Senate Republicans failed to pass a simple, far-reaching bill repealing major provisions of the Affordable Care Act in the wee hours Friday morning, a stunning rebuke of their leaders’ strategy to deliver on a core promise made to their voters for the past seven years.
The defeat lay at the hands of every Democratic senator and Republican Sens. Lisa Murkowski of Alaska, Susan Collins of Maine, and John McCain of Arizona, who voted against the bill despite last-minute lobbying from Vice President Mike Pence and others.
“I do my job as a senator,” said McCain, who recently returned to the Senate after being diagnosed with brain cancer. “I thought it was the right vote.”
Instead of celebrating a victory on their top legislative priority, Republican senators were dispirited by a disjointed procedure that led to a bill few loved and wanted to ultimately pass. It followed a winding, six-month process that saw more ambitious proposals fail in the face of criticism from both the conservative and centrist wings of the party.
“This is a disappointment,” said Senate Majority Leader Mitch McConnell. “A disappointment indeed.”
The smaller measure was a desperate attempt by the Senate Republicans to keep the process alive. But the bill was far from ideal for many Republicans. Many of the senators who did support the legislation wanted a guarantee from the House that they would continue debating the bill, rather than pass the measure out of Congress.
The Senate bill, called the Health Care Freedom Act or the “skinny bill,” would have repealed the unpopular penalty urging individuals to purchase insurance and revise waivers under Obamacare to make it easier for states to set their own minimum benefits, among other changes. The Congressional Budget Office estimated that 16 million more people would be uninsured under this legislation.
In a process perhaps without precedent, the Republican senators tried to pass its preeminent promise without a hearing, and without the support of major industry groups affected, including doctors, patients, seniors, and—according to multiple polls—the public. The senators attempted to pass the health care bill without bipartisan support, in the middle of the night, to get to a conference with the House, a manner they criticized when the Democrats passed Obamacare.
The legislation would have increased premiums by 20 percent, according to CBO, after the GOP had pledged for years—and in recent months—that their mission was to decrease costs.
Before voting for the Senate Republican bill, some experienced legislators said they hoped Congress could return to the usual committee process, in which rank-and-file members and the public can have more power to write legislation.
“We have had seven years of almost intractable political differences of opinion,” said Sen. Lamar Alexander, the Republican chairman of the health care panel. “This may be the only way we could get a bill through the Congress that would actually repeal and replace major parts of Obamacare. But I much prefer the process that we usually use—and I hope that in the future, we resume that kind of process.”
The three Republican senators opposed the bill both for what it did and how it was made. In a statement, McCain said it would not increase competition, lower cost, or improve health care outcomes. But he also sharply criticized the process created by McConnell. “We must now return to the correct way of legislating and send the bill back to committee, hold hearings, receive input from both sides of aisle, heed the recommendations of nation’s governors, and produce a bill that finally delivers affordable health care for the American people,” he said.
Even Republican senators who voted for the bill criticized both the policy and the politics of it, saying it was an unsavory but necessary way to get to a debate with the House, when they could write something new. In a press conference Thursday, Sen. Lindsey Graham of South Carolina warned the House to not pass the bill he voted for, on the condition that the House would change it under further negotiations.
“The skinny bill as a replacement for Obamacare is a fraud,” he said. “Not only do we not replace Obamacare, we politically own the collapse of health care. I’d rather get out of the way and let it collapse, then have a half-ass approach where it is now our problem.”
On Thursday, House Speaker Paul Ryan and Republican senators said they did not want to repeat the same process on health care for future major legislation. Rank-and-file Republicans have criticized McConnell for rushing to release the Senate’s broader Obamacare replacement plan, which failed this week by a wider margin, without enough time to for the public to weigh in or for analysis from the official budget score. “This is not something I’d want to repeat,” said one GOP senator. “No.”
Congress has passed major legislation before on party-line votes, including cutting taxes in the Bush administration and aspects of Obamacare, through a process known as budget reconciliation, which limits debate.
But the Republicans’ health care process was much different than the Democratic one that lead to the passage of Obamacare. Bill Dauster, a senior policy staffer under former Senate Democratic leader Harry Reid, noted that Democrats held 60 hours of meetings among a group of bipartisan senators, spent hours revising the bill in two committees and accepted “dozens” of Republican amendments.
“All this is in contrast to Senator McConnell’s mode this time, which has been hyper-partisan and secretive,” said Dauster.
After the legislation was killed, McConnell said it will be interesting to see Democratic ideas, but appeared hesitant to support them, portraying the liberal alternative as a government takeover of the health care system. “Bailing out insurance companies with no thought of any kind of reform is not something I want to be part of,” he said.
Republicans could always revisit their intraparty efforts; Sen. Bill Cassidy of Louisiana immediately touted a plan that he developed with Graham that would provide states the authority and money to build their own systems.
President Trump suggested a different strategy, reviving a threat he has made repeatedly. “3 Republicans and 48 Democrats let the American people down,” Trump tweeted. “As I said from the beginning, let ObamaCare implode, then deal. Watch!”
But Republicans will likely agree with McConnell in that “it’s time to move on.” In an interview on FOX Business Network’s Mornings with Maria on Thursday, House Speaker Paul Ryan promised that on the next major legislative battle—the first overhaul of the tax code in 30 years—Republicans would do business “better and differently.”
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"The Senate voted Monday to reimpose the U.S. ban on Chinese telecom giant ZTE, in a rebuke to President Donald Trump and his efforts to keep the company in business. The provision targeting ZTE was part of the National Defense Authorization Act, a must-pass defense spending bill that cleared the Senate by a vote of 85-10. It must now be reconciled with the House version of the measure, which takes a narrower approach to ZTE." Separately, Trump is directing U.S Trade Representative Robert Lighthizer to identify $200 billion more in tariffs on Chinese products.
"Two longtime associates of President Donald Trump are now acknowledging a previously undisclosed contact in May 2016 with a Russian who they say offered dirt on Hillary Clinton. Roger Stone and Michael Caputo say they forgot to tell investigators about their contact with a Russian national who goes by the name Henry Greenberg — even though they say Greenberg offered to sell incriminating information to the Trump campaign for $2 million."
"As Trump signed a joint statement with Kim Jong Un that offered few details on how the North Korean leader would make good on his vow to denuclearize, Republicans on Capitol Hill said Tuesday that they want and expect the White House to submit any final agreement for their approval." Senate Majority Leader Mitch McConnell called for any agreement to be in the form of a treaty.
President Trump announced that the United States will suspend "war games" with South Korea, which are "inappropriate" given his meeting with North Korean leader Kim-Jong Un. "We will be stopping the war games which will save us a tremendous amount of money," said Trump, "unless and until we see the future negotiation is not going along like it should." The military exercises "carried out each year by the US and South Korean militaries have been consistently cited by Pyongyang as a US rehearsal for war, and a reason it needs to build a nuclear arsenal."


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Share this Recipe[ssba]
Broccoli and Parmesan Gratin
COOK TIME: 30 Minutes
PREP TIME: 20 Minutes
MAKES: 6 Servings
3 cups DOLE® Broccoli cut into florets (570 g)
1 can (12 oz.) fat-free evaporated milk (360 mL)
1 cup shredded Parmesan cheese (90 g)
¼ teaspoon salt (1 mL)
¼ teaspoon ground black pepper (1 mL)
1 cup panko Japanese-style bread crumbs (150 g)
1 tablespoon olive oil 15 (mL)
¼ teaspoon paprika (1 mL)
Preheat oven to 350° F (175° C).
Place broccoli florets and water in microwave-safe dish. Cover with plastic wrap, venting one corner for steam to escape. Cook on HIGH for 4 minutes. Drain broccoli well; place in 2-quart (2L) baking dish, sprayed with nonstick cooking spray.
Whisk together evaporated milk, Parmesan cheese, eggs, salt and pepper in medium bowl; pour over broccoli.
Mix bread crumbs, oil and paprika in small bowl; sprinkle over broccoli mixture.
Bake 25 to 30 minutes or until egg mixture is set and bread crumbs are golden brown. Let stand 5 minutes before serving.

domain: food


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News › Weekly Alert Article
Tax update: Recent developments in Romanian tax legislation
The Romanian Government has recently made various updates to tax legislation. Taxand Romania discovers the key highlights.
International tax: Ratification of the Convention between Romania and Uruguay
The Law ratifies the Convention between Romania and the Oriental Republic of Uruguay for the avoidance of double taxation and the prevention of fiscal evasion with respect to taxes on income and capital, which will be effective from 1 January of the year following the one during which it entered into force.
Compensation tax: Clarifications regarding the 85% tax for certain types of compensatory income
According to the provisions of the present Law, the 85% tax on income representing salaries/ allowances/compensatory amounts granted upon termination of the employment contract, the labour relation or of the mandate agreement applies only to employees holding management positions or to persons who were appointed as members in the Council of the Financial Supervisory Authority or of the National Securities Commission, the Insurance Supervisory Commission and the Supervisory Committee of the Private Pension System.
Indirect tax: Procedure for VAT registration upon request
According to the new provisions, in order to proceed with the VAT registration, upon request in the case of companies regulated by Law no. 31/1990, it shall be checked if the current majority shareholders or, as the case may be, the sole shareholder have registered in their fiscal record offenses and/or situations of joint and several liability as provided under art. 2 para. (2) letter a) of the Government Ordinance no. 75/2001 regarding the organisation and operation of the fiscal record.
Energy tax: Taxation for exploitation of natural resources
The present Law approves GO no. 6/2013 with certain amendments and completions. Therefore the exploitation permits issued according to the law are also treated as exploitation licenses. Moreover, the law introduces different computation methods for the revenues obtained by economic operators falling under the scope of this tax, based on how the natural resources are used and on whether their price is listed or not on the exchange market. The tax is due also for resources exploited before 1 February 2013 and sold after this date.

domain: finance


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The Dermaroller is a cylindrical drum, studded with high grade, fine, stainless steel needles. It is a medical device used in micro needling to stimulate proliferation of the skin cells. This cell multiplication results, in the formation of new tissue layers of elastin and collagen fibres, as well as new capillaries for an improved blood supply. This procedure is called Collagen Induction Therapy (CIT). The Dermaroller improves the thickness of the dermis as well as the epidermis. This in return improves skin texture and reduces wrinkles. It also has a positive effect on hyper-pigmentation as well as on hypo-pigmentation, new collagen fibres, an improved blood supply and harmonises pigmentation.
Since Dermaroller does not use thermal heat (unlike IPL and Lasers), it is safe to use on all skin types and all skin colours. There is no risk of post-inflammatory hyperpigmentation.
Collagen Induction Therapy
Dermaroller Collagen Induction Therapy procedure works by stimulating your own body’s collagen. Your body will start to naturally regenerate the skin, working below the surface in the dermis. New skin cells are generated and blood supply is enhanced. It will only take up to 6 weeks before visible signs of regeneration are seen and the process will continue over the following months, providing you with a gradual & long lasting enhancement.
Scar Reduction Therapy
Dermaroller Scar Reduction Therapy procedure works by releasing old fibrotic scar tissue deep within the skin. Scar tissue is what creates visible scars on the skin’s surface. The treatment will repair the skin naturally, working below the surface in the dermis. This takes time as new collagen and elastin fibres are formed, softening scars, generating new skin cells & enhancing blood supply. Results can be seen within 6 weeks, and a total of 70-80% improvement of scars.

