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When purchasing a new car, one considers many things: is it reliable? How is the gas mileage? How much does it cost? What will the upkeep be?
However, many would-be car buyers are failing to factor in the larges expense associated with purchasing a new ride: depreciation, according to AAA.
AAA's "Your Driving Costs" study found that the largest expense associated with purchasing a new vehicle is depreciation, which accounts for nearly 40 percent of the cost of owning a new car - more than $3,000 a year - and is influenced by a number of factors.
"New vehicles offer the latest designs, cutting-edge technologies and warranties that offer peace of mind," AAA Managing Director of Automotive Engineering and Repair John Nielsen noted. "But, car owners that like to change vehicles frequently should be thinking about the resale value – not just the purchase price – when choosing their next ride."
According to AAA, buyers tend to only give priority to purchase price and monthly payment when choosing a new car, sometimes selecting a vehicle based on the best deal available. The length of car ownership, however, is of equal importance.
"Consumers who plan to keep a vehicle for only a few years should be cautious of deep discounts and incentives offered by automakers and dealers," AAA officials noted. "These are often designed to sell less popular models and directly influence depreciation. Low down payments and extended finance terms can also have a similar effect. Stretching a car loan over five, six or even seven years may be an effective way to lower payments, but owners may quickly find themselves owing more than the vehicle is worth."
The 2018 "Your Driving Costs" study found the average cost to own and operate a new vehicle this year will be $8,849 per year, which was calculated based on the cost of fuel, maintenance, repairs, insurance, taxes, loan interest and depreciation.
Small sedans have the lowest annual cost ($6,777), while pickup trucks come in at the highest annual cost to own a new vehicle ($10, 215).
"While the latest technology, style and options make them attractive to car buyers, a new car may not be the most economical choice for some buyers," AAA officials stated. Vehicle owners looking for alternatives to new car ownership or ways to minimize their operating costs can consider the following tips from AAA:
Click here to sign up for Daily Voice's free daily emails and news alerts.


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By TODD RICHMOND
Associated Press
MADISON, Wis. (AP) – Wisconsin Republicans inched closer Wednesday to passing their divisive mining measure, moving the proposal to the state Senate floor for a vote and triggering hours of debate with minority Democrats who contend the measure would open the door to devastating pollution.
Powerless to stop the bill, Democrats chose to stall. They started to rail against the measure around midday and still were at in the late afternoon. It was unclear when the Senate would vote on the measure.
Republicans hold an 18-15 edge in the chamber, making the bill's passage all but inevitable. It would next go to the Republican-controlled Assembly, where a vote is scheduled for next week. From there it would move to GOP Gov. Scott Walker.
Republicans have been working for nearly two years to help Gogebic Taconite dig an open-pit mine in the Penokee Hills just south of Lake Superior. Their bill would make sweeping changes to the state's mining rules to clear the company's regulatory path.
Republicans insist the measure would help the company create hundreds of jobs at the mine and pave the way for thousands more around the state. Walker, eager to deliver on job creation promises, wants to get the bill on his desk.
Democrats and conservationists, though, maintain the company's job promises are exaggerated. They say the bill loosens environmental protections and would allow the mine to pollute one of the state's last pristine areas.
Senate Democrats on Wednesday sounded the same themes they have for months. They complained the bill would clear the way for mining waste to contaminate area waters and rob local governments of mining tax revenue. They also ripped Republicans for not holding a public hearing in northwestern Wisconsin, blasted the measure as a sweetheart deal for Gogebic Taconite and predicted it would end up in court.
"The company's intent can be summed up in five words. Give us what we want," said Sen. Bob Jauch, D-Poplar, whose district includes the mine site. "Our job is not to be Santa Claus for a mining company."
The bill's author, Sen. Tom Tiffany, R-Hazelhurst, insisted the measure doesn't compromise the environment. He pointed out mining issues have been thoroughly vetted in a dozen hearings. Wary of prolonging the proceedings, Republicans said little else.
"There are so many misrepresentations, but I don't feel like giving a 25-minute speech," Sen. Glenn Grothman, R-West Bend, told the chamber.
Under the bill, the state Department of Natural Resources would have up to 480 days to make a permitting decision. Right now, the process is open-ended. The public couldn't challenge a DNR permit decision until after it was made, and damage a mine might cause to wetlands would be presumed necessary. Applicants would have to submit a plan to compensate for damage, however, which would include a proposal for creating up to an acre and a half of new wetlands for every acre impacted.
A mining company's permit application fees would be capped at $2 million plus the DNR's expenses for delineating wetland boundaries. Tax on a company's revenue would be split 60-40 between local governments and the state. Current law imposes no cap on application fees and calls for all taxes on revenue to go to local governments to offset mining impacts.
The bill also exempts mining companies from the state's $7 per ton recycling fee on waste materials. That exemption could result in a potential loss of up to $171 million annually the state would collect from Gogebic Taconite for environmental protection programs under current law, according to the Legislative Fiscal Bureau.
One of the bill's staunchest opponents is the Bad River Band of Lake Superior Chippewa. The tribe's reservation lies just north of the mine site, and members fear the mine would pollute the reservation's water and destroy their wild rice beds.
Tribal members gathered in a hearing room before the Senate session began in a show of opposition, pinning felt water-droplet cut-outs to their clothing. As debate began in the Senate, they joined with the usual daily group of about 100 protesters two floors down in the Capitol to sing songs, play drums, dance and hold anti-mine signs.
"It's a sad day," said Annie Maday, a 60-year-old Bad River tribal council member. "Scary is not the word for it. It's devastating. They're going to destroy my home."

subset: commoncrawl


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Kamaaina Loan – Maui Hawaii
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"Three crowns in James's charter": An Irish poem for the new king
This Sunday is March 24, the date in 1603 when Queen Elizabeth I died, and James VI of Scotland became king of England. When James succeeded Elizabeth to the English monarchy, he also inherited the crown of Ireland.
Although a high king of Ireland was both a legal and historical reality, the concept of an Irish crown was innovative in this period. The bardic poet Fearghal Óg Mac an Bhaird may be the first to have articulated the notion of 'the crown of Ireland' as part of a triple monarchy, which he did in his famous poem "Tri coróna i gcairt Shéamais" (Three crowns in James's charter), an inaugural poem celebrating James's accession to the triple monarchy of England, Scotland, and Ireland.
The current Folger exhibition, Nobility and Newcomers in Renaissance Ireland, which I curated with Thomas Herron, Associate Professor of English at East Carolina University, includes a facsimile manuscript of Mac an Bhaird's poem, in the original Irish language. The poem is one of several Irish-language works on display. A fundamental, and immensely interesting, part of the exhibition, these works have been a way for us to show how the Irish language itself was a powerful cultural and social force in the world of Renaissance Ireland. You can hear a spoken passage from the poem, in Irish, in this online audio stop from our exhibition audio tour (the passage from the poem begins after my spoken introduction) and see an image of the poem here as well.
The poem's fame derives in part from its seeming curiosity: why would a Gaelic Irishman welcome a "British" king? One answer is that Gaelic intellectuals were not absolutely averse to political connection to the monarchy in London, and currying favor with the new king was simply good politics.
On a more basic level, the bardic class hoped to enjoy greater protection and rights under a Scottish king than they had under an English queen: Elizabeth may have had an interest in the Irish language, but she did next to nothing to arrest the destruction of Gaelic culture. James, by contrast, had successfully ruled Scotland for decades and had managed his Gaelic and Catholic subjects with relative tolerance. Many Irish—bards, lords, and even churchmen—held out hope that his reign would usher in better days.
