effects of various therapeutic protocols thereon.
[0028] In one aspect of the invention method, biological fluid from a subject or subjects with a known abnormal condition may be used to establish a baseline pattern of alteration in the responder cell components. It is not necessary to know in what manner the components of the biological fluid are altered by the abnormal condition as the pattern obtained from the responder cells is diagnostic therefor. In one embodiment, the pattern of components of the responder cells in the abnormal condition is compared with the pattern of components in the responder cells when in the presence of or having been incubated with normal biological fluid that does not exhibit the abnormal condition or that are incubated in the absence of biological fluid. The differential pattern exhibited by the responder cells, then, can be used as a standard for comparison to similar results from test samples. Thus, a differential pattern can be established by identifying those elements of a response pattern exhibited as a result of contact with bodily fluids representing an abnormal condition from those elements in patterns established by contacting fluids derived from subjects that are normal, and creating a pattern that includes these elements or that are not contacted by such fluids. This embodiment has the advantage of leaving out parameters that are not indicative of the disease itself. However, as further discussed below, it is possible simply to directly compare the pattern of parameters obtained from a subject with known abnormal conditions or stage of disease with that of a test subject.
[0029] Thus, a "differential pattern" refers to a pattern obtained by comparing the pattern from a subject with a known condition or stage of condition with a normalizing pattern or a pattern obtained from a test subject with a normalizing pattern. The normalizing pattern is typically a pattern obtained when the responder cells are contacted with fluids or fractions from one or more normal subjects. Other types of normalizing patterns could also be used, such as a pattern obtained when the cells are simply cultured in the absence of any extraneous fluids other than typical culture media. In both cases, the differences can be evaluated statistically depending on the number of subjects included in any of these groups. Thus, if sufficient numbers of independent patterns are used, statistically significant differences can be evaluated, and if desired, used as a criterion for including a specific parameter in the final pattern or profile. In some cases, only a single subject may be used to create a standard for abnormal conditions or disease stage and a single normal control may be used.
[0030] While theoretically any cells could be used as responder cells, the information obtainable from the assay may be improved if the responder cells are selected from those cells that are expected to show a response to whatever changed compositions are associated with an abnormal condition. For example, for conditions that are associated with abnormalities in the neuronal system, neuronal cells may be used on responders. For assessment of abnormal conditions that exhibit their biological effects on the hematopoietic system, hematopoietic cells are favored. This nexus, however, is not necessary, and any cell that provides a sufficiently nuanced pattern in response to an abnormal condition could be used.
[0031] In general, the differential response pattern can be established by comparing responses obtained from fluids obtained from abnormal subjects with those obtained from fluids of normal subjects. The responses can be compared by identifying individual transcripts that are significantly differentially expressed between the two responses, or by generating and testing a more complex disease classification model using approaches such as support vector machines or random forest algorithms (Statnikov, A., et al., BMC Bioinformatics (2008) 9:319). Such algorithms can identify diagnostic signatures composed of sets of candidate transcripts and disease classification decision rules (which can be based on more complex aspects of the data such as the relative intensities of two different transcripts in the same sample).
[0032] The analysis can be expanded to obtain a longitudinal or cross sectional set of disease signatures, by obtaining complex multicomponent readouts from responder cells (e.g., gene expression microarrays) after exposure to sera from normal or diseased subjects taken at various stages of disease progression. In longitudinal studies, a single subject at various disease stages is assessed, whereas in cross-sectional diagnoses, multiple subjects at various disease stages can be used as subjects.
[0033] One illustrative embodiment to handling data showing disease progression is to construct a differential response pattern made up of log2 expression ratios (disease serum exposure/normal serum exposure) obtained of responder-cell genes in the cultured cells that are good indicators of disease progression. Expression values of various genes for disease subjects at each stage of progression are evaluated relative to matched normal subjects (e.g., subjects can be matched with respect to genetics, age and/or environment). This sets a standard pattern to which expression level data obtained similarly with respect to fluids of a test subject can be compared. If a large number of genes make up the signature, it is possible, if desired, to cluster genes in some way; for example, genes with similar response profiles (data in rows) could be clustered. Categorizing genes in this way enables recognition of higher-level disease signatures such as response characteristic of a cellular process rather than expression level of an individual gene.