domain: medical


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February 26, 2009
In 1999, UCLA Professor Franklin D. Gilliam wrote a report for Harvard University’s Nieman Foundation for Journalism. That paper concerned a study he had done regarding public perception of the “welfare queen” stereotype and how that perception had been shaped by the media. He discussed how the term had been introduced by Ronald Reagan during the 1976 Presidential campaign. Reagan told the story of a woman from Chicago’s South Side, who had been arrested for welfare fraud. The term became widely used in reference to a racist (and sexist) stereotype of an iconic African-American woman, enjoying a lavish lifestyle and driving a Cadillac while cheating the welfare system.
Ten years after the publication of Gilliam’s paper, we have a new group of “welfare queens”: the banks. The banks have already soaked over a trillion dollars from the federal government to remedy their self-inflicted wounds. Shortly after receiving their first $350,000,000,000 in payments under the TARP program (which had no mechanism of documenting where the money went) their collective reputation as “welfare queens” was firmly established. In the most widely-reported example of “corporate welfare” abuse by a bank, public outcry resulted in Citigroup’s refusal of delivery on its lavishly-appointed, French-made, Falcon 50 private jet. Had the sale gone through, Citi would have purchased the jet with fifty million dollars of TARP funds. Now, as they seek even more money from us, the banks chafe at the idea that American taxpayers, economists and political leaders are suggesting that insolvent (or “zombie”) banks should be placed into temporary receivership until their “toxic assets” are sold off and their balance sheets are cleaned up. This has been referred to as “nationalization” of those banks.
Despite all the bad publicity and public outrage, banks still persist in their welfare abuse. After all, they have habits to support. Their “drug” of choice seems to be the lavish golf outing at a posh resort. The most recent example of this resulted in Maureen Dowd’s amusing article in The New York Times, about a public relations misstep by Sheryl Crow.
The New Welfare Queens have their defenders. CNBC’s wildly-animated Jim Cramer has all but pulled out his remaining strands of hair during his numerous rants about how nationalization of banks “would crush America”. A number of investment advisors, such as Bill Gross, co-chief investment officer at Pacific Investment Management Company, have also voiced objections to the idea of bank nationalization.
Another defender of these welfare queens appears to be Federal Reserve Chairman Ben Bernanke. In his latest explanation of Turbo Tim Geithner’s “stress test” agenda, Bernanke attempted to assure investors that the Obama administration does not consider the nationalization of banks as a viable option for improving their financial health. As Craig Torres and Bradley Keoun reported for Bloomberg News on February 25, the latest word from Bernanke suggests that nationalization is not on the table:
. . . while the U.S. government may take “substantial” stakes in Citigroup Inc. and other banks, it doesn’t plan a full- scale nationalization that wipes out stockholders.
Nationalization is when the government “seizes” a company, “zeroes out the shareholders and begins to manage and run the bank, and we don’t plan anything like that,” Bernanke told lawmakers in Washington today.
The only way to deal with The New Welfare Queens is to replace their directors and managers. The Obama administration appears unwilling to do that. During his February 25 appearance on MSNBC’s Countdown, Paul Krugman (recipient of the Nobel Prize in Economics) expressed his dread about the Administration’s plan to rehabilitate the banks:
I’ve got a bad feeling about this, as do a number of people. I was just reading testimony from Adam Posen, who is our leading expert on Japan. He says we are moving right on the track of the Japanese during the 1990s: propping up zombie banks — just not doing resolution.
. . . The actual implementation of policy looks like a kind of failure of nerve.
* * *
On the banks — I really can’t see — there really seems to be — we’re going to put in some money, as we’re going to say some stern things to the bankers about how they should behave better. But if there is a strategy there, it’s continuing to be a mystery to me and to everybody I’ve talked to.
You can read Adam Posen’s paper: “Temporary Nationalization Is Needed to Save the U.S. Banking System” here. Another Economics professor, Matthew Richardson, wrote an excellent analysis of the pros and cons of bank nationalization for the RGE Monitor. After discussing both sides of this case, he reached the following conclusion:
We are definitely caught between a rock and a hard place. But the question is what can we do if a major bank is insolvent? Sometimes the best way to repair a severely dilapidated house is to knock it down and rebuild it. Ironically, the best hope of maintaining a private banking system may be to nationalize some of its banks. Yes, it is risky. It could go wrong. But it is the surest path to avoid a “lost decade” like Japan.
As the experts report on their scrutiny of the “stress testing” methodology, I get the impression that it’s all a big farce. Eric Falkenstein received a PhD in Economics from Northwestern University. His analysis of Geithner’s testing regimen (posted on the Seeking Alpha website) revealed it to be nothing more than what is often referred to as “junk science”:
Geithner noted he will wrap this up by April. Given the absurdity of this exercise, they should shoot for Friday and save everyone a lot of time. It won’t be any more accurate by taking two months.
On a similar note, Ari Levy wrote an illuminating piece for Bloomberg News, wherein he discussed the stress testing with Nancy Bush, bank analyst and founder of Annandale, New Jersey-based NAB Research LLC and Richard Bove of Rochdale Securities. Here’s what Mr. Levy learned:
Rather than checking the ability of banks to withstand losses, the tests outlined yesterday are designed to convince investors that the firms don’t need to be nationalized, said analysts including (Nancy) Bush and Richard Bove from Rochdale Securities.
* * *
“I’ve always thought that this stress-testing was a politically motivated approach to try to defuse the argument that the banks didn’t have enough capital,” said Bove, in an interview from Lutz, Florida. “They’re trying to prove that the banks are well-funded.”
Will Turbo Tim’s “stress tests” simply turn out to be a stamp of approval, helping insolvent banks avoid any responsible degree of reorganization, allowing them to continue their “welfare queen” existence, thus requiring continuous infusions of cash at the expense of the taxpayers? Will the Obama administration’s “failure of nerve” — by avoiding bank nationalization — send us into a ten-year, “Japan-style” recession? It’s beginning to look that way.


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Chuck E. Weiss, a longtime presence on the Los Angeles music scene and the topic of a Prime 5 hit for Rickie Lee Jones, died yesterday at age 76. Particulars on his loss of life weren’t instantly out there, however buddies mentioned he died after an extended sickness.
A staple performer on Mondays at The Central nightclub on the Sundown Strip for 11 years with his band, The Goddamn Liars, Weiss satisfied buddy Johnny Depp to /assist revive it when it fell on laborious instances. It grew to become the Viper Room, one of many hottest music venues within the metropolis. He additionally grew to become related to the Kibitz Room, an offshoot of Canter’s Deli on Fairfax, the place a sales space was named in his honor for his presence on the room’s frequent jams.
An outpouring on social media got here as information of Weiss’s loss of life unfold. “Please let individuals know he was a lot greater than the title of a track,” mentioned Sheryl Northrop, the publicist for 2 of his albums.
Robby Steinhardt Dies: Violinist, Founding Member Of Rock Band Kansas Was 71
Weiss didn’t wish to journey by airplane, so confined his actions to Southern California. However he reached nationwide fame on songwriter and buddy Rickie Lee Jones debut album in 1979.
Jones and her boyfriend Tom Waits had been buddies with Weiss, and steadily frolicked with him on the legendary punk motel The Tropicana in Los Angeles. Weiss disappeared for a time, however then phoned Waits to inform him he moved to his hometown of Denver as a result of he had fallen in love with a cousin situated there.
Waits purportedly hung up the cellphone and declared, “Chuck E.’s in love.” Jones beloved the sound of that and wrote the track that rose to No. 4 on the Billboard Scorching 100, making it her largest hit. The track’s ending, although, modifications the article of Chuck E.’s affection to Jones herself, which was by no means the case.
As a performer, Weiss was recognized for his eclectic mixture of rock ‘n roll and his means to insert all the pieces from poetry to zydeco into his music. A consummate frontman, he was a grasp at turning a membership room into an on the spot get together.
Born in Denver, Colorado, to oldsters who owned a report story, Weiss met blues legend Lightnin’ Hopkins at a neighborhood nightclub. His drum expertise impressed Hopkins, who took him on tour, permitting him to play with blues giants like Muddy Waters, Howlin’ Wolf and Dr. John, amongst others.
Weiss later grew to become a disc jockey on different rock station KFML-FM in Denver within the early Nineteen Seventies.
Leaving the Denver scene behind, Weiss headed to Los Angeles His first album was an EP, The Different Facet of City, was launched in 1981.
Weiss contributed to the L.A. Ya Ya compilation of Los Angeles blues artists, and appeared on the choice rock band album P, that includes Depp and Gibby Haynes of the Butthole Surfers.
From that, Weiss launched his first full-length album, 1999’s Extraordinarily Cool, that includes contributions from Tony Gilkyson and Tom Waits, the latter the co-producer. The opening monitor, Satan With Blue Suede Sneakers, was produced by Depp and was utilized in Depp’s movie The Courageous.
The follow-up album, 2001’s Previous Souls and Wolf Tickets, was highlighted by a monitor that includes Willie Dixon. In 2006, Weiss launched the album twenty third & Stout,
Steaming forward within the teenagers, Weiss wrote and recorded “Anthem for Previous Souls” for the compilation album Son of Rogues Gallery, then got here out with 2014’s Purple Beans and Weiss on the Anti label.
Along with his music ventures, Weiss appeared on a 2002 episode of the collection Gilmore Women because the proprietor of a New York Metropolis report retailer. He additionally did cameos in two 1990 Married…with Kids episodes, taking part in a homeless man, and likewise appeared within the collection Brotherly Love and My Spouse and Children.
Info on survivors was not instantly out there. Buddies are within the early levels of placing collectively memorial plans, however no particulars have been launched.