Mac an Bhaird's poem details the grounds of James's claim for all three kingdoms. In the case of Ireland, legitimacy derived from blood (Scottish kings having been descended of Irish Gaels) rather than conquest. In traditional motif, then, Ireland was portrayed in the poem as the feminized 'spouse' of the rightful king, a union that would bring peace and plenty to the land.
Great hopes, however, were quickly dashed. A mere six years later, in 1609, James's government would commence the largest and most ambitious of English/British colonial schemes in Ireland, the Plantation of Ulster, and thus the repeopling of Ireland's northern province with English and Scottish settlers.
Brendan Kane is Associate Professor of History at the University of Connecticut, Associate Director of the University of Connecticut Humanities Institute, and the curator, with Thomas Herron, of the Folger exhibition Nobility and Newcomers in Renaissance Ireland, January 19 to May 19, 2013.


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Chemigation and fertigation systems can be individual but from where we stand the principles are the same every time: Safe, precise dosing and a uniform mix in the water line can be felt on the bottom line. With Grundfos dosing and metering equipment, efficient and effective use of chemicals is made simple.
For handling any chemical, Grundfos has pump technology that is easy to adjust and maintain. Our corrosion-resistant dosing pumps are ideal for injecting nutrients and pesticides as well as acid for the removal of organic material. They dose concentrated and ready-to-use chemicals evenly and precisely into the line, and will automatically adjust dosages in response to changing flow.
Out in the field, the crop will receive the desired and uniform concentration of nutrient and pest-control chemical. The right amount in the right place at the right time. We can also advise on relevant safety procedures, back-flow prevention, etc. in relation to local regulatory constraints.


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Let me just say that I like the Penderwicks best when they are on vacation. Jeanne Birdsall's first book, The Penderwicks, tracked four kids and a big sloppy dog named Hound through a summer holiday on a country estate. Plenty of misadventures and great adventures behind the hedgerows in a very charming, old-fashioned story with wonderful characters, terrific humor and the sort of British country setting (although the tale is set in New England) that enchanted me in the books I read as a child.
Birdsall's second in the series follows them home to Gardam Street in Massachusetts and it is sweet and simple but not nearly as much fun as the summer escapades. In book three, The Penderwicks at Point Mouette, the three youngest girls, Skye, Jane and Batty, are vacationing in Maine while their eldest sister Rosalind gets a much needed break from minding all of them and is off to the Jersey Shore with a friend. Their newly-married father, stepmother and her young son are on an academic business trip to England and the girls are in the care of second-eldest Penderwick, Skye, and their Aunt Claire.
This makes Skye all kinds of nervous because Rosalind has always cared for Batty, the baby, since their mother died and keeps the rest of the team in line. Skye isn't sure she has the right stuff for the job. Their best friend Jeffrey, collected on the first summer jaunt, joins them for this one and mild disasters accumulate faster than shells on the strip of beach outside their vacation cottage. Skye studies astrophysics and black holes and practices soccer moves. Jane, next in line, is a serious writer struggling with a bad case of writer's block over her latest Sabrina Starr novel that deals with the perils of falling in love. Small problem—the author is eleven and lacks the requisite research to get past the opening sentence. Batty (Elizabeth) has given up the fairy wings she once wore everywhere but still drags her stuffed elephants along for the trip. She must wear a bright orange life preserver whenever she gets anywhere NEAR the water, which hampers her style.
Jeffrey is happy to have eluded his difficult mother's summer plans and sets about having adventures with the girls. A neighbor next door is a musician and invites musical prodigy Jeffrey to use his grand piano for practice. Five-year-old Batty, who trails Jeffrey everywhere and intends to marry him when she grows up, displays a surprising musical talent. Love rears its unpredictable head among the lobster rolls. Aunt Claire winds up on crutches. The girls find out that errant golf balls from the nearby club are a boon to piggy banks. And a long lost parent surfaces, causing havoc for kids and grown-ups alike.
I like the Penderwicks books. They are innocent and fun; the writing is good; the characters are real people with lots of interesting quirks; the adventures are lively. I know a few young readers who devoured the first book and still like the series, even though they are older now and the books are middle grade level. Then again, middle grade is one of my favorite book categories and I am far from the target audience. Penderwicks at home—not so fascinating. Penderwicks up to summer mischief—a recipe for a delightful read.
The Penderwicks at Point Mouette Jeanne Birdsall | Alfred A. Knopf 2011


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Discussion in 'BMW' started by rwhgme, Dec 23, 2009.
title says it all
You know we have BMW Ombudsman that can help with these type of situations. You might want to call the National Office for more details.
Sorry to hear that. What is going on, what is the problem?
Some problems it might appear, with some BMW Performance Products. A thread with the title "2010 335i BMW factory hp mod redux" details it.
I have escalated the situation to Group 1 Automotive. The possible new answer to this situation is that the muffler has a vacuum operated valve in it. It either isn't supposed to have it or it does and needs to be disabled(I think). I should know something by tomorrow which will be day 30 and 12. I really do hope that I will be enjoying the "Ultimate driving experience" soon. I have owned a number of American performance cars in my 54 years but this is what happens when we let our cars get smarter that the technicians who repair them...makes me wish that the techs were old school mechanics who can think outside of the computer box.
Okay, back up here for a second.
I have to admit that this statement is not fair to say. Working with computers myself, I have heard my fair share of customers comlaining about techs not being able to immediately diagnose a problem. It isn't fair to blame the technicians for not being able to diagnose a vehicle that is as complex and advanced as yours. There are a number of things that could go wrong, and like when I'm diagnosing a computer, I won't know for sure what it is that's wrong until I test certain parts. Some things you just won't know until you take a look at it and test it, so once again,thinking they're incompetant for not magically knowing what the problem is the first time is not fair to them or the engineers that designed the vehicles. BMW-trained and certified techs are very higly trained and very qualified to perform work on these advanced automobiles, so please let them do their jobs and not bash them for not yet having figured out a potentially complicated and/or elusive [intermittent] problem.
I'd be annoyed as well if I were you, having just bought an expensive car, but please do know where to draw the line as far as insulting their intelligence goes.
Thank you.
I am not certain that anything I said implied that any technician was incompetent. What I said was that MY cars' problem was not related to the computer as much as it was that a PERSON designed this performance modification at BMW and was not available to brainstorm a solution like we used to be able to do. Computers are a wonderful advancement to our way of life, but, at times, they can also absolve you of guilt(IT MUST BE A COMPUTER GLITCH) or a logical, human derived answer. In a car, sometimes we need to rely on an old school answer to a piece of machinery that in one form or another has been around for more than 100 years. With that said I have my car back today and I hope to only post good news stories as tomorrow is a day reserved for the TRUE good news for those who believe in Him and peace for all people no matter who or what you believe! When we get to God, the path you took matters less to Him than the fact that you at least tried! Shalom, Salaam, and Peace!
My apologies if this is not what you meant. This is the line that made me think that's what you meant:
Let's just hope you won't have this issue again. It's about time you got to enjoy your car.
I think he meant the cars are getting too complicated, can't say I disagree
For my two cents worth modern cars are far too complicated. Batteries now need to programmed by the dealer for replacement? How is this possibly a positive attribute? I hope I'm not coming cross as painfully stuck in the past but how does this pass any test of logical design?
This is an old thread, but can't say I disagree - nuisance to have to hit up the dealer instead of being able to go anyplace and get a battery replaced the instant you need to.