[0034] To apply the results obtained above, the responder cell assay is performed with serum from the subject and disease state is assessed by mapping to the longitudinal progression pattern. This is done by obtaining readouts from responder cells after exposure to query serum of a test subject and control serum using the same experimental conditions that were used to generate the longitudinal (progression) data. Here the control is non-disease serum from a genetically matched subject of the same age, but for other disease applications, it could be serum taken from the subject itself before disease onset.
[0035] Alternatively, the pattern obtained from various subjects representing the stages of disease progression could be compared directly with the expression pattern obtained from a test subject to match directly the expression pattern in the respondent cells from fluids in the test subject with the corresponding pattern in the known disease stage.
[0036] Assessing disease stage can also be useful in evaluating treatment. The expression pattern of responder cells characteristic of a particular stage of a disease, whether or not normalized to that of normal subjects can be compared to the pattern obtained from a test subject before and after treatment, again, either directly or where both patterns have been normalized to normal subjects. Effectiveness of the treatment is reflected in finding that the pattern in the test subject represents an earlier stage of progression than was exhibited before treatment. Thus, if before treatment the subject exhibits the pattern characteristic of stage 5, successful treatment would be indicated if the pattern after treatment is representative of disease stage prior to 5, such as disease stage 3.
[0037] Even with respect to conditions that are not directly specific to a particular type of cell, such as malignancies, secretions from malignant cells to, for example, the bloodstream, permit the blood to be used as a test sample in which case the responder cells could be those of normal tissue associated with the cancer itself.
[0038] None of these pairings are required; the complexity of metabolism makes it possible to use virtually any cells as responder cells regardless of whether they are primary targets of the altered composition of the biological fluid.
[0039] It is also possible to identify components of a biological fluid that effect the complex changes in the responder cells if it is the case that only one or two or a few markers are responsible. The biological fluid can be fractionated and each fraction evaluated separately with respect to changes in the responder cell pattern. On the other hand, if it is a complex mixture of altered components in the biological fluid that is responsible for the pattern alterations in the responder cell, the task becomes more complex.
[0040] The following examples are included to illustrate but not to limit the invention.
Use of Motor Neurons to Detect Amyotrophic Lateral Sclerosis (ALS) Susceptibility in Serum
[0041] ALS is a human neurodegenerative disease caused by progressive degeneration of motor neurons that control voluntary muscle movement. The molecular mechanisms of disease progression are not known, but evidence suggests that ubiquitous activation of the subcellular misfolded protein response cascades to a dysfunctional interaction between motor neurons and supporting astrocytes, resulting in neuronal death. Most ALS cases are sporadic, while 10% of cases are inherited. Dominant mutations of the SOD1 gene are among the genetic defects that have been reported to cause ALS. A transgenic mouse model of ALS has been generated, which is hemizygous for G93A mutant form of human SOD1.
[0042] Although the unfolded protein response is detectable in motor neurons of the mice by about 7 weeks of age, the mice have only minor symptoms of disease until about 11 weeks of age when body weight starts to significantly decline. Symptoms progress to neuromuscular dysfunction at about 13 weeks, followed by paralysis by 19-20 weeks.
[0043] In this example, the ALS signature in blood serum of mice developing ALS was determined using motor neurons as detector cells. Motor neurons have been shown to be targeted by the disease in a non cell autonomous manner (Nagai, M, et al., Nature Neuroscience (2007) 10:615-622), and therefore may be responsive to disease-specific signatures in serum.
[0044] Disease serum was taken from 5 transgenic ALS susceptible mice (SOD1; G93A) at 9 weeks of age and control serum was taken from 5 non-carrier mice of the same age from the same colony.