domain: artistic


How the California Governor’s Race May Be a Boon for House Democrats
A controversial Republican gubernatorial candidate in California could cause the GOP headaches in down-ballot House races in a state crucial to the party’s hopes of increasing its House majority.
State Assemblyman Tim Donnelly led Neel Kashkari, a former U.S. Treasury official and the establishment-preferred candidate, by 5 points in a poll this month from the Public Policy Institute of California. That has top Republican operatives in California and Washington, D.C., concerned that Donnelly’s controversial comments and ties to the California Minutemen , a group that fights illegal immigration on the border, could mobilize Hispanic and other Democratic-base voters who otherwise might fall off in this midterm year.
Their fear is that the increased turnout to oppose Donnelly could boost the re-election hopes of vulnerable House Democrats and perhaps even improve the party’s chances to add to its ranks in the already Democrat-heavy delegation. A consultant with ties to Kashkari is among the Republicans sounding the alarm. “It would be a fucking disaster,” said Jason Roe, a San Diego-based consultant who has worked with numerous congressional campaigns and whose firm is consulting for Kashkari.
Democratic Gov. Jerry Brown is expected to finish way ahead of both Republicans in the June 3 top-two primary and is favored to win re-election in November. House Majority Whip Kevin McCarthy of California, who has led the party’s efforts at the congressional level in the state, noted in a brief interview Thursday that the GOP always has difficulty running statewide, so Republican House candidates must work to individualize their races.
“Neither of them are polling really well,” McCarthy said. “I always think people run in their own districts. Statewide it’s tough.”
Still, the party’s concern has been voiced over national airwaves. Karl Rove, founder of the GOP-aligned super PAC American Crossroads, which is spending on congressional races, said on the Hugh Hewitt radio show this month that the “outrageous things” Donnelly has said and done “will be used to tarnish” the state party and Republican candidates.
While the gubernatorial race is an issue for the GOP, Republicans are expected to get three top-tier House candidates through primaries in competitive districts represented by freshman Democrats.
Former Rep. Doug Ose is favored to take on Rep. Ami Bera in the 7th District; former Capitol Hill aide Brian Nestande is expected to face Rep. Raul Ruiz in the 36th District; and former San Diego City Councilman Carl DeMaio will take on Rep. Scott Peters in the 52nd District.
They represent some of the party’s best pickup opportunities in the country, though President Barack Obama won all three in 2012. Crucial to Republicans winning back these seats is Democratic drop-off and a motivated GOP base. Republicans say Donnelly could keep moderate Republicans home, and motivate Democrats to head to the polls — effectively vanquishing a GOP advantage in the fall.
“There were some seats that Republicans narrowly lost in 2012,” California Republican strategist Kurt Bardella said. “And Donnelly could have a harmful effect if he’s on the top of the ticket.”
Similarly, Democrats have a prime pickup opportunity in the 21st District, where freshman GOP Rep. David Valadao is likely to face former Capitol Hill aide Amanda Renteria in a district Obama carried by 11 points in 2012. Higher Hispanic turnout in that district could be a boon for Renteria, whom Democrats tout as one of their top recruits this cycle.
While Donnelly leads in polling of the gubernatorial race, Kashkari has the infrastructural advantages. He pulled in high-profile endorsements in the final weeks of the contest from Mitt Romney, former Secretary of State Condoleezza Rice and Rep. Darrell Issa. Plus, the San Francisco Chronicle reported Thursday that Kashkari’s investment of personal funds gave him a significant financial edge over Donnelly, while a super PAC formed to help Kashkari get through the primary received a massive influx of cash this week.
Democrats have their own problems in the state, stemming from the top-two primary — where the two highest vote recipients advance to the general regardless of party.
In the 31st District, it could be déjà vu for Democrats in a district they should have won in 2012. Retiring Republican Rep. Gary G. Miller and another Republican advanced there in 2012, when four Democrats splintered the vote, taking a seat off the map even though Obama carried it with 57 percent. Democrats are working to avoid a similar scenario this year.
Still, Democrats in the state are jubilant about the possibility of a Donnelly candidacy as June 3 draws near. After a four-seat gain in 2012, the party is looking to increase its majority in the delegation.
“I think Donnelly’s victory would motivate true blue Democrats, while Kashkari’s victory has the potential to change the dialogue for who votes Republican,” Los Angeles Democratic Party Chairman Eric Bauman said.
Kyle Trygstad contributed to this report


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Let me introduce you to Melika Payne, a fabulous 54-year-old American actress, and a proud mom to the talented Landon Brown, who’s made a name for himself in the world of American film and television.
Melika, born Melika Michem Williams on April 10, 1969, hails from the city of Angels, Los Angeles, California. She had her elementary and high school education in some secret schools within the beautiful state of California. Shhh, it’s a mystery!
Moreover, Payne has not only pursued her passion for acting but has also raised a star in her own family. With her intriguing background and connection to the entertainment industry, it’s no wonder that talent runs in the family.
Who Is Melika Payne?
The gorgeous woman Melika is a true star in the entertainment world. She’s not only an amazing actress but also a producer and director who knows how to captivate audiences with her talent and charm. Growing up in Hollywood, the lady discovered her passion for performing arts early on and pursued a degree in Theater Arts, specializing in Acting.
Melika Payne is the mother of Landon Payne.
With her talent and hard work, the Afro-American origin actress is making a difference in the world. She supports various charitable causes that are close to her heart and believes in giving back to society.
Throughout her career, the beautiful woman has had some impressive achievements. She’s had recurring roles on popular TV shows like George Lopez, The Game, For Richer or Poorer, and Step Up High Water. On top of that, she’s a talented director, having worked on a film called Misguided Behavior in 2017, and even directed episodes of Tyler Perry’s Young Dylan.
But wait, there’s more! the 5 feet 7 inches tall lady is not just a talented actress and director, she’s also a hilarious standup comedian. She travels around the country, bringing laughter to audiences with her comedy. And let’s not forget her iconic role in the show Martin, which she starred in from 1992 to 1997. It’s a true legacy she’s proud of.
Melika Payne and Bobby Brown’s Short-Lived Romance
Well, the TV star Melika made headlines for her romantic involvement with American R&B singer and dancer, Bobby Brown, whose full name is Robert Barisford Brown.
At that time, Payne and her former partner Brown’s love story caught the media’s attention. They had an exclusive and open relationship, dating each other in 1984. During their time together, they welcomed their son, Landon, into the world on June 22, 1986. However, their relationship didn’t stand the test of time, and after three years, the love mates decided to part ways in 1987.
Even though Landon was just a young child when his parents ended their relationship, his father, Bobby, has always been supportive and actively involved in his upbringing. Despite the ex-couple separation, The Game actress and Bobby share the responsibility of raising their son, showing a mutual commitment to his well-being.
Who Is Bobby Brown? Is He Married?
Get ready to groove to the music of Brown, the “Prince of Pop“! This talented American singer, songwriter, and dancer is a true icon in the music industry. He’s known for his incredible skills in blending hip-hop and R&B, creating a style called new jack swing.
Bobby Brown is married to Alicia Etheredge.
Moreover, the media personage started his musical journey as part of the R&B and pop group called New Edition. They were a hit! But the star didn’t stop there. He went on to have an amazing solo career, and his second album called Don’t Be Cruel (1988) was a huge success. Can you believe it? He has sold over 50 million copies of his music all around the world. That’s a lot of fans dancing to his catchy tunes! See another musician Ahmet Zappa.
Not only is Bobby a talented musician but he’s also dabbled in acting. You might have seen him in movies like A Thin Line Between Love and Hate and Two Can Play That Game. He’s got the moves, the voice, and the charm to captivate audiences wherever he goes. Brown is truly a legend in the world of music and entertainment.
Now, let’s talk about the big-name’s love life! On June 18, 2012, the talented musician tied the knot with Alicia Etheredge, and their journey as a married couple began. They’ve been blessed with three beautiful children: Cassius Brown, Bodhi Jameson Rein Brown, and Hendrix Estelle Sheba Brown.
Melika Payne Is A Blissfully Married Woman
Meet Payne, a woman of Afro-American heritage, who shares a strong and enduring bond with her husband, Carl Anthony Payne II. You might recognize Carl from his notable role in The Cosby Show. They’ve been through thick and thin in their relationship, but their love has stood the test of time.
Melika Payne with her spouse Carl Anthony Payne.Source: Instgaram@thecarlpayne
In December 1992, Melika and her now husband Anthony tied the knot, surrounded by their loved ones. They preferred to keep their wedding celebration intimate and away from the public eye. During their marriage, they were blessed with two wonderful sons, Carl Anthony Payne III, and Malek Payne.
Similarly, the details of how the pretty woman and Carl first met and fell in love remain a delightful mystery, keeping their fans intrigued and eager to know more.
Unfortunately, in 2012, Payne filed for divorce, and the case even went to the LA County Superior Court in 2014. However, this resilient couple decided to give their marriage another chance, choosing to mend and reconcile their relationship.
It’s truly remarkable that Melika and her spouse have been living a blissful married life for decades, with no rumors of any separation. As an exciting milestone, they will soon celebrate their thirty-first wedding anniversary in December 2023, marking a remarkable journey of love and togetherness.
Melika Payne’s Impressive Net Worth
Despite her preference for privacy and absence from social media platforms including Instagram, Facebook, and Twitter, the lovely lady has managed to build a successful career and accumulate a considerable income.
Although the stunning Payne keeps her earnings under wraps, it is estimated that her net worth stands at $1 million. Alongside her professional achievements, Carl, her hubby, has also made a name for himself in the entertainment industry. In contrast, Suge Knight holds a net worth of $200,000.
Likewise, the charming guy has acted in popular shows like The Rickey Smiley Show and Single Drunk Female, portraying memorable characters. With his acting career and social media presence, including over 404 thousand followers on Instagram, Carl has further boosted his net worth to $1 million.
Stay Tuned at Featured Biography for similar content!