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Moderate to Severe Psoriasis: "Off-label" Systemic Medications
There are additional systemic medications that are not approved by the FDA for treating psoriasis and psoriatic arthritis. However, some doctors prescribe them off-label—a common and accepted medical practice.
Download the Systemics Medication Booklet (pdf) »
Hydrea (hydroxyurea) is an oral cancer medication found to be effective for psoriasis in the late 1960s. Hydrea can produce significant improvement in stable plaque psoriasis, but it also has potentially dangerous side effect, including bone marrow toxicity. Long-term use has been associated with skin cancer.
Isotretinoin is an oral retinoid approved as a treatment for severe cystic acne. The most common side effects of isotretinoin are eye and lip dryness, and nosebleeds. Bone spurs and hair loss occur to a lesser degree.
Isotretinoin has the potential for severe birth defects if a woman becomes pregnant while the drug is still in her system. A woman on isotretinoin should use reliable birth control one month before treatment, during treatment and for at least one month afterward.
Mycophenolate mofetil is used for the prevention of organ transplant rejection, as well as in the treatment of several inflammatory or autoimmune skin diseases. It has been used in combination with cyclosporine. Because it is an immunosuppressive agent, people with compromised immune systems should not take it.
Sulfasalazine is a combination anti-inflammatory and antibiotic commonly used for treating psoriatic arthritis. Many people cannot tolerate sulfasalazine because of allergy to sulfa, or because of side effect, including nausea, vomiting and loss of appetite.
6-thioguanine is an oral medication approved for treating certain types of leukemia. 6-Thioguanine has been reported to be effective for psoriasis, including treatment of pustular psoriasis. 6-Thioguanine must be used under close supervision due to the potential side effects associated with suppression of the bone marrow.


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If you notice discomfort when wearing them, red eyes or do not see correctly, remove the contact lenses immediately and discard them, consult your optician or trusted ophthalmologist. Otherwise, you may have eye problems.
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More money means more problems for GOP
Mitt Romney and Newt Gingrich's fight for Florida and the states beyond stayed at a high boil Tuesday as Romney released tax returns showing annual income topping $20 million — including a now-closed Swiss bank account — and Gingrich insisted his high-paid consulting work for a mortgage giant that contributed to the housing crisis didn't include lobbying.
After a night of mutual sniping in a debate, the two leading GOP presidential candidates tried to turn the arguments over their various business dealings to his own advantage. Romney's release of two years' worth of tax documents, showing him at an elite level even among the nation's richest 1 percent, kept the focus on the two men's money and how they earned it.
Romney's income put him in the top 0.006 percent of Americans, according to Internal Revenue Service data from 2009, the most recent year available. His net worth has been estimated as high as $250 million.
As the former Massachusetts governor relented to pressure and released more than 500 pages of tax documents, Gingrich kept up the heat, saying Romney was "outrageously dishonest" for accusing him of influence peddling for government-backed mortgage giant Freddie Mac.
The former House speaker said Romney's charges were ironic, given that it was revealed after Monday's debate that Romney himself was an investor in both Freddie Mac and its sister entity, Fannie Mae.
"I don't own any Fannie Mae and Freddie Mac stock. He does, so presumably he was getting richer," Gingrich told Fox News on Tuesday.
The specter of well-off Gingrich and wealthier Romney feuding over money matters pleased Rick Santorum, who lags in polls for next Tuesday's Florida primary but hopes to benefit from the dust-up as the race moves on. He told MSNBC: "The other two candidates have some severe flaws."
Striking out in two directions, Romney planned to offer advance criticism of President Barack Obama's Tuesday night State of the Union address, then focus on Florida's housing woes in an event sure to again highlight Gingrich's $25,000 monthly retainer from Freddie Mac.
Gingrich, starting a busy day of campaign rallies along Florida's Gulf Coast, also took on Obama, criticizing him on foreign policy, energy plans and other issues. "I'm not so much interested in trying to tear Obama down as I am interested in getting the country to see clearly who he is," he said in St. Petersburg.
Records released by Romney's campaign show he closed a bank account in Switzerland in 2010, as he was entering the presidential race. He also kept money in the Cayman Islands, another spot popular with investors sheltering their income from U.S. taxes. But Benjamin Ginsberg, the Romney campaign's legal counsel, said Romney didn't use any aggressive tax strategies to help reduce or defer his tax income.
"Gov. Romney has paid 100 percent of what he owes," Ginsberg said Tuesday.
Romney paid about $3 million on nearly $22 million in income in 2010 and indicated his 2011 taxes would be about the same, $3.2 million on nearly $21 million in income.
During the debate, Romney predicted his tax information would generate chatter but not any surprises, saying what he paid was "entirely legal and fair."
Romney had declined to disclose any tax releases until he came under mounting criticism from his rivals.
In 2010, he donated a combined $3 million to the Mormon Church and other charitable causes. His effective tax rate was about 14 percent, the records showed. For 2011, he'll pay an effective tax rate of about 15.4 percent, a level far lower than standard rates for high-income earners, reflecting the lower rate for long-term capital gains.
The tax records may silence Gingrich and others who argued that Republican voters should know the details of Romney's wealth before they select their presidential nominee and not after. But it also could open up new lines of attack.
After Gingrich's overwhelming victory in South Carolina last Saturday, Romney can ill afford to lose Florida's Jan. 31 primary, and he showcased a new aggression from the opening moments of the debate. He said Gingrich had "resigned in disgrace" from Congress after four years as speaker and then had spent the next 15 years "working as an influence peddler."
In particular, he referred to the contract Gingrich's consulting firm had with Freddie Mac, a government-backed mortgage giant that Romney said "did a lot of bad for a lot of people and you were working there."
"I have never, ever gone and done any lobbying," Gingrich retorted emphatically, adding that his firm had hired an expert to explain to employees "the bright line between what you can do as a citizen and what you do as a lobbyist."
Rep. Ron Paul, who's bypassing Florida in favor of smaller, less expensive states, returned to Texas after Monday's debate. Santorum will appeal to the tea party to help revive his candidacy, appearing at two tea party events.
Associated Press writers Kasie Hunt in Florida and Connie Cass, Jack Gillum, Stephen Braun and Stephen Ohlemacher in Washington contributed to this report.


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Challenge to pension reform moves forward
Labor unions have scored two separate legal victories in the past week in their push to invalidate San Diego's pension reform initiative, which voters overwhelmingly approved earlier this month.
The two wins essentially lead to the same result: The state's Public Employment Relations Board can now move forward with its investigation into a labor complaint that city leaders violated state law by helping craft Proposition B as a citizens' initiative.
Proposition B calls for replacing pensions with 401(k)-style plans for most new city hires and proposes a five-year freeze on the pensionable pay of current employees.
Unions are challenging that latter provision, saying it flies in the face of the city's legal requirement to negotiate with labor over benefit changes.
Proposition B proponents say the initiative was placed on the ballot through a signature drive – not by city officials – and therefore no such negotiations were necessary.
The legal tussle began in February when an initial review of labor's complaint by PERB found there was "reasonable cause" to believe the law had been broken. The board then sought a court injunction — which was denied — to block Proposition B from appearing on the June ballot. The city then successfully sought a delay in the PERB administrative hearings, set to begin April, until after the public vote.
The 4th District Court of Appeal ruled Tuesday that "the trial court erred" by delaying the PERB proceedings and that they should proceed. The ruling slapped down the city's arguments that PERB wasn't the proper venue to determine whether Proposition B was legal and said an unfair labor practice allegation falls within PERB's exclusive jurisdiction.