[0045] Spinal motor neurons (MNs) were derived from HGB3 embryonic stem cells expressing a fluorescently labeled motor neuron marker (HB9-eGFP) by a method previously described (Wichterle, H., et al., Cell (2002) 110:385-397) as described below. Unless otherwise specified, growth of ES cells was in differentiation medium (consisting of equal parts Advanced® DMEM/F12 (Invitrogen) and Neurobasal® medium (Invitrogen) supplemented with penicillin/streptomycin, 2 mM L-Glutamine, 0.1 mM 2-mercaptoethanol, and 10% KnockOut® serum replacement (Invitrogen)). ES cells were plated at ˜105 cells per mL and grown in aggregate culture for 2 days to form embryoid bodies (EBs) in a 10 cm2 dish. EBs were split 1:4 into four 10 cm2 dishes and exposed to 1 μM each retinoic acid and sonic hedgehog agonist (Hh-Ag1.3, Curis, Inc.) for two days, to caudalize spinal character and ventralize into MN progenitors, respectively. Medium was changed and EBs were grown for an additional 3 days in differentiation medium to generate MNs. Two dishes of EBs were pooled, washed with PBS and resuspended in 1 mL of differentiation medium. 100 μL it of these EBs were inoculated in each of 10 wells of a 3.8 cm2 12-well dish. EBs were incubated for 24 h in 2 mL differentiation medium containing either 5% serum from 9 week-old ALS susceptible mice or 5% serum from normal mice. Each experiment (disease or control) was done five times with serum from five different mice.
[0046] RNA was isolated using TRIzol® reagent, and cDNA was synthesized from polyA RNA, labeled and hybridized to Affymetrix GeneChip® mouse exon arrays according to manufacturer's recommendations.
[0047] Probe intensities for ten experiments (five replicates each of control and disease serum) were normalized together and data from probes representing a continuous stretch of putatively transcribed genomic sequence were merged into probe sets (using RMA algorithm of the Affymetrix Expression Console software). Two filters were applied to exclude probe sets that did not meet the criteria below: 1. Probe sets map to the genome and thus levels are annotated as "core", "full", "free" or "extended" by Affymetrix. 2. Probe sets have high confidence of detection over background in at least 5 of the 10 experiments (P<0.001 determined using the DABG algorithm of the software). After application of these two filters, the data set consisted of 135,181 probe sets.
[0048] Probe-level expression values were analyzed for significant differential expression between cells exposed to control serum and those exposed to disease serum using Significance Analysis for Microarrays (SAM) of MeV component of TM4 microarray software (by running a two-class paired analysis using default parameters and the 32 possible unique permutations of the data to calculate the statistic). This analysis generated an ALS disease signature consisting of 441 probe sets that significantly increased in expression in response to disease serum compared to normal serum with q-values and false discovery rates <15%.
[0049] The differential response pattern need not reflect the underlying biology of disease progression in order to be used as a classifier of disease state; however significant overrepresentation of disease relevant genes in the signature supports the contention that the measured cellular response is specific to the disease rather than background or noise. The disease signature of 441 exons contained four probe sets in three genes that are causatively associated with ALS including SETX (Chen, Y -Z., et al., Am J. Hum. Genet. (2004) 76:1128-1135), TARDBP (Sreedharan, J., et al., Science (2008) 319:1668-16672; Yokoseki, A., et al., Ann. Neurol. (2008) 63:538-542; Rutherford, N. J., et al., PLoS Genetics (2008) 4:e1000193), and VCP (Johnson, B. S., et al., Neuron (2010) 68:857-864). Nine genes have been identified to have mutations linked to ALS (Strong, M. J., J. Neurological Sciences (2010) 288:1-12; Johnson, B. S., et al., Neuron (2010) 68:857-864), and three of them are in the disease signature (representing 400 of the 30,000-40,000 total genes). This overrepresentation of disease genes in the signature is significant with HGD p-value of ˜1×10-6).
[0050] This cell-mediated disease signature could be used to classify the disease states of test subjects from their serum. A possible approach is to generate more replicates of the data with known disease and normal samples, and apply support vector machines or random forest algorithms (Statnikov, A., et al., BMC Bioinformatics (2008) 9:319) to build and test a disease classification model. The models can be tested using cross-validation and then validated using independent test sets of mice. In this case, test subjects would be analyzed by measuring the global expression response of responder cells to serum as was done to build the model, but in other applications, test subject analysis might involve measuring only the response of transcripts that comprise the diagnostic signature using targeted approaches for quantification of selected transcripts.