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text:
Diffractive micro bar codes for encoding of biomolecules in multiplexed assays.
ABSTRACT Microparticles incorporating micrometer-sized diffractive bar codes have been modified with oligonucleotides and immunoglobulin Gs to enable DNA hybridization and immunoassays. The bar codes are manufactured using photolithography of a chemically functional commercial epoxy photoresist (SU-8). When attached by suitable linkers, immobilized probe molecules exhibit high affinity for analytes and fast reaction kinetics, allowing detection of single nucleotide differences in DNA sequences and multiplexed immunoassays in <45 min. Analysis of raw data from assays carried out on the diffractive microparticles indicates that the reproducibility and sensitivity approach those of commercial encoding platforms. Micrometer-sized particles, imprinted with several superimposed diffraction gratings, can encode many million unique codes. The high encoding capacity of this technology along with the applicability of the manufactured bar codes to multiplexed assays will allow accurate measurement of a wide variety of molecular interactions, leading to new opportunities in diverse areas of biotechnology such as genomics, proteomics, high-throughput screening, and medical diagnostics.
- SourceAvailable from: onlinelibrary.wiley.com[Show abstract] [Hide abstract]
ABSTRACT: Advances in lab-on-a-chip technologies enabled programmable, reconfigurable, and scalable manipulation of a variety of laboratory procedures. Samples, reagents, and fluids can be precisely controlled; buffer temperature, pH, and concentration control systems as well as a variety of detection systems can be integrated on a small chip. These advantages have attracted attention in various fields of clinical application including leukemia diagnosis and research. A lot of research on lab-on-a-chip based diagnosis has been reported and the field is rapidly expanding. This review describes recent developments of lab-on-a-chip technologies as solutions to challenges for high-throughput leukemia diagnosis.International journal of laboratory hematology 02/2013; · 1.30 Impact Factor
- Advanced Materials 06/2009; 21(40):4020 - 4024. · 14.83 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: A multiplexed suspension array platform, based on SU8 disks patterned with machine-readable binary identification codes is presented. Multiple probe molecules, each attached to individual disks with different unique codes, provide multiplexed detection of targets in a small sample volume. The experimental system consists of a microfluidic chamber for arraying the particles in a manner suitable for high throughput imaging using a simple fluorescent microscope, together with custom software for automated code readout and analysis of assay response. The platform is demonstrated with a multiplexed antibody assay targeting 3 different human inflammatory cytokines. The suitability of the platform for other bio-analytical applications is discussed.Biomedical Microdevices 03/2012; 14(4):651-7. · 2.72 Impact Factor
Diffractive Micro Bar Codes for Encoding of
Biomolecules in Multiplexed Assays
Graham R. Broder,†Rohan T. Ranasinghe,†Joseph K. She,†Shahanara Banu,†,‡Sam W. Birtwell,§,|
Gabriel Cavalli,†Gerasim S. Galitonov,§David Holmes,‡Hugo F. P. Martins,†Kevin F. MacDonald,§
Cameron Neylon,†,⊥Nikolay Zheludev,§Peter L. Roach,*,†and Hywel Morgan*,‡
School of Chemistry, School of Electronics and Computer Science, Optoelectronics Research Centre, and School of Physics
and Astronomy, University of Southampton, Highfield, Southampton, SO17 1BJ, UK, and STFC, Rutherford Appleton
Laboratory, Didcot, OX11 0QX, UK
Microparticles incorporating micrometer-sized diffractive
bar codes have been modified with oligonucleotides and
immunoglobulin Gs to enable DNA hybridization and
immunoassays. The bar codes are manufactured using
photolithography of a chemically functional commercial
epoxy photoresist (SU-8). When attached by suitable
linkers, immobilized probe molecules exhibit high affinity
for analytes and fast reaction kinetics, allowing detection
of single nucleotide differences in DNA sequences and
multiplexed immunoassays in <45 min. Analysis of raw
data from assays carried out on the diffractive micropar-
ticles indicates that the reproducibility and sensitivity
approach those of commercial encoding platforms. Mi-
crometer-sized particles, imprinted with several super-
imposed diffraction gratings, can encode many million
unique codes. The high encoding capacity of this technol-
ogy along with the applicability of the manufactured bar
codes to multiplexed assays will allow accurate measure-
ment of a wide variety of molecular interactions, leading
to new opportunities in diverse areas of biotechnology
such as genomics, proteomics, high-throughput screen-
ing, and medical diagnostics.
Rapid and multiplexed molecular detection with high sensitivity
and specificity is of great importance in gene profiling, clinical
diagnostics, and environmental monitoring. A key requirement
for multiplexing is the identification of each molecule set within
the library of probes used in an assay. In this context, microarrays
have revolutionized DNA expression profiling1,2and show great
promise for proteomics3-6and DNA sequencing.7In these
systems, a large number of different biomolecules are identified
by their position on a two-dimensional grid. However, microarrays
have certain disadvantages, including slow diffusion of molecules
to their binding sites and the inability to perform large numbers
of reactions simultaneously over a wide dynamic range.
Bead or suspension arrays, in which probes are attached to
the surface of microparticles, are an attractive alternative for
multiplexed analysis and have found applications in profiling DNA
or proteins from complex biological samples.8,9Such arrays offer
a number of advantages, including fast reaction kinetics and
flexibility of library content. High-throughput analysis by flow
cytometry makes it possible to interrogate vast numbers of probes
or to perform large numbers of replicate measurements.
In suspension arrays, sets of beads carry a unique bar code.10,11
To fully exploit the potential for multiplexed bead-based arrays, a
robust and reliable method of manufacturing encoded beads is
required. For high-throughput analysis, the decoding system must
identify each and every code quickly and reliably. A number of
encoding strategies have been proposed and demonstrated includ-
ing the use of nanoscale metallic bar codes that are read by
reflectance,12quantum dot-encoded mesoporous beads,13spatially
resolved photobleaching of fluorescent particles,14direct optical
reading of the shape of micromachined particles,15incorporation
of Raman-active organic compounds,16or optical resonance
(whispering gallery mode) in micrometer-sized silica spheres.17
* To whom correspondence should be addressed. E-mail: email@example.com
†School of Chemistry, University of Southampton.
‡School of Electronics and Computer Science, University of Southampton.
§Optoelectronics Research Centre, University of Southampton.
|School of Physics and Astronomy, University of Southampton.
⊥Rutherford Appleton Laboratory.
(1) Gresham, D.; Ruderfer, D. M.; Pratt, S. C.; Schacherer, J.; Dunham, M. J.;
Botstein, D.; Kruglyak, L. Science 2006, 311, 1932-1936.
(2) Abdullah-Sayani, A.; Bueno-de-Mesquita, J. M.; van de Vijver, M. J. Nat.
Clin. Pract. Oncol. 2006, 3, 501-516.
(3) Becker, K. F.; Metzger, V.; Hipp, S.; Hofler, H. Curr. Med. Chem. 2006,
(4) Balboni, I.; Chan, S. M.; Kattah, M.; Tenenbaum, J. D.; Butte, A. J.; Utz, P.
J. Annu. Rev. Immunol. 2006, 24, 391-418.
(5) Barbulovic-Nad, I.; Lucente, M.; Sun, Y.; Zhang, M. J.; Wheeler, A. R.;
Bussmann, M. Crit. Rev. Biotechnol. 2006, 26, 237-259.
(6) Wingren, C.; Borrebaeck, C. A. K. Omics 2006, 10, 411-427.
(7) Bentley, D. R. Curr. Opin. Genet. Dev. 2006, 16, 545-552.
(8) Meza, M. B. Drug Discovery Today 2000, 38-41.
(9) Nolan, J. P.; Mandy, F. Cytometry, Part A 2006, 69A, 318-325.
(10) Braeckmans, K.; De Smedt, S. C.; Leblans, M.; Pauwels, R.; Demeester, J.
Nat. Rev. Drug Discovery 2002, 1, 447-456.
(11) Wilson, R.; Cossins, A. R.; Spiller, D. G. Angew. Chem., Int. Ed. 2006, 45,
(12) Nicewarner-Pena, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pena, D. J.;
Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294,
(13) Han, M. Y.; Gao, X. H.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-
(14) Braeckmans, K.; De Smedt, S. C.; Roelant, C.; Leblans, M.; Pauwels, R.;
Demeester, J. Nat. Mater. 2003, 2, 169-173.
(15) Zhi, Z. L.; Morita, Y.; Yamamura, S.; Tamiya, E. Chem. Commun. 2005,
(16) Jun, B. H.; Kim, J. H.; Park, H.; Kim, J. S.; Yu, K. N.; Lee, S. M.; Choi, H.;
Kwak, S. Y.; Kim, Y. K.; Jeong, D. H.; Cho, M. H.; Lee, Y. S. J. Comb. Chem.
2007, 9, 237-244.
Anal. Chem. 2008, 80, 1902-1909
Analytical Chemistry, Vol. 80, No. 6, March 15, 2008
10.1021/ac7018574 CCC: $40.75© 2008 American Chemical Society
Published on Web 02/14/2008
Nucleic acids have also been used as coding elements; these are
read in situ by sequential hybridizations of labeled oligonucle-
otides18,19or via hybridization to a conventional DNA chip after
removal of the tag sequence from the beads.20
Among these, spectral encoding, based on fluorescence
wavelength and intensity, appears to be the most promising.
However, this approach limits the number of bead-based assays
that can be performed to the number of spectrally resolvable
fluorophores that can be incorporated into a bead. Current spectral
encoding technology (e.g., Luminex xMAP) provides ∼100 unique
codes. In principle, the possibilities are far greater; for example,
6 colors at 10 intensity levels gives ∼1 million codes. However,
practical encoding capabilities are much lower (due to spectral
overlap, fluorescence intensity variations, signal-to-noise ratio, etc.)
and realistically five to six colors with six intensity levels could
yield ∼10 000-40 000 codes.10A major drawback of this method
is that most bead-based biochemical assays rely on fluorescence
readout, so that the additional use of fluorescence for encoding,
particularly where multiple fluorophores are used, places limits
on the spectral bandwidth available for fluorescent-based assays
or the number of individual codes that can be identified. Recently,
a method for fabricating graphical bar codes within microfluidic
channels was reported. Using laminar flow, separate coding and
probing regions were simultaneously assembled during a polym-
erization step.21The potential for encoding ∼106unique codes in
particles of length 180-270 µm was demonstrated.
We have developed a new and robust encoding system,
orthogonal to fluorescence-based assays, based on microparticles
that incorporate diffractive elements as identifiers.22Noninvasive
and noncontact reading of codes can be accomplished by analyzing