In a separate ruling last week, the same appellate court denied City Attorney Jan Goldsmith's request for it to consolidate the various legal challenges to Proposition B, skip a PERB hearing and rule on the issue.
Goldsmith said he hoped to avoid going before PERB because he believes the panel is labor-friendly and the outcome is predetermined. Any decision PERB makes would likely be appealed in court anyway and he hoped to expedite the legal process by skipping ahead.
"We tried to short circuit what could be months, years of who knows how many hearings," Goldsmith said. "It's pretty clear to us that PERB is a stacked deck."
Goldsmith also said the PERB process won't affect implementation of Proposition B because only a court can stop the city from moving forward.
Michael Zucchet, head of the Municipal Employees Association, which filed the initial PERB complaint, said it's ironic that city officials are blaming unions for the delays when the PERB process would have been completed by now if the city hadn't blocked it.
"I think it's fair to say the city has expended a tremendous amount of resources over the last three or four months to avoid what apparently is going to be the inevitable of an administrative process at PERB," he said. "So they may be downplaying it, but it doesn't match up with the efforts they've put into it."
PERB is expected to begin its hearing before an administrative law judge in July or August. The judge will make a ruling based on evidence from both sides and any decision can be appealed to the board. That ruling can then be appealed in court, all the way up to the state Supreme Court, if necessary. A final resolution could be years away.
Proposition B — crafted by Mayor Jerry Sanders and City Council members Carl DeMaio and Kevin Faulconer — was approved by 66 percent of city voters.
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Page Contents
The BODIPY fluorophores have spectral characteristics that are often superior to those of fluorescein, tetramethylrhodamine, Texas Red and longer-wavelength dyes. With derivatives that span the visible spectrum (Figure 1.4.1), BODIPY dyes are extremely versatile. We use them to generate fluorescent conjugates of proteins, nucleotides, oligonucleotides and dextrans, as well as to prepare fluorescent enzyme substrates, fatty acids, phospholipids, lipopolysaccharides, receptor ligands and polystyrene microspheres.
BODIPY dyes are unusual in that they are relatively nonpolar and the chromophore is electrically neutral (Figure 1.4.2). These properties tend to minimize dye-induced perturbation of conjugate functional properties. BODIPY dyes are therefore often the preferred choice for labeling nucleotides, amino acids and other low molecular weight ligands. BODIPY dye conjugates of low molecular weight molecules also tend to be more permeant to live cells than are conjugates of charged fluorophores (Membrane-Permeant Reactive Tracers—Section 14.2). With their high peak intensity, reactive BODIPY dyes are among the most detectable amine-derivatization reagents available for HPLC and capillary electrophoresis. BODIPY dyes are also more useful than most other long-wavelength dyes, including fluoresceins and carbocyanines, for assays that measure fluorescence polarization (Fluorescence Polarization (FP)—Note 1.4), and they have large cross-sections for excitation by multiphoton excitation sources (Fluorescent Probes for Two-Photon Microscopy—Note 1.5).
Figure 1.4.1 Normalized fluorescence emission spectra of 1) BODIPY FL, 2) BODIPY R6G, 3) BODIPY TMR, 4) BODIPY 581/591, 5) BODIPY TR, 6) BODIPY 630/650 and 7) BODIPY 650/665 fluorophores in methanol.
Figure 1.4.2 The structure and numbering of the BODIPY fluorophore, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene.
The core structure of the BODIPY fluorophore is shown in Figure 1.4.2. Solutions of the alkyl-substituted derivatives have a green, fluorescein-like fluorescence. However, when substituents that yield additional conjugation are added to the parent molecule, both the absorption and emission spectra of the resulting derivatives can shift to significantly longer wavelengths, with emission maxima of greater than 750 nm now possible with some BODIPY derivatives. Our goal has been to develop BODIPY dyes that are optimal for the major excitation sources and that match the common optical filter sets. Accordingly, our recommended BODIPY substitutes for the fluorescein, rhodamine 6G, tetramethylrhodamine and Texas Red fluorophores are named BODIPY FL, BODIPY R6G, BODIPY TMR and BODIPY TR, respectively (Figure 1.4.3). Because there are so many different BODIPY dyes, we have had to develop a systematic strategy for naming them. Except for BODIPY FL, BODIPY R6G, BODIPY TMR and BODIPY TR, we identify these dyes with the registered trademark BODIPY followed by the approximate absorption/emission maxima in nm (determined in methanol); for example, the BODIPY 581/591 dye.
Amine-reactive BODIPY dyes (Amine-reactive BODIPY dyes—Table 1.7) are discussed below; thiol-reactive BODIPY dyes are included in Thiol-Reactive Probes Excited with Visible Light—Section 2.2. Other reactive BODIPY dyes useful for derivatizing aldehydes, ketones and carboxylic acids are described in Reagents for Modifying Aldehydes and Ketones—Section 3.3 and Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4. Applications of some thiol-reactive BODIPY dyes for cell tracing are discussed in Membrane-Permeant Reactive Tracers—Section 14.2.
BODIPY FL Dye: A Substitute for Fluorescein
With the most fluorescein-like spectra of the BODIPY dyes, the green-fluorescent BODIPY FL fluorophore () (excitation/emission maxima ~503/512 nm) has several characteristics that make it potentially superior to fluorescein in some applications. These include:
- High extinction coefficient (EC >80,000 cm-1M-1) and high fluorescence quantum yield (often approaching 1.0, even in water)
- Lack of ionic charge and spectra that are relatively insensitive to solvent polarity and pH
- Narrow emission bandwidth (Figure 1.4.3), resulting in a higher peak intensity than that of fluorescein
- Red shift in fluorescence emission at high dye concentrations—a property that can be used to detect regions of high probe density ()
- Relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization–based assays (Fluorescence Polarization (FP)—Note 1.4)
- Little or no spectral overlap with longer-wavelength dyes such as tetramethylrhodamine and Texas Red dye (Figure 1.4.3), making BODIPY FL one of the preferred green-fluorescent dyes for multicolor applications
- Greater photostability than fluorescein in some environments
- Large two-photon cross-section for multiphoton excitation (Fluorescent Probes for Two-Photon Microscopy—Note 1.5)
Figure 1.4.3 Normalized fluorescence emission spectra of goat anti–mouse IgG antibody conjugates of fluorescein (FL), tetramethylrhodamine (TMR) and the Texas Red (TR) dyes, shown by dashed lines (---), as compared with goat anti–mouse IgG antibody conjugates of BODIPY FL, BODIPY TMR and BODIPY TR dyes, respectively, shown by solid lines (—).
Longer-Wavelength BODIPY Dyes
It is possible to synthesize BODIPY fluorophores with altered spectral properties by simply changing the substituents on the parent molecule. This discovery has led to creation of a series of longer-wavelength BODIPY dyes with fluorescence spectra that span the visible spectrum (Figure 1.4.1). The BODIPY R6G (excitation/emission maxima ~528/547 nm), BODIPY TMR (excitation/emission maxima ~543/569 nm) and BODIPY TR (excitation/emission maxima ~592/618 nm) fluorophores are spectrally similar to the rhodamine 6G, tetramethylrhodamine and Texas Red fluorophores, respectively, and are thus compatible with standard optical filter sets designed for these important dyes.