Use of Endothelial Progenitor Cells (EPCs) and Other Vascular Progenitor Cells as Biosensors of Malignant Tumors, Proliferative Retinopathy and Ischemia
[0051] Angiogenesis or blood vessel formation is a fundamental step in malignant tumor formation and is also associated with other health concerns including proliferative retinopathy associated with diabetes, and ischemia associated with stroke and heart disease. It is mediated by mobilization and recruitment of bone marrow-derived endothelial precursor cells (including endothelial progenitor cells (EPCs), and hematopoietic stem and progenitor cells). Recruitment of these cells to the target location is mediated by signaling molecules in the serum (including cytokines, angiogenic factors, platelet-derived growth factors, and as of yet uncharacterized factors), which likely comprise organ-specific signatures. In addition, it is thought that upon reaching the target tissue, differentiation of the progenitor cells is influenced by specific signaling molecules including local extracellular matrix components. Therefore a potential biosensor assay for tumor development and other cell proliferative conditions includes the use of detector cells that are EPCs and other vascular progenitor cells (which can be isolated from bone marrow, or derived from embryonic stem cells), and the use of blood serum as the biofluid. We anticipate that this assay will be particularly valuable for detection and characterization of malignant tumors as they have been linked to local secretion of destructive proteases (both matrix metalloproteases and serine and threonine proteases) that break down extracellular matrix components releasing locally confined growth factors and polysaccharides that regulate cell behavior into the blood stream.
Use of Stromal Cells and Other Cells Involved in Desmoplastic Reaction as Biosensors of Various Malignant Tumors
[0052] Desmoplastic reaction is the growth of fibrous or connective tissue in response to neoplasm, resulting in the formation of dense fibrosis around a tumor. This is usually associated specifically with malignant neoplasms. The reaction involves stromal cells that are considerably diverse depending on environmental cues including heterologous interactions with other cell types in the stroma and differential regulation by tumor cells. For example, tumor cells signal to various stromal cells including fibroblasts, which are transformed into myofibroblasts that produce collagen and other ECM proteins (Desmouliere, et al., Int. J. Dev. Biol. (2004) 48:509-517), inflammatory cells that have a role in tumor immunity (Coussens, et al., Nature (2002) 420:860-887), and endothelial cells that have roles in neoangiogenesis and tumor progression (Carmeliet, et al., Nature (2000) 407:249-257). Therefore, potential cell-based assays to detect malignant neoplasm and possibly the evolutionary stage and location of the tumor involve the use of stromal cells involved in desmoplastic reaction (either in isolation or in a heterologous mixture to leverage cell-cell communications to amplify the signal). For example, a potential biosensor of hepatocellular carcinoma is a mixture of involved stromal cells, including hepatic stellate cells (fibroblasts), sinusoidal endothelial cells and inflammatory Kupffer cells (reviewed in Desmouliere, et al., supra). In this heterologous stromal cell detection system, serum from diseased patients is expected to transform fibroblasts into myofibroblasts among other responses that make up a specific signature of hepatocellular carcinoma that is different from other desmoplastic responses to other tumor types and wound repair. In breast tissue, the fibrous tissue is fatty and is presumably derived from adipose cells. Therefore, a potential biosensor assay for breast cancer development includes the use of stromal cells including adipocytes as responder cells to detect molecular breast cancer signature in blood serum. Supporting these approaches, exposure of adipose cells to media conditioned by different human breast cancer cell lines induces reversion of adipocytes to fibroblastic cells. In addition, exposure of hepatic stellate cells to media conditioned by rat hepatocellular carcinoma cell lines, increased cell proliferation, alpha-smooth muscle actin expression and gelatinase A secretion. Faouzi, et al., Lab. Invest. (1999) 79:485-493.
Use of Neurons as Biosensors of Neurodegenerative Diseases Including Alzheimer's Disease, Amyotrophic Lateral Sclerosis and Parkinson's Disease
[0053] Several neurodegenerative diseases result in the selective destruction a specific neuronal population. The cause of neuronal loss is not known, but in some cases it has been linked to altered environment of the neuron including signaling from other cells, rather than the neurons themselves. Additionally, it has been shown that biomarkers of disease in cerebral spinal fluid can also be found in the blood. For these reasons, neurons that are targeted by neurodegenerative diseases are potential biosensors of disease-specific signatures in blood serum and cerebral spinal fluid. Specifically motor neurons, glutamatergic and cholinergic neurons, and dopamine neurons are potential biosensors of amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease, respectively. These neurons can be derived from embryonic stem cells or neuroblastoma cell lines. Hill and Robertson, Brain Res. Prot. (1998) 2:183-190.
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