the spatial distribution of the diffracted light. This is shown
schematically in Figure 1, which demonstrates the principle of
encoding using microdiffraction gratings. A single grating diffracts
the incident light into a unique diffraction pattern as shown in
Figure 1a. The “code” is read by measuring the spatial distribution
of this diffracted light. In the simplest implementation, information
is encoded in the pitch of the grating, a. When the grating is
illuminated with light at wavelength λ, a series of diffracted beams
is created at angles θ, according to eq 1, where m is the order of
the diffracted beam. Measurement of the first order (m ) (1)
diffracted beam position (with respect to the zero order) gives
direct information about the pitch a. This is shown in Figure 1a
a sinθ ) mλ
We have previously demonstrated that ∼109unique codes can
be created from a library of five times superimposed gratings
fabricated on a 100-µm-long particle.22In fact, the encoding has
recently been extended to two dimensions, greatly amplifying the
encoding capacity to huge numbers; up to 1018unique codes.23
Shrinking the gratings reduces the encoding capacity; the value
quoted above is for a 100-µm particle read with a laser light of
wavelength 633 nm. The lower limit of grating size is ∼3 times
the wavelength of the reading laser (in our case, ∼1.9 µm);
however, gratings of this size only have one possible code. The
encoding capacity increases rapidly as the grating size is in-
creased, already reaching ∼104codes for 10-µm gratings.
In this paper, we assess the functionality of probe biomolecules
immobilized onto these diffractive micro bar codes, namely,
immunoglobulin Gs (IgGs) and DNA. We characterize the
thermodynamics and kinetics of their molecular interactions and
go on to demonstrate their use in multiplexed assays.
Optical Hardware. Simultaneous diffraction and fluorescence
measurements were made using a custom-made optical system
with an infinity corrected high numerical aperture objective lens
and appropriate optical band-pass filters and dichroics (Supporting
Information Figure S-2). Diffraction patterns were generated using
plane incident light. The diffracted light was collected by a CCD
camera and the diffraction pattern analyzed by dedicated computer
Particle Fabrication. Diffractive bar codes were fabricated
from photoactive epoxy SU-8-5 (Chestech Ltd. Rugby, UK). The
resist was spin-coated onto Si wafers (525 ( 25 µm thick) onto
which a thin layer of Al had been evaporated. After spinning onto
the primed wafers, the SU-8 was soft baked, exposed (using an
EVG 620 mask aligner with photomask from Compugraphic, UK),
and postexposure baked. The wafers were then developed in EC
solvent poly(propylene glycol) methyl ether acetate (Chestech
Ltd., Rugby, UK) for 2 min with agitation. The wafers were
thoroughly rinsed with isopropyl alcohol and blow dried. The Al
sacrificial layer was removed by sonicating the wafers in Microp-
osit MF-319 (tetramethylammonium hydroxide (2.2% w/v solution
in water), Chestech Ltd., Rugby, UK) at room temperature for 10
min. The microparticles were collected by centrifugation (12500g,
1 min), washed in methanol (1 mL × 8), and dried under vacuum
at room temperature for 4 h.
Reagents. Oligonucleotides (P1, 5′-biotin-AAAAAGTTGGATCC-
3′; P2, 5′-biotin-AAAAACTTGGATCC-3′ C1; 5′-Cy5-GGGATC-
CAAGTTTTTT-3′; FP1, 5′-Cy5-CTAGTTACTCTTGTTC-biotin-3′)
were purchased from ATDBio (Southampton, UK) or synthesized
using a MerMade 192 DNA synthesizer (Bioautomation Inc.), for
which all reagents were obtained from Link Technologies (Bellshill,
UK). Cy3-labeled goat polyclonal anti-human IgG and Cy5-labeled
goat polyclonal anti-rabbit IgG, anti-mouse IgG, and anti-guinea
pig IgG were purchased from Abcam plc. (Cambridge, UK).
Conjugation of Oligonucleotides and IgGs to Encoded
Microparticles. SU-8 microparticles for use in both hybridization
assays and immunoassays were functionalized by reaction of
avidin-DN (Vector laboratories, Burlingame, CA) or protein A
(Cambridge Bioscience, Cambridge, UK), respectively, with car-
boxyl-functionalized particles according to standard amide cou-
pling protocols.24Carboxyl groups were introduced by succiny-
lation of primary amine groups resulting from ring opening of
(17) Vollmer, F.; Arnold, S.; Braun, D.; Teraoka, I.; Libchaber, A. Biophys. J. 2003,
(18) Gunderson, K. L.; Kruglyak, S.; Graige, M. S.; Garcia, F.; Kermani, B. G.;
Zhao, C. F.; Che, D. P.; Dickinson, T.; Wickham, E.; Bierle, J.; Doucet, D.;
Milewski, M.; Yang, R.; Siegmund, C.; Haas, J.; Zhou, L. X.; Oliphant, A.;
Fan, J. B.; Barnard, S.; Chee, M. S. Genome Res. 2004, 14, 870-877.
(19) Li, Y. G.; Cu, Y. T. H.; Luo, D. Nat. Biotechnol. 2005, 23, 885-889.
(20) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884-1886.
(21) Pregibon, D. C.; Toner, M.; Doyle, P. S. Science 2007, 315, 1393-1396.
(22) Galitonov, G. S.; Birtwell, S. W.; Zheludev, N. I.; Morgan, H. Opt. Express
2006, 14, 1382-1387.
(23) Birtwell, S. W.; Galitonov, G. S.; Morgan, H.; Zheludev, N. I. Opt. Commun.
In press. Doi:10.1016/j.optcom.2007.04.066.
(24) Hermanson, G. T. Bioconjugate Techniques; Academic Press: London, 1996.
Analytical Chemistry, Vol. 80, No. 6, March 15, 2008
residual surface epoxide groups of SU-8 with Jeffamine, as
described previously.25Detailed protocols for all synthetic steps
are available as Supporting Information.
Determination of Equilibrium Dissociation Constants.
Thermodynamics of molecular interactions were determined using
titration of analytes and flow cytometry. Human IgG-coated SU-8
microparticles (1 mg), suspended in storage buffer (10 mM NaH2-
PO4, 150 mM NaCl, 0.1% Tween-20, pH 7.8, 100 µL, giving a 0.75
µM concentration of immobilized human IgG) were incubated with
Cy5-labeled detector antibody (3 pM) at room temperature for
15-30 min. An aliquot of the sample (5 µL) was withdrawn,
immediately washed with PBS (200 µL), and stored at 4 °C in the
dark. The microparticles were separated from the reaction mixture
by centrifugation (12500g, 1 min), and an aliquot of the supernatant
(5 µL) was removed. A further aliquot of Cy5-labeled anti-IgG was
added, and the above procedures were repeated. The fluorescence
intensity of the samples was measured by fluorescence-activated
(25) Cavalli, G.; Banu, S.; Ranasinghe, R. T.; Broder, G. R.; Martins, H. F. P.;
Neylon, C.; Morgan, H.; Roach, P. L. J. Comb. Chem. 2007, 462-472.
Figure 1. (a) First-order (m ) (1) diffraction from a grating with pitch a1. (b) First-order diffraction from a grating with pitch a2, where a1> a2.
The two codes are distinguishable if the beam separation is greater than the beam width. (c) Four different SU-8 microparticles with their
corresponding diffraction patterns immersed in water. Insets show micrographs of the particles manufactured by optical lithography. The separations
between the first-order diffraction lines, which vary with the inverse of the grating period, are indicated. (d) Histogram showing the distribution
of observed diffraction angles from particles used in multiplexed assays.
Analytical Chemistry, Vol. 80, No. 6, March 15, 2008
Solution-phase data for the human IgG/anti-human IgG inter-
action were obtained by measuring fluorescence polarization. To
a solution of Cy5-labeled goat anti-human IgG (0.67 nM) in buffer
(10 mM NaH2PO4, 150 mM NaCl, 0.01% NaN3, pH 7.8) was added
human-IgG (final concentration 0-666 nM), and the reaction
mixture was incubated at room temperature in the dark for 16 h.
The fluorescence polarization of each of the samples was recorded
using a microplate reader (Safire2, Tecan, Switzerland, λex) 635
nm, λem) 666 nm).
Avidin-DN coated SU-8 microparticles (0.4 mg), suspended in
SSPE (5×) buffer (0.75 M NaCl, 50 mM NaH2PO4, 5 mM EDTA,
0.02% Tween-20, pH 7.0, 250 µL, giving a 0.1 µM concentration of
immobilized avidin-DN), were incubated with Cy5-labeled bioti-
nylated oligonucleotide, FP1 (2.5 nM) at room temperature for
15-30 min. An aliquot of the sample (10 µL) was withdrawn and
stored at 4 °C in the dark. The microparticles were separated from
the reaction mixture by centrifugation (12500g, 1 min), and an
aliquot of the supernatant (5 µL) was removed. A further aliquot
of FP1 was added, and the above procedures were repeated. The
fluorescence intensity of the samples was measured by fluorescence-
activated flow cytometry (FACS).
The data (Figure 2) were fitted to a ligand-binding function
(eq 2, where [Bound], [Free], and [Nonspecific] refer to concen-
trations of specifically bound, free, and nonspecifically bound Cy5-
labeled analyte) using commercially available software (SigmaPlot
9, Systat Inc., San Jose, CA), to determine the total number of
binding sites (Bmax) and Kd.
Kinetic Analysis of Avidin/Biotin Binding. Avidin-coated
SU-8 microparticles, (0.08 mg, bearing 5.7 pmol of avidin-DN) were
suspended in SSPE (5×) buffer (45.5 µL). Biotinylated Cy-5
labeled oligonucleotide (FP1) was added (0.3 nmol, final concen-
tration 6.5 nM). The suspension was agitated and aliquots (5 µL)
were removed periodically for immediate analysis, without wash-
ing. The fluorescence intensity of the samples was measured by
Solution-phase data were obtained by measuring fluorescence
polarization. A solution containing biotinylated Cy-5-labeled oli-
gonucleotide (FP1, 0.2 pmol, final concentration 1 nM), biotin (1.8
pmol, final concentration 9 nM), and avidin-DN (2 pmol, final
concentration 10 nM) in SSPE (5×) buffer (200 µL) was incubated
at room temperature. (Note: Solutions containing only FP1 and
avidin-DN were found to undergo self-quenching upon binding,
presumably due to the proximity of four fluorophores to each other
in the protein tetramer.) The fluorescence polarization was
recorded periodically using a microplate reader (Safire2, Tecan,
Switzerland, λex) 635 nm, λem) 666 nm).
Both data sets were fitted to eq 3 using commercially available
software (SigmaPlot 9, Systat Inc., San Jose, CA) in order to
determine the rate constant, kon.
Kinetic Analysis of Human IgG/Anti-Human IgG Binding
on Encoded Microparticles. Human IgG immobilized SU-8
microparticles (1 mg), suspended in storage buffer (100 µL), were
incubated with Cy5-labeled detector antibody (6.67 pmol, final
concentration 67 nM) at room temperature. After fixed time
intervals, samples (5 µL) were withdrawn, immediately washed
with storage buffer (200 µL), and stored at 4 °C in the dark. The
Figure 2. Saturation binding curves for Cy5-anti human/human IgG interaction in solution (a) on SU-8 microparticles, (b) and for avidin-DN/
Cy5-labeled biotinylated oligonucleotide FP1 on SU-8 particles (c). Time-course measurements of avidin/biotin binding in solution (d) and with
avidin-DN immobilized on SU-8 microparticles (e), with insets showing linear plots of the integrated rate equation. Panel f shows fluorescence
time-course measurement of human IgG/anti-human IgG binding on encoded microparticles.