The BODIPY 630/650-X and BODIPY 650/665-X fluorophores are the longest-wavelength amine-reactive BODIPY fluorophores currently available. The spectral properties of these longer-wavelength BODIPY derivatives retain most of the advantages of the BODIPY FL fluorophore, including narrow bandwidths, high extinction coefficients, good fluorescence quantum yields and relatively long excited-state lifetimes (>3 nanoseconds for the BODIPY 630/650 dye ). Like the BODIPY FL fluorophore, however, most of these dyes have a small Stokes shift, which may require that they be excited or detected at suboptimal wavelengths. The spectral characteristics of 13 different red-fluorescent fluorophores, including the Alexa Fluor 647 (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3) and BODIPY 630/650 dyes, have been evaluated in different surrounding media to assess the influence of polarity, viscosity and detergent concentration and to facilitate probe choice in fluorescence-based assays.
BODIPY Succinimidyl Esters
We offer an extensive selection of amine-reactive BODIPY dyes (Amine-reactive BODIPY dyes—Table 1.7). These include succinimidyl esters of several BODIPY propionic acids and of BODIPY FL pentanoic acid:
- BODIPY FL C3 succinimidyl ester (D2184)
- BODIPY FL C5 succinimidyl ester (D6184)
- BODIPY R6G C3 succinimidyl ester (D6180)
- BODIPY 493/503 C3 succinimidyl ester (D2191)
- BODIPY 530/550 C3 succinimidyl ester (D2187)
- BODIPY 558/568 C3 succinimidyl ester (D2219)
- BODIPY 564/570 C3 succinimidyl ester (D2222)
- BODIPY 576/589 C3 succinimidyl ester (D2225)
- BODIPY 581/591 C3 succinimidyl ester (D2228)
We have also prepared reactive BODIPY X succinimidyl esters that contain an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and the succinimidyl ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated and making it more accessible to secondary detection reagents such as anti-dye antibodies. These BODIPY X succinimidyl esters include:
BODIPY succinimidyl esters are particularly useful for preparing conjugates of peptides, nucleotides, oligonucleotides, drugs, toxins, sphingolipids and other low molecular weight ligands that contain aliphatic amines. Several BODIPY succinimidyl esters have been conjugated to aminoacyl tRNAs for metabolic incorporation into proteins through in vitro translation.
BODIPY TMR-X SE has been reacted with a series of peptide ligands for use in a high-throughput fluorescence polarization assay of ligand binding to G protein–coupled receptors. The red fluorescence of the BODIPY 581/591 fluorophore shifts to green fluorescence upon peroxidation, a unique feature that has been exploited for ratiometric measurements of lipid oxidation in live cells (Generating and Detecting Reactive Oxygen Species—Section 18.2). BODIPY 630/650-X and BODIPY 650/665-X succinimidyl esters (D10000, D10001) are quite fluorescent when conjugated to nucleotides and oligonucleotides and can be excited with near-infrared excitation sources.
For amplifying the BODIPY FL dye signal or converting it into an electron-dense signal, we offer an unlabeled anti–BODIPY FL rabbit polyclonal antibody (A5770, Anti-Dye and Anti-Hapten Antibodies—Section 7.4). This antibody crossreacts with some other BODIPY dyes, but not with other fluorophores, and therefore should not be used for simultaneous detection of more than one dye based on the BODIPY fluorophore.
Water-Soluble BODIPY Sulfonated Succinimidyl Esters
The moderate lipophilicity of the BODIPY propionic acid succinimidyl esters discussed above requires their dissolution in an organic solvent before use in conjugations. Although these reactive dyes are very useful for preparing conjugates of amines in organic solvents, they are less suitable for reaction with proteins.
To address the solubility in aqueous solution, we have prepared the sulfosuccinimidyl ester of BODIPY FL propionic acid (BODIPY FL, SSE; D6140), as well as the STP ester of BODIPY FL propionic acid (B10006). STP esters, which are prepared by coupling a carboxylic acid and 4-sulfo-2,3,5,6-tetrafluorophenol (S10490, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4, Figure 1.4.4), are more readily purified than sulfosuccinimidyl esters but equally amine reactive. BODIPY FL SSE and BODIPY FL STP ester are quite soluble in water and more suitable than the corresponding BODIPY succinimidyl esters for amine conjugation in aqueous solution. These sulfonated succinimidyl esters are useful for preparing conjugates of proteins, amine-modified oligonucleotides and other biomolecules.
Figure 1.4.4 Reaction of a primary amine with an STP ester.
BODIPY Carboxylic Acids
Two green-fluorescent BODIPY carboxylic acids (D2183, D3834) are available. These carboxylic acid derivatives can be converted to fluorescent esters, acid halides or amides using standard chemical techniques.
The versatility of the BODIPY fluorophore is demonstrated by its incorporation into literally hundreds of products listed in this Handbook, including many of our FluoSpheres and TransFluoSpheres microspheres (Microspheres—Section 6.5), enzyme substrates (Enzyme Substrates and Assays—Chapter 10) and several of our imaging and flow cytometry standards (Fluorescence Microscopy Accessories and Reference Standards—Section 23.1, Flow Cytometry Reference Standards—Section 23.2). Some examples of our BODIPY dye conjugates are described below.
BODIPY Peptide, Protein and Polysaccharide Conjugates
As is common with many fluorescent dyes, conjugation of BODIPY dyes to proteins is sometimes accompanied by significant fluorescence quenching. Because of this potential problem, we do not recommend using the simple BODIPY propionic acid succinimidyl esters discussed above for preparing most protein conjugates, although peptides labeled with a single BODIPY dye can be quite fluorescent and are quite useful for fluorescence polarization–based assays (Labeling Small Peptides with Amine Reactive Dyes in Organic Solvents—Note 9.2). We prepare conjugates of its BODIPY dyes with an exceptionally wide variety of peptides, proteins and polysaccharides, including:
- Pepstatin A, a membrane-permeant analog of this important cathepsin D inhibitor (P12271, Detecting Peptidases and Proteases—Section 10.4)
- Antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4; Summary of Molecular Probes secondary antibody conjugates—Table 7.1)
- Phallacidin and phalloidin for staining F-actin filaments (B607, B3475, B12382; Probes for Actin—Section 11.1; Spectral characteristics of Molecular Probes actin-selective probes—Table 11.2)
- Bovine serum albumin, for use as a tracer (A2750, Protein Conjugates—Section 14.7)
- Acetylated and non-acetylated low-density lipoproteins (L3485, L3483; Probes for Following Receptor Binding and Phagocytosis—Section 16.1)
- Escherichia coli and Staphylococcus aureus BioParticles conjugates (E2864, S2854; Probes for Following Receptor Binding and Phagocytosis—Section 16.1; BioParticles fluorescent bacteria and yeast—Table 16.3)
- Lipopolysaccharide (L23350, Probes for Following Receptor Binding and Phagocytosis—Section 16.1; Fluorescent lipopolysaccharide conjugates—Table 16.1)
- Dextran (D7168, Fluorescent and Biotinylated Dextrans—Section 14.5)
In addition, we prepare conjugates of proteins (and of starch) that are so heavily labeled that they are almost nonfluorescent (Figure 1.4.7). Use of the EnzChek Kits and DQ reagents that incorporate these bioconjugates as fluorogenic enzyme substrates is described later in this section and in Detecting Peptidases and Proteases—Section 10.4.
BODIPY Nucleotide and Oligonucleotide Conjugates
With the exception of guanosine nucleotides (see below), fluorescence quenching is usually not a problem if the BODIPY derivative is conjugated to nucleotides, oligonucleotides, peptides or low molecular weight amines in which the stoichiometry of modification is 1:1. BODIPY FL dye–labeled oligonucleotide primers also have lower photodestruction rates than fluorescein-labeled primers, improving the detectability of labeled DNA in sequencing gels. Oligonucleotide conjugates of several of our BODIPY dyes have been shown to be useful for DNA sequencing (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Molecular Probes nucleic acid labeling kits—Table 8.6), in part because the dye exhibits minimal effect on the mobility of the fragment during electrophoresis. We also offer a Chromatide BODIPY FL-14-dUTP for enzyme-mediated incorporation into nucleic acids (C7614, Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Characteristics of ChromaTide and aha labeled nucleotides—Table 8.5).
In addition, we have prepared BODIPY FL conjugates of ATP and GTP that are labeled through the ribose moieties and serve as structural probes of nucleotide-binding proteins (A12410, G12411; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3). The fluorescence of BODIPY dyes is quenched by photoinduced electron transfer from proximal guanosine bases. BODIPY FL GTP therefore shows significant fluorescence quenching (Figure 1.4.5) that is relieved by binding to GTP-binding proteins (G-proteins). Longer-wavelength BODIPY TR conjugates of ATP and GTP are also available (A22352, G22351; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3).
For protein-binding studies that require nonhydrolyzable nucleotides, we offer the BODIPY FL fluorophore linked through the γ-thiol of ATP-γ-S (A22184, ) and the BODIPY FL, BODIPY 515/530 and BODIPY TR fluorophores linked through the γ-thiol of GTP-γ-S (G22183, G35779, G35780; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3). Like BODIPY FL GTP, the fluorescence of the BODIPY GTP-γ-S thioesters is quenched ~90% relative to that of the free dye but is recovered upon protein binding to at least some G-proteins. The green-fluorescent BODIPY FL GTP-γ-S has been used to detect GTP-binding proteins separated by capillary electrophoresis. BODIPY 515/530 GTP-γ-S thioester also exhibits green fluorescence and has a greater fluorescence increase upon protein binding, as compared with the BODIPY FL GTP-γ-S thioester. The BODIPY TR GTP-γ-S thioester is a red-fluorescent analog with spectral properties similar to those of the Texas Red dye.
We also offer the green-fluorescent BODIPY FL GTP-γ-NH amide (G35778, Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3) as another choice for protein-binding studies. Although this analog exhibits less fluorescence enhancement upon protein binding, it is reportedly the best of the three green-fluorescent GTP-γ analogs for directly monitoring nucleotide exchange. The different linker lengths of the green-fluorescent GTP-γ analogs (six-carbon for BODIPY FL GTP-γ-NH amide, four-carbon for BODIPY FL GTP-γ-S and one-carbon for BODIPY 515/530 GTP-γ-S) may be useful for understanding protein active-site geometries.
In addition to their potential use for binding studies, the BODIPY FL ATP-γ-S and BODIPY FL GTP-γ-S thioesters are important substrates for Fhit (Figure 1.4.6), a member of the histidine triad superfamily of nucleotide-binding proteins that bind and cleave diadenosine polyphosphates. Fhit, one of the most frequently inactivated proteins in lung cancer, functions as a tumor suppressor by inducing apoptosis. These BODIPY nucleotides should be especially useful for screening potential Fhit inhibitors and activators.
Figure 1.4.5 Fluorescence emission spectra of (1) free BODIPY FL dye in phosphate-buffered saline, pH 7.2; (2) BODIPY FL ATP (A12410); and (3) BODIPY FL GTP (G12411). Samples were prepared with equal absorbance at the excitation wavelength (488 nm). The areas under the curves are therefore proportional to the relative fluorescence quantum yields, clearly showing the quenching effect caused by interaction of the BODIPY FL fluorophore with the guanine base of GTP.
Figure 1.4.6 Principle of fluorescence-based detection of the diadenosine triphosphate hydrolase activity of Fhit using BODIPY FL GTP-γ-S thioester (G22183) as a substrate analog.
BODIPY Lipids and BODIPY Receptor Ligand Conjugates
BODIPY dye conjugates of lipids, toxins, steroids, drug analogs and receptor probes typically have quantum yields approaching unity, especially in organic solvents. The low polarity of the BODIPY fluorophore makes probes containing these dyes excellent analogs of biological lipids (Probes for Lipids and Membranes—Chapter 13). Consequently, these probes are well tolerated by enzymes that metabolize lipids, including phospholipases and sphingomyelinases (Probes for Lipid Metabolism and Signaling—Section 17.4, Fluorescence-based phospholipase assays—Table 17.3). In most cases, lack of a spectral shift in the metabolic product's fluorescence requires use of an easy extraction and chromatographic separation step to detect product formation, with quantitation possible by photography or with a fluorescence- or absorption-based scanner. BODIPY derivatives of lipids and low molecular weight ligands include analogs of:
- Phospholipids and fatty acids, numerous versions of which are described in Fatty Acid Analogs and Phospholipids—Section 13.2
- Sphingolipids, including several ceramide derivatives (Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3)
- Cholesterol (C3927MP, Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3)
- Cytochalasin D, an actin-polymerization inhibitor (C12377, Probes for Actin—Section 11.1)
- Paclitaxel (Taxol), for staining tubulin in isolated preparations (P7500, P7501; Probes for Tubulin and Other Cytoskeletal Proteins—Section 11.2)
- Vinblastine, a microtubule-disrupting agent (V12390, Probes for Tubulin and Other Cytoskeletal Proteins—Section 11.2)
- Vancomycin, an antibiotic (V34850, Viability and Cytotoxicity Assay Reagents—Section 15.2)
- Penicillin V (B13233, B13234; Viability and Cytotoxicity Assay Reagents—Section 15.2)
- Verapamil, for investigating multidrug resistance (B7431, Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6)
- Forskolin, an adenylate cyclase activator (B7469, Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6)
- Prazosin, an α1-adrenergic receptor probe (B7433, Probes for Neurotransmitter Receptors—Section 16.2)
- Ouabain, a cardiac glycoside (B23461, Probes for Ion Channels and Carriers—Section 16.3)
- Glibenclamide, a probe for potassium channels (E34250, E34251; Probes for Ion Channels and Carriers—Section 16.3)
- Dihydropyridine derivative that is selective for Ca2+ channels (D7443, S7445; Probes for Ion Channels and Carriers—Section 16.3)
- Thapsigargin, which promotes Ca2+ release by inhibiting the endoplasmic reticulum Ca2+-ATPase (B7487, B13800; Calcium Regulation—Section 17.2)
In addition to the BODIPY dye conjugates of receptor ligands in the list above, we have utilized BODIPY dyes for synthesis of several LysoTracker and LysoSensor dyes, as well as BODIPY FL histamine (B22461, ), which are extremely useful probes for labeling acidic organelles in live cells. These products are discussed in Probes for Lysosomes, Peroxisomes and Yeast Vacuoles—Section 12.3.