Kd+ [Free]+ [Nonspecific][Free](2)
∂[Avidin - Biotin Complex]
kon[Free Avidin][Free Biotin] (3)
Analytical Chemistry, Vol. 80, No. 6, March 15, 2008
fluorescence intensity of the samples was measured by fluorescence-
Multiplexed DNA Hybridization Assay. Biotinylated oligo-
nucleotides P1 and P2 were captured onto encoded particles by
immobilized avidin in 5× SSPE buffer in separate reactions. A
mixture of the encoded particles underwent hybridization to C1
(1 pmol, 3 nM) in 5× SSPE buffer at room temperature for 40
min, followed by centrifugation and resuspension in 2 M aqueous
urea for 2 min. The microparticles were then washed twice with
5× SSPE buffer before analysis.
Mutliplexed Immunoassays. Polyclonal IgGs from human,
rabbit, guinea pig, or mouse IgG were captured onto encoded
particles by immobilized protein A in PBS in separate reactions.
Labeled anti-IgGs (Cy3- or Cy5-labeled, supplied by Abcam,
Cambridge, UK, 13 pmol, 260 nM) were added to the appropriate
mixture of the encoded particles in PBS at room temperature for
1 h. with occasional gentle agitation. The microparticles were then
washed twice with PBS before analysis.
Statistical Analysis of Fluorescence Measurements and
Estimate of Sensitivity of Multiplexed Assays. Student’s t-test
was used to calculate values of P for measured fluorescence
intensities for positive and negative data sets (using Microsoft
Excel). The sensitivity of assay for anti-human IgG was estimated
according to Kd, Bmax, and the number of required replicate
measurements as follows. The ratio of complexed to free protein
concentration is determined through eq 4:
[Free Analyte] ) Kd[Complex]/[Free Probe]
Assuming 20 particles are measured (P <0.0003 for all data sets
measured), then [Free Probe] + [Complex] ) 0.37 nM, and at
saturation [Complex] ) 0.37 nM. Hence [Complex] required for
a signal/noise ratio of five ) (5 ÷ 7.7) × 0.37 nM ) 0.24 nM, and
[Free Probe] ) 0.37 nM - 0.24 nM ) 0.13 nM. Substituting into
eq 4 gives [Free Analyte] ) 16.6 nM. Therefore, the total
concentration of analyte in the sample ) [Free Analyte] +
[Complex] ) 16.6 + 0.37 ) 16.97 nM.
RESULTS AND DISCUSSION
Particle Fabrication. The multiplexed encoding principle was
demonstrated using bar-shaped microparticles fabricated using
conventional photolithography from a photoactive epoxy resin (SU-
8).26This material has been previously shown to be suitable for
multistep solid-phase peptide and oligonucleotide synthesis.25
Micrometer-sized diffraction gratings were created using particles
with serrated edges, as show in Figure 1c. The advantage of this
method is that it involves a single photolithographic step. Large
numbers of particles (2.5 × 106particles/silicon wafer) can be
manufactured with identical chemical and physical properties
using this method. More complex and higher density gratings
can be made by nanoembossing.26,27The diffracted first-order
beams from the four codes used for multiplexed assays are
sufficiently separated in angular space to permit unambiguous
decoding, with a small overlap between codes 2 and 3 (Figure
1d). Data from particles with diffraction angles within the overlap
region were discarded.
Thermodynamic and Kinetic Characterization of Probes
Attached to Encoded Microparticles. One of the advantages
of particle-based suspension arrays over planar array formats is
that reaction kinetics and thermodynamics resemble those ob-
served in solution phase, enabling higher throughput and sensitiv-
ity.28In order to demonstrate these principles, we measured
binding thermodynamics and kinetics on our encoded micropar-
ticles via titrations of analyte binding and fluorescence time-course
We determined the equilibrium dissociation constants for key
molecular interactions by carrying out binding titrations (Figure
2a-c). These data confirm that both anti-IgG/IgG (Kdin solution
) 15 ( 6 nM; Kdon SU-8 microparticles ) 9 ( 3 nM) and avidin/
biotin (Kdin solution ) 1 × 10-6nM;29Kdon SU-8 microparticles
) 1 ( 0.3 nM) interactions retained high affinity when transferred
to the solid phase, though in the case of avidin/biotin, a significant
reduction in affinity was observed, which may be due to the use
of recombinant avidin-DN or the bulky oligonucleotide-biotin
conjugate. From Bmax, the number of analyte molecules bound to
each microparticle at saturation was calculated. For Cy5-labeled
anti-IgG, this value was ∼107molecules (or 17 amol) per particle,
while ∼0.8 × 107molecules (or 13 amol) of Cy5-labeled biotiny-
lated oligonucleotide were bound to each microparticle.
The kinetics of binding of Cy5-labeled biotinylated oligonucle-
otide to immobilized avidin-DN, and Cy5-labeled anti-human IgG
to human IgG were analyzed by carrying out fluorescence time-
course experiments. For the avidin-DN-biotinylated oligonucle-
otide interaction (Figure 2d), the data were fitted to eq 3 in order
to calculate kon, with the linear plots of the integrated rate equation
used to confirm the second-order nature of the reaction. The value
obtained on SU-8 particles (4.4 × 106M-1s-1) was very close to
that observed in solution for avidin-DN/biotinylated oligonucle-
otide FP1 (7.4 × 106M-1s-1) and within the range previously
reported for the native biotin-avidin interaction (kon) 5 × 105-
8.2 × 107M-1s-1).29For the kinetically more complex sandwich
system used for binding of labeled detection IgGs, we did not
attempt to parse individual rate constants but simply measured
the time taken for a system containing a 67 nM concentration of
analyte to reach equilibrium (Figure 2f), which was found to be
<10 min. We were therefore able to confirm that binding assays
on our encoded microparticles can be carried out in short time
Multiplexed Biomolecular Assays. Multiplexed analysis was
demonstrated using the four different photolithographically fab-
ricated bar codes (designated codes 1-4) shown in Figure 1c.
These codes can easily be differentiated with the aid of a light
microscope, but to increase throughput, the bar codes were read
using an automated recognition and analysis system that captured
the diffraction image on a CCD camera. Image processing software
identifies a particle from the captured image, assigns a direction
vector to the principal axis, and then performs a Fourier analysis
of the first-order spots to determine the particle code. This process
is performed at high speed, taking less than 1 ms.
A multiplexed DNA hybridization assay was performed using
sequences corresponding to a polymorphic locus (N1303K) on
the gene encoding the cystic fibrosis transmembrane conductance
(26) Banu, S.; Birtwell, S. W.; Galitonov, G. S.; Chen, Y.-F.; Zheludev, N. I.;
Morgan, H. J. Micromech. Microeng. 2007, 17, S116-S121.
(27) Guo, L. J. J. Phys. D 2004, 37, R123-R141.
(28) Finkel, N. H.; Lou, X.; Wang, C.; He, L. Anal. Chem. 2004, 76, 352A-
(29) Green, N. M. Biochem. J. 1963, 89, 585.
Analytical Chemistry, Vol. 80, No. 6, March 15, 2008
regulator protein (Figure 3a).30Two sets of encoded particles were
functionalized with avidin and used to capture biotinylated probe
oligonucleotides. Wild type sequence P2 was bound to code 3 and
the mutant sequence, P1, differing by a singe nucleotide, was
attached to code 2. Figure 3b shows a microscope image and
fluorescence image of the two particle types, demonstrating that
the perfectly matched sequence hybridizes but the mismatched
sequence does not. The observed fluorescence shows a degree
of inhomogeneity across the particle although the physical basis
for this observation has not yet been determined. The particles
were assayed using conventional flow cytometry, and the scatter
plot for the two different populations is shown in Figure 3c. Sorting
on the basis of the fluorescence signal, followed by decoding of
a representative sample of particles (50 from each fraction) showed
that the hybridized population consisted of 96% code 3, with the
unhybridized population B consisting of 96% code 2. These results
demonstrate that the use of diffractively encoded microparticles
permits discrimination of a single mismatch from a Watson-Crick
base pair within a short DNA duplex, confirming their applicability
to SNP genotyping applications.
To demonstrate the applicability of the technology to immu-
nodiagnostics, a multiplexed immunoassay was implemented for
the detection of IgGs. Two sets of encoded particles were
functionalized with two different IgGs using protein A capture.
The particles were incubated with complementary antibodies as
shown schematically in Figure 4a. The fluorescence images
(Figure 4b) show the selective binding of the differentially labeled
detection antibodies to the correct encoded particle. The labeled
particles were again analyzed using flow cytometry and sorted
into two populations on the basis of relative fluorescence (Figure
4c). A total of 50 particles from each of these two sorted
populations were then decoded, showing that particles from the
Cy5-labeled population (A) were correctly identified as code 2,
and the Cy3-labeled population (B) as code 4 with greater than
A prerequisite for reliable multiplexed immunoassays is the
demonstration of selective antigen-antibody binding against a
background of other potentially competing interactions. Therefore,
a small library consisting of four sets of different encoded particles
were each functionalized with a different antigen. Mixtures of
fluorescently labeled detection antibodies were added to aliquots
of the library (Figure 4d). For the experiments described above,
a two-step sorting/decoding process was used to read the code
and measure the fluorescence from each particle. Since this
approach is not amenable to high-throughput analysis, the mi-
croparticles from these quadruplexed experiments were dispersed
on a microscope slide and analyzed using a modified optical
microscope setup capable of reading diffraction and fluorescence
intensity. Manual reading of each particle using this apparatus
took ∼10-30 s. Binding of the complementary antibody gave
fluorescence intensities in the range 250 000-430 000 (au, cor-
rected for the quantum yield of the detection antibodies). Control
particles, whose corresponding anti-IgG was not present in the
sample, had intensities in the range 13 000-58 000 (au), demon-
strating very little cross-reaction.
Replicate Measurements within Multiplexed Assays and
Analyte Sensitivity. The throughput and sensitivity of micropar-
ticle-based assays is determined in practice by the detectable
amount of analyte per particle and the number of replicate samples
that must be measured to satisfy a set confidence level in the
results. The latter in turn is determined by the variation of signal
(in this case fluorescence) between particles of the same type that
have been subject to the same assay conditions. We analyzed data
(30) Osborne, L.; Santis, G.; Schwarz, M.; Klinger, K.; Dork, T.; McIntosh, I.;