DQ Reagents: Heavily Labeled BODIPY Dye Conjugates as Fluorogenic Enzyme Substrates
We have found BODIPY dye conjugates to be very useful reagents for numerous bioanalytical screening applications. In particular, we have utilized the tendency of BODIPY dyes to quench their fluorescence on conjugation to certain biopolymers to our advantage (Figure 1.4.7) in the following enzyme-assay kits and reagents:
- EnzChek Protease Assay Kits, which contain almost nonfluorescent casein derivatives that are heavily labeled with either the green-fluorescent BODIPY FL dye (E6638; Detecting Peptidases and Proteases—Section 10.4) or red-fluorescent BODIPY TR-X dye (E6639, R22132; Detecting Peptidases and Proteases—Section 10.4)
- EnzChek Elastase Assay Kit (E12056, Detecting Peptidases and Proteases—Section 10.4), with DQ elastin, a quenched BODIPY FL conjugate
- EnzChek Ultra Amylase Assay Kit (E33651, Detecting Glycosidases—Section 10.2), containing a highly quenched BODIPY FL starch derivative
- DQ Green BSA (D12050, Detecting Peptidases and Proteases—Section 10.4) and DQ Red BSA (D12051, Detecting Peptidases and Proteases—Section 10.4), heavily labeled and almost nonfluorescent BODIPY BSA conjugates that yield intense green or red fluorescence upon proteolytic digestion
- DQ ovalbumin (D12053, Detecting Peptidases and Proteases—Section 10.4, DQ Ovalbumin), a heavily labeled and almost nonfluorescent BODIPY FL ovalbumin conjugate
Conjugation of either the BODIPY FL dye (excitation/emission maxima ~500/506 nm) or BODIPY TR dye (excitation/emission maxima ~589/617 nm) to a biopolymer at high degrees of substitution (DOS) results in almost total quenching of the conjugate's fluorescence; they typically exhibit <3% of the fluorescence of the corresponding free dyes. Enzyme-catalyzed hydrolysis relieves this quenching, yielding brightly fluorescent BODIPY FL dye– or BODIPY TR-X dye–labeled peptides (Figure 1.4.7), or, in the case of the BODIPY FL amylase substrate in the EnzChek Ultra Amylase Assay Kit, BODIPY FL dye–labeled carbohydrates. The increase in fluorescence, which can be measured with a spectrofluorometer, minifluorometer or fluorescence microplate reader, is proportional to enzymatic activity. The DQ BSA and DQ ovalbumin substrates are particularly suitable for the study of receptor labeling and antigen processing. DQ BSA conjugates can be targeted to Fc receptors after they are complexed with our anti-BSA antibody (A11133, Anti–Epitope Tag and Anti-Reporter Antibodies—Section 7.5). Ovalbumin is efficiently processed through mannose receptor–mediated endocytosis by antigen-presenting cells and is widely used for studying antigen processing. Upon endocytosis and proteolysis, highly fluorescent peptides are released within intracellular vacuoles. DQ ovalbumin appears to be an excellent indicator of macrophage-mediated antigen processing in flow cytometry and microscopy assays.
Figure 1.4.7 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hydrolysis of the heavily labeled and almost totally quenched substrates provided in our EnzChek Protease Assay Kits relieves the intramolecular self-quenching, yielding brightly fluorescent reaction products.
BODIPY Dye Conjugates for Fluorescence Polarization–Based Assays
When a fluorescent molecule tethered to a protein is excited by polarized fluorescent light, the polarization of fluorescence emission is dependent on the rate of molecular tumbling. Upon proteolytic cleavage of the fluorescently labeled protein, the smaller peptides that result tumble faster and the emitted light is depolarized relative to the light measured from the intact conjugate (Fluorescence Polarization (FP)—Note 1.4). Fluorescence polarization technology is more sensitive than many other nonradioactive assays for proteases and allows measurements to be taken in real time, permitting the collection of kinetic data.
The relatively long fluorescence lifetimes (typically >5 nanoseconds) at visible wavelengths, good anisotropy properties, high molar absorptivity and fluorescence intensity and lack of pH sensitivity in the spectra of the BODIPY dyes have been shown to make these dyes the preferred fluorophores for high-throughput fluorescence polarization–based assays. The EnzChek Polarization Assay Kit for Proteases (E6658, Detecting Peptidases and Proteases—Section 10.4) contains green-fluorescent BODIPY FL casein with an optimal degree of labeling for fluorescence polarization–based protease assays. BODIPY dye conjugates of nucleotides, peptides and drug analogs are available or are readily prepared from the chemically reactive BODIPY dyes. Fluorescence polarization–based assays for G-protein–coupled receptors, kinases and phosphatases and for high-affinity receptors are particularly important when screening for new drug candidates.
BODIPY Substrates for Chloramphenicol Acetyltransferase
Chloramphenicol acetyltransferase (CAT), an enzyme that is encoded by an important reporter gene, can acetylate chloramphenicol derivatives that incorporate the BODIPY fluorophore (Figure 1.4.8). The acetylated products are readily separated from the substrate by thin-layer chromatography () and quantitated by photography, fluorometry or with a plate scanner. Our original FAST CAT Chloramphenicol Acetyltransferase Assay Kit (F2900) and improved FAST CAT (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6616) utilize a green-fluorescent BODIPY FL substrate, and the FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6617) employs a yellow-fluorescent BODIPY TMR 1-deoxychloramphenicol substrate. These products are described in detail in Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes—Section 10.6.
Figure 1.4.8 The green-fluorescent BODIPY FL 1-deoxychloramphenicol substrate in our FAST CAT Green (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6616). CAT-mediated acetylation of this substrate and of the BODIPY TMR 1-deoxychloramphenicol in our FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6617) results in single fluorescent products because these substrates contain only one hydroxyl group that can be acetylated. In contrast, the BODIPY FL chloramphenicol substrate in our original FAST CAT Kit (F2900) contains a second hydroxyl group at the 1-position (indicated by the labeled arrow). This hydroxyl group undergoes a nonenzymatic transacetylation step, restoring the original hydroxyl for a second acetylation. CAT-mediated acetylation of this chloramphenicol substrate produces three fluorescent products, thus complicating the analysis.
Additional Methods of Analysis Using BODIPY Dye Conjugates
In addition to their general utility for the intensity-based and fluorescence polarization–based assays described above, the BODIPY dyes are near optimal for a variety of other bioanalytical techniques:
- The spectral variety and high absorbance of the BODIPY dyes (Figure 1.4.1) permits their use as efficient donor or acceptor dyes for numerous assays that use fluorescence resonance energy transfer, including internally quenched endopeptidase substrates (Detecting Peptidases and Proteases—Section 10.4), nucleic acid hybridization assays and receptor-binding assays (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).
- BODIPY dye conjugates of peptides are readily separated by chromatographic means and can be used to detect the activity of enzymes that catalyze secondary modifications, such as phosphorylation/dephosphorylation, glycosylation/deglycosylation, oxidation/reduction, myristoylation, farnesylation and peptide–peptide crosslinking.
- Hydrolysis of peptides that are singly labeled with BODIPY dyes to smaller peptides can be detected chromatographically with extremely high sensitivity.
- With their high peak intensity and narrow emission spectra, reactive BODIPY dyes are among the most detectable amine-derivatization reagents available for HPLC and capillary electrophoresis; thus, amine-containing metabolites can be derivatized with succinimidyl esters of the BODIPY dyes (Amine-reactive BODIPY dyes—Table 1.7) for ultrasensitive analysis.
|Cat #
|1, 2
|DMSO, MeCN
|DMSO, MeCN
|1, 3
|DMSO, MeCN
|DMSO, MeCN
|DMSO, MeCN
|DMSO, MeCN
|DMSO, MeCN
|DMSO, MeCN
|DMSO, MeCN
|DMSO, MeCN
|DMSO, MeCN
|1, 5
|DMSO, MeCN
|1, 6
|DMSO, MeCN
|DMSO, MeCN
|DMSO, MeCN
|1, 7
|DMSO, MeCN
For Research Use Only. Not for use in diagnostic procedures.