Schwartz, M.; Nunes, V.; Macek, M.; Reiss, J.; Highsmith, W. E.; McMahon,
R.; Novelli, G.; Malik, N.; Burger, J.; Anvret, M.; Wallace, A.; Williams, C.;
Mathew, C.; Rozen, R.; Graham, C.; Gasparini, P.; Bal, J.; Cassiman, J. J.;
Balassopoulou, A.; Davidow, L.; Raskin, S.; Kalaydjieva, L.; Kerem, B.;
Richards, S.; Simonbouy, B.; Super, M.; Wulbrand, U.; Keston, M.; Estivill,
X.; Vavrova, V.; Friedman, K. J.; Barton, D.; Dallapiccola, B.; Stuhrmann,
M.; Beards, F.; Hill, A. J. M.; Pignatti, P. F.; Cuppens, H.; Angelicheva, D.;
Tummler, B.; Brock, D. J. H.; Casals, T.; Macek, M.; Schmidtke, J.; Magee,
A. C.; Bonizzato, A.; Deboeck, C.; Kuffardjieva, A.; Hodson, M.; Knight, R.
A. Hum. Genet. 1992, 89, 653-658.
Figure 3. Assay used to differentiate oligonucleotides differing by a single nucleotide. (a) Two different oligonucleotides (P1 and P2) that vary
only at a single position are bound to encoded microparticles. After hybridization to a third oligonucleotide (C1) that is a perfect match for one
sequence, the particles were washed in 2 M urea and then imaged. (b) White light microscope image (left panel) and fluorescence image (right
panel). The particles bearing the perfectly matched sequence fluoresce and those with the single mismatch do not. (c) Scatter plot of the two
populations (A and B). These were sorted by FACS on the basis of Cy5 fluorescence and the codes of 50 particles in each population read.
Analytical Chemistry, Vol. 80, No. 6, March 15, 2008
from the quadruplex immunoassay and single base mismatch
DNA hybridization assay, since these assays are the most complex
and have the highest probability of cross-reactions and high
background fluorescence, allowing the most realistic estimation
of these parameters. Mean fluorescence intensities with standard
deviations for representative positive (complementary anti-IgG or
oligonucleotide present) and negative (complementary anti-IgG
or oligonucleotide absent) data sets are shown in Figure 5a and
c. Though the standard deviations of positive and negative
populations are well separated, the coefficients of variance (47-
60%) are larger than those typical of optimized ELISAs (5-10%).31
A possible contribution to these relatively large coefficients of
variance may be the observed inhomogeneity of the fluorescence
(Figures 3b and 4b). There is therefore a nonzero probability of
any given particle within either positive or negative population
giving a fluorescent signal that falls within the range attributed
to the other population, i.e., generating a false positive or false
Student’s t-test was used to compare the difference between
“negative” and “positive” data sets from the quadruplexed immu-
noassay and DNA hybridization assay. Cumulative P values from
three positive data sets are plotted against number of particles
read, Figure 5b and d. As expected, as more particles are read,
the value of P falls, indicating greater confidence in the difference
between data sets. After reading 11 particles, the probability of
any of the positive data sets being indistinguishable from the
negative data set is <0.01, and after reading 16 particles, the
probability is <0.0003. Therefore, for multiplexed platforms where
thousands of molecular interactions are assayed, measurement
of 15-20 replicates is necessary to satisfy a confidence level of
>99.97%. This level of degeneracy is greater than that necessary
for ELISAs, but is comparable to that used in Illumina BeadArrays
where ∼30 replicate beads are used for each oligonucleotide to
enable averaging and thorough statistical analysis of results.32
From Bmax, the number of analyte molecules bound to each
microparticle at saturation can be calculated. For Cy5-labeled anti-
IgG, this was ∼107molecules (or 17 amol) per particle; a similar
number (∼0.8 × 107molecules, or 13 amol) of Cy5-labeled
biotinylated oligonucleotide were bound to each microparticle.
Given the number of replicate particles required for analysis, we
can estimate the amount of bound analyte required to generate a
positive signal. The maximum binding capacity of a single particle
is ∼107molecules (17 amol); therefore, 20 particles will be
saturated by 340 amol of bound (labeled) IgG or DNA analyte.
The sensitivity of individual assays will be determined by the
signal/noise ratio (SNR) obtained for the analyte and its affinity
for the immobilized complementary probe. For example, Figure
5a shows that the human IgG/anti-human IgG interaction, has a
SNR (at saturation) of 7.7, with Kd) 9 ( 3 nM. Assuming that a
SNR of 5 is sufficient for detection, and that the assay is carried
out in a volume of 1 µL, eq 4 can be used to determine the total
concentration of anti-human IgG required to produce a signal. This
predicts that the total concentration of analyte in the sample
is16.97 nM, giving an estimate of sensitivity of approximately 17
fmol, or 1010molecules. This is a relatively conservative estimate
of sensitivity; if a cutoff value equal to twice background fluores-
cence is used, as for other bead-based assays,33the sensitivity
increases to a detection limit of 5.35 fmol (in a 1-µL volume), or
3.2 × 109molecules. To demonstrate this experimentally, we
(31) de Jager, W.; Rijkers, G. T. Methods 2006, 38, 294-303.
(32) Novak, J. P.; Miller, M. C.; Bell, D. A. Biol. Direct In press. DOI 10.1186/
(33) Dunbar, S. A.; Vander Zee, C. A.; Oliver, K. G.; Karem, K. L.; Jacobson, J.
W. J. Microbiol. Methods 2003, 53, 245-252.
(34) Marie, R.; Schmid, S.; Johansson, A.; Ejsing, L. E.; Nordstrom, M.; Hafliger,
D.; Christensen, C. B. V.; Boisen, A.; Dufva, M. Biosens. Bioelectron. 2006,
Figure 4. Multiplexed immunoassays with encoded microparticles.
(a) Rabbit and human antibodies are attached to encoded micropar-
ticles, which are then mixed with detection antibodies in a single
reaction. Detection antibodies were Cy3-labeled anti-human and Cy5-
labeled anti-rabbit IgG, respectively. (b) Microscope images of two
particles from the multiplex reaction, imaged with white light (top),
Cy3 fluorescence (middle) and Cy5 fluorescence (bottom). The faint
fluorescence from code 2 in the second image (Cy3 filter) is due to
the intrinsic fluorescence of the SU-8 polymer34and not to cross-
reaction of the Cy3-labeled antibody. (c) Scatter plot for two popula-
tions of particles, which were then sorted on the basis of Cy3 and
Cy5 fluorescence and the codes of 50 particles in each population
read. Population A: (bar code 2, 49 (98%); bar code 4, 0 (0%);
unreadable/incorrectly identified, 1 (2%). Population B: (bar code 2,
0 (0%); bar code 4, 50 (100%); unreadable/incorrectly identified, 0
(0%). (d) Results from the quadruplex immunoassay showing mean
particle fluorescence intensity (n ) 15-25) for a small library of four
different IgG functionalized bar codes after addition of Cy5-labeled
anti-IgGs to an aliquot of the library. Sample A: only anti-mouse IgG
was added, binding selectively to code 3. The mean fluorescence
following binding (and washing) of ∼100 particles is shown (with
standard error). Sample B: anti-guinea pig, anti-mouse, and anti-
rabbit IgG were all added to an aliquot of the codes, giving a
fluorescent “readout” as for sample A. Sample C: all four comple-
mentary fluorescently labeled antibodies were added to the sample
of four codes.
Analytical Chemistry, Vol. 80, No. 6, March 15, 2008
attempted to detect 5.35 fmol of IgG in an immunoassay. The IgG
was successfully detected with an average SNR of 4 (for a sample
of 20 particles, data shown in Supporting Information Figure S-1).
We have demonstrated a new method for encoding biomo-
lecular probes, based on fabricating micrometer-sized diffraction
gratings. The technology has the potential to provide vast numbers
of unique codes, up to 1018for a 50-µm particle using 5 times
superimposed 2-D gratings.23The particles can be rapidly decoded
(in <1 ms) by automated software. The encoded particles are
fabricated using basic photolithography and are made from a
functional polymer (SU-8) that is compatible with multistep
chemical synthesis25and multiplexed assays. Statistical analysis
of the signal variation from particle to particle in assays indicates
that 15-20 replicate measurements are required to achieve a
>99.97% confidence in the discrimination between positive and
negative samples. Biochemical assays carried out on these
microparticles exhibit favorable binding kinetics and thermody-
namics, allowing small amounts (<20 fmol in 1-µL volume) of
target analyte to be detected, thus enabling massively parallel
determination of molecular interactions with high sensitivity and
accuracy. While manual operation of the combined diffraction/
fluorescence microscope described herein requires 10-30 s to
analyze each particle, the throughput could be greatly increased
by coupling decoding optical hardware to a fluidic system. Since
analysis of diffraction can be accomplished in an automated
manner on the millisecond time scale, we anticipate throughput
comparable to conventional flow cytometers. We envisage using
the same devices for directed sorting of particles during multistep
chemical synthesis to provide an integrated system for the high-
speed manufacture of encoded libraries of molecules (oligonucle-
otides, peptides, etc.) and analysis of their molecular interactions.
This project was supported by Research Councils UK through
the Basic Technology Programme. We also thank the cleanroom
staff at EPFL, Switzerland, for their assistance with fabrication
and Andrew Whitton for technical assistance. G.R.B., R.T.R. and
J.K.S. contributed equally to this work.
SUPPORTING INFORMATION AVAILABLE
Detailed experimental protocols for functionalization of SU-8
microparticles with oligonucleotides/antibodies and detection of
femtomole quantities of anti-human IgG, as well as schematic
representation of the modified microscope setup for simultaneous
measurement of diffraction and fluorescence from encoded mi-
croparticles. This material is available free of charge via the
Internet at http://pubs.acs.org.
November 21, 2007.
for review September 4,2007. Accepted
Figure 5. Mean fluorescence intensities with standard deviations for representative positive (complementary anti-IgG or labeled oligonucleotide
present) and negative (complementary anti-IgG or labeled oligonucleotide absent) particle sets functionalized with different probes, (a) and (c),
and cumulative P values from positive data sets plotted against read number, (b) and (d). Data from particles functionalized with human IgG
(blue squares), guinea pig IgG (green triangles), and rabbit IgG (pink circles) are compared in (b), while data from P2- and P1-functionalized
particles (differing by a single nucleotide) are compared in (d). The 0.03% probability that the difference in mean fluorescence of positive and
negative populations could arise by chance is shown by the horizontal dotted line in (b) and (d).
Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

domain: chemistry


But if there was one thing a slew of strong series confirmed it’s that we’ve officially entered the golden age of streaming.
Starting the year off strong, by proving we DID in fact need a series adaption of the beloved movie classics, Mr and Mrs Smith and One Day, 2024 has not disappointed.
From the long-awaited return of Bridgerton and The Bear for their third respective seasons to the highly-anticipated arrivals of Star Wars spin-off Acolyte and the glamorous 1960’s drama, Palm Royale.
So add these upcoming series to your binge-list and indulge your eyes, ears and soul.
Featuring a star-studded line up, including Kristen Wiig, Laura Dern, Carol Burnett, Alison Janney, Kaia Gerber and Ricky Martin, Palm Royale follows the story of an ambitious woman as she muscles her way into the elite world of Miami high society in the 60s.
Release date: 20 March on Apple TV.
A conspiracy theory thriller, Manhunt navigates the aftermath of the first American presidential assassination and the fight to preserve and protect the ideals of Abraham Lincoln’s Reconstruction plans.
Release date: 15 March on Apple TV.
Bridgerton Season 3
Bodices, balls, buzz – oh my! The return of the beloved period dramas, Bridgerton is fast approaching. The third season will focus on the love story between Penelope (also knows as Lady Whistledown) and Colin.
Release date: Part 1 — 16 May, Part 2 — 13 June on Netflix.
Set against the backdrop of post-apocalyptic L.A., Prime Video’s buzzy new series Fallout is anything but bleak. With Ella Purnell (best known for Yellow Jackets) bringing wit and humour to a dire landscape of a post-nuclear war wasteland the series combines ‘50s Americana style with corrosive comedy and action-packed drama.
Release date: 12 April on Prime Video.
With more mystery shrouding the series that the inner workings of Mr Ripley himself, Andrew Scott’s small-screen adaptation of the infamous 1999 film starring Gwyneth Paltrow, Matt Damon and Jude Law, has already created some serious hype.
Release date: 4 April on Netflix.
The Bear Season 3
Yes Chef! The fast-paced drama starring the internet’s man of the moment, Jeremey Allan White (Carmy) is officially returning. After leaving us on an emotional and sensory high last season, we’re expecting plenty of fires to be put out as the restaurant officially opens.
Release date: June TBA on Disney Plus.
You Season 5
Hello, you! You’re favourite romantic killer, Joe Goldberg is back, this time for his final chapter. After last season saw Joe finally hind his happy ending, the final chapter will reportedly see a familiar face returning to haunt Joe.
Release date: TBA on Netflix.


text:
Title: Exploring the Dr. Glen Brown’s Market Expected Moves Hypothesis: A New Framework for Optimized Risk Management in Financial Markets
This paper investigates the innovative approach proposed by Dr. Glen Brown’s Market Expected Moves Hypothesis (MEMH), a quantitatively-driven model for managing risk and potential rewards in financial trading. The hypothesis presents a novel method of leveraging Dynamic Adaptive ATR Trailing Stops (DAATS) to predict and manage the potential extent of price movements of financial assets. We elaborate the theoretical underpinnings of MEMH, explore its potential applications, and suggest areas for future research.
In the rapidly evolving and often volatile financial markets, effective risk management is paramount. Dr. Glen Brown’s MEMH presents an innovative approach to this challenge, combining insights from historical volatility data and statistical probabilities to inform trading strategies.
2. Theoretical Background:
The MEMH proposes that a financial asset is likely to reach 75% of its DAATS 85% of the time, effectively providing an exit strategy that balances risk and reward. Further, MEMH suggests that the optimal break-even point for any financial asset is this same 75% of DAATS, guiding traders to adjust stop losses at a statistically informed level.
3. MEMH and Dynamic Adaptive ATR Trailing Stops:
The DAATS concept underpinning MEMH is a refinement of the standard Average True Range (ATR) volatility measure. It provides adaptive, timeframe-specific trailing stops that reflect market conditions more accurately than fixed-value stops. By integrating DAATS into MEMH, Dr. Brown harnesses a deeper understanding of market volatility to optimize trading outcomes.
4. Practical Applications in Financial Markets:
The MEMH offers clear applications for individual and institutional traders seeking to improve their risk management strategies. Its statistically-derived exit points and break-even targets could contribute to more robust decision-making processes in various trading contexts.
Furthermore, the MEMH provides guidance on when to adjust stop losses, potentially limiting loss exposure and locking in profits more effectively. The use of a break-even stop informed by the MEMH allows traders to protect their positions from loss while leaving room for further potential gains.
5. Empirical Validation and Future Research:
While the MEMH provides a compelling theoretical framework, further empirical testing and validation are necessary to ascertain its efficacy across different market conditions and asset classes. Future research could focus on backtesting the hypothesis using historical data and testing it in real-time trading environments.
The Dr. Glen Brown’s Market Expected Moves Hypothesis presents a sophisticated, data-driven approach to risk management in financial trading. By integrating insights from market volatility and empirical statistical probabilities, MEMH offers a promising framework for optimizing trading outcomes. As with any financial model, the efficacy of MEMH depends on its integration into broader trading strategies and its alignment with individual risk tolerance and trading goals.
Keywords: Dr. Glen Brown, Market Expected Moves Hypothesis, MEMH, Dynamic Adaptive ATR Trailing Stops, DAATS, risk management, financial markets, trading strategies.

domain: literature


text:
Oils may be partially hydrogenated to produce various ingredient oils. Lightly hydrogenated oils have very similar physical characteristics to regular soy oil but are more resistant to becoming rancid. Margarine oils need to be mostly solid at 32 °C, so that the margarine does not melt in warm rooms, yet it needs to be completely liquid at 37 °C , so that it doesn’t leave a “lardy” taste in the mouth.
Hardening vegetable oil is done by raising a blend of vegetable oil and a catalyst in near vacuum to very high temperatures and introducing hydrogen. This causes the carbon atoms of the oil to break double-bonds with other carbons, each carbon forming a new single-bond with a hydrogen atom. Adding these hydrogen atoms to the oil makes it more solid, raises the smoke point, and makes the oil more stable.


The National Highway Traffic Safety Administration is starting to announce the expanded Takata airbag inflator recalls. FCA has over 5 million vehicles affected worldwide, and BMW has over 420,000 in the US. Honda's expansion includes 350,000 more Civics and Accords.
The BMW X2 reportedly has a green light for production, possibly for the second half of 2017. The compact, coupe-like crossover is closely based on the next-gen, front-wheel drive X1, and shares its drivetrains and platform.
BMW is building a concept that pays tribute to the original 3.0 CSL, and it's being unveiled at the Concorso d'Eleganza Villa d'Este on May 22. Judging by the teaser image pictured here, it's going to be hot.
The BMW M2 is reportedly going into production in November with 365 horsepower on tap from a tuned version of the N55 3.0-liter inline-six engine. The color palette is rumored to include just four shades.
The BMW 3 Series is getting a refresh for the 2016 model year, although you might not be able to tell by looking at it. Underneath the slight styling tweaks is a revised suspension and automatic gearbox. Plus, there are two new models: the 340i and 330e plug-in hybrid.


Deer Island extended, providing new fish habitat
Published: June 5,2012
BILOXI — The Mississippi Department of Marine Resources has finished a project that extends the eastern end of Deer Island by about three acres.
The agency says in a news release Monday that 1,500 cubic yards of limestone were added in May to the nearshore reef for Deer Island North.
Just south of Biloxi, Deer Island is the closest island to the Mississippi Gulf Coast.
Kerwin Cuevas, the agency’s artificial reef bureau director, says enhancing the reef provides additional fish habitat. He also says in a few years, it could be used as an oyster tonging reef for commercial harvest.
To sign up for Mississippi Business Daily Updates, click here.
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text:
Essay on the May 4th Movement
Geoffrey Lee 6. 16 Macedonia History Essay To what extent do you agree with the assertion that May 4th 1919 was one of the most significant dates in Chinese history. The May 4th movement in 1919 was a significant date in Chinese history to a very large extent. This essay will attempt to justify this claim by explaining the short-term effects of the M4M and more importantly the long-term effects. The year 1919 is a significant year as it was the year right after World War 1 (WW1) had ended; hence many war-settlement treaties were signed. One of these would be the Treaty of Versailles (TOV) which involved China.
The M4M was a movement that had begun from the rise of sentiments of democracy and nationalism. Previously, China had been ruled by a single ruler where traditional culture and Confucianism dominated the nation. However after WW1 and the influx of western ideas and ideologies, thus many youth in China begin to call for a demolition of the previous culture and Confucianism, and to replace it with a new culture and direction. This movement was headed by large forms of mass advertising through the use of the New Youth and many other magazines.
This increase of national sentiments led to the Chinese demanding that their government to oppose the Japanese 21 demands on Yuan Shikai’s government and it also saw the emergence of the new merchants and labour class. With all these demonstrations and national sentiments, China was in a period of disorder and lawlessness, which was similar to that during the warlord era in the 1800s. In the short-term, the M4M was a significant date in Chinese history to a small extent as it resulted in many strikes and demonstrations throughout China to occur.
Students demonstrated in the capital of Beijing while many other occupants such as shopkeepers, industrial workers and other urban employees joined in the strikes and demonstrated in other key cities in China such as in Shanghai. The demonstration also saw a nationwide boycott of Japanese goods in an attempt to show Yuan Shikai to reject the Japanese demands to make China a Japanese protectorate. Also, the government of China was forced into rejecting the terms of the TOV which angered the West. This resulted in massive violence throughout the nation which did the
Chinese no good and it aggravated ties between China and the West. In the long-term, the M4M was a significant date in Chinese history to a very large extent as it resulted not only in many cultural changes, but most importantly; it changed the political scene in China. The M4M gave birth to political activism where the people called for the end of a single ruler, but instead to have a democratic government. This surge of political activism was propelled by the education of the youths who believed that they would transform China.
The youths emphasized heavily on the destruction of China’s traditional culture which was based on Confucianism, and they assumed that Western thought and civilisation was the way forward. This led to the reorganisation of the Guomindang (GMD) which saw Sun Yatsen calling on the Soviet Union. This also led to the founding of the Chinese Communist Party (CCP). The CCP was founded as a result of the increased appeal of Marxism-Leninism, which proved to be very successful in the Soviet Union.
These 2 political groups would later take over the whole of the Chinese political scene and would later oppose one another in their attempt to dominate the Chinese government, which would result in massive killings of one another. However these 2 parties would combine forces during the World War 2 (WW2) period to fight off the Japanese imperialism which ensured that China would not fall into the hands of the Japanese. Hence this proved that the M4M was indeed a significant date in the history of China to the largest extent.
In conclusion, it can be agreed that the M4M was one of the most significant dates in the history of China simply due to the fact that it was through the M4M that both the GMD underwent a massive reform, and at the same time, the birth of the CCP was witnessed. The combination of these 2 factors proved to be significant as without the formation of either party, China would have suffered much more detrimentally in WW2 as it was only through the surge of nationalism of the Chinese, and the combined forces of the GMD and the CCO that China managed to fight off the Japanese.

domain: sociology


text:
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Begin the journey of seeing physiologic birth with a free class that will introduce you to the 3 P's of physiologic birth that help my students and clients avoid tearing in only 15 minutes so you will know why you can trust your body to give birth.
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