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|Sun 19 Jul
|Strawberries and Prosecco
|Sat 25 Jul
|Sparkle & Steam
|Sat 1 Aug
|Haworth Haddock
|Sat 15 Aug
|Sparkle & Steam
|Sat 22 Aug
|Haworth Haddock
|Sat 29 - Mon 31 Aug
|The Railway Children 50th Anniversary
The KWVR is a heritage railway which was reopened in 1968 and runs from Keighley to Oxenhope through beautiful Brontë country. Board our steam train and enjoy the sound of the engine climbing the steep sides of the valley, while great clouds of steam and smoke add drama to the scene. The five mile journey is a powerful reminder of our industrial heritage, as well as being a unique way of enjoying the beautiful countryside immortalised by Charlotte, Emily and Anne Brontë.
The Railway hosts a number of events throughout the year including two Steam Gala's and the ever popular Beer and Music Festival. You can also enjoy our evening events such as the Haworth Haddock and Keighley Korma, travel on the train and enjoy your meal before alighting at the station and dancing the night away to live bands.
The Railway is perhaps most famous for its role in the 1970 film version of Edith Nesbit's story The Railway Children but
over the years, the Railway has appeared in many TV and film productions. You can visit Rail Story at Ingrow and see the engines and carriages used in these films.
The Railway really does have something for everyone so why not come and visit us!
Opening details
Please see the website for full timetable information.
Day Rover: £16 (Adult)
Family Day Rover: £40. This ticket is the same as the Day Rover, although is ideal for Families and Small Groups travelling together. Valid for 2 Adults and up to 3 Children or Concessions. (eg - 2 adults + 1 child + 1 OAP).
Line return, concession fares, student rates & Local resident reductions available.
Also see
- Top things to do Brontë Country
- Fun Things to do on a Rainy Day
with charge
- Coach parties accepted
- Gift shop
- Picnic site
- Public toilets
- Accepts groups
- Children welcome
- Disabled access
- Dogs accepted


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Move over Flopsy (Vampire Bill), Mopsy (Vampire Eric), and Cottontail (Jason Stackhouse)! The real deal is (rumored) to be coming to True Blood. That's right folks, the man who invented the steely-eyed-furrowed-brow-head-tilt and is filled with so much testosterone he can impregnate ladies with a gaze is ready to punch first and ask questions later, Bon Temps-style. If this rumor is correct, then True Blood is back!
Admit it, last year's True Blood was the worst. The villains stunk, the threeway vampire sex session was never really a threeway vampire sex session, Hoyt turned into a lanky whiner, and don't even get us started on the castration of Vampire Eric. Sigh. But if Meloni is cast, that's all about to change.
According to TV Line, Meloni is rumored to be circling a role of a very powerful vampire for the fifth season. Who do you think it is? A possible new Alan Ball character or someone from the books? We have no idea, we're too busy running around in circles on the floor Make 'Em Laugh style. The last time we saw Meloni walking the streets of New York we had to stop ourselves from running up and begging to be punched in the face by this Wet Hot American Summer and Oz legend.
Next season of True Blood might just be the most bad-ass or ridiculously over-the-top thing we've ever seen. We'll take either! But one thing is for certain, it's not going to be boring. Because no one puts Meloni in the corner.


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Some Orthodox Christians have proposed something like the following scenario for a healing of the breach between Orthodoxy and Catholicism:
1. Bishops ought to do nothing of consequence without the consent of the pope.
2. The pope cannot do anything of momentous significance without the consent of the consensus of the bishops and the totality of the Church.
If the pope, however, can only do what is consented to by the council or the whole body of the faithful, in all particulars, then the group truly sets the standard and that is pure conciliarism (because the pope has no more authority than the collective), which has been condemned as heresy by the Catholic Church. He would merely become one bishop among many, with little or nothing distinguishing him in authority. If there is no real distinction in power and jurisdiction (mere "primacy of honor" logically reduces to that, in my opinion), then this is Orthodoxy, and hardly a compromise "solution."
The group doing what the pope consents to sounds more "Catholic" to me, so I have little problem with #1; it is #2 that I think runs into problems. Making #1 and #2 equal in application simply reduces to conciliarism / Gallicanism, in which case our ecclesiology is hardly distinguishable from Orthodox ecclesiology (not to mention, contrary to Vatican I and II).
In practice, of course, both are usually held together, and there need not be mutual hostility or competition at all, but there is still theoretically, or dogmatically, at least, papal headship or supremacy, in which the pope can act alone. I would contend that this occurred, e.g., with Humanae Vitae in 1968 (which is an infallible document, far as I can determine), though that didn't involve a council.
Virtually all of the pope's advisers (if not all) argued against the continuing ban on contraception, but that was false to the history of Catholic teaching (reiterated in 1931), so the pope acted on his own and reinforced it. If he had to agree with that group, let alone a council, then evil may have been promulgated as good, as in Anglicanism after 1930, in virtually all other Protestant groups, and increasingly in practice (to my great distress), even in Orthodoxy.
This is one of the three main reasons I am Catholic rather than Orthodox. I wanted the morality of the Apostles and early Church, and since the Orthodox were compromising on contraception (e.g., three successive editions of Metr. Kallistos Ware's book, The Orthodox Church have increasingly weaker statements on it), I concluded that Orthodoxy was non-apostolic in that regard, and there was no question of joining them, since that issue was the first major issue in which I changed my mind.
I would say that the solution to the different conceptions of the relationship of pope and Church is for the Orthodox to grant the pope dogmatic singularity and supremacy, with the pope, for his part, making every effort to act directly in concert with councils and bishops, with the understanding that he voluntarily does so but is not duty-bound or required to do so, at least in some cases.
We know, e.g., that there were great inquiries sent out to bishops before the definitions of the Immaculate Conception and Assumption, so they were by no means isolated papal decisions.
A Catholic can never say that the pope does not have supremacy in some sense. If all he can do is agree with councils and bishops and be a mere figurehead (sort of like the Queen of England, with all the "Byzantine" pomp and circumstance), that is not Catholicism; it is Orthodoxy; in which case it is no compromise at all. I wouldn't expect ecumenical Orthodox to disregard all of our distinctives anymore than I would want to neglect their biggest concerns.
My proposal basically boils down to the day-to-day function and practice of the pope being essentially in accord with Orthodox conceptions, with the principle of the thing (supremacy, papal infallibility) being more "Catholic." If the pope has no final say in some real, concrete sense (as he did in the first millennium, in many instances), he is not a pope.
I think my proposal for how a reunited papacy would work is quite ecumenical and respectful of Orthodox concerns (I believe it is a true compromise), and in line with what I have read from Blessed Pope John Paul II. It was just off the top of my head, too. I'm sure I could hone and refine it further upon more reflection.
I agree totally with "the law of love" in how the papacy operates and "gives orders," and I think the present Holy Father and his Blessed predecessor followed that to an extraordinary degree. I oppose all strong-arm tactics of any sort and am in favor of "subsidiarity" economically, politically, and ecclesiastically. In fact, I detest all "Church politics" so much that I have very little to do with it.
Accordingly, I was in favor of freedom of all Catholics to worship at a Tridentine Mass if they so chose, long before this was decreed by Pope Benedict XVI. I've attended a (historically German) parish that has always offered a Latin Mass: never stopped (Novus Ordo and sometimes Tridentine). We were the only cluster in the Detroit archdiocese that did so, when it was rare, and there are still very few even now, after it has been allowed. I think freedom of rite and custom is crucial to preserve, provided it is all orthodox.
The fact remains that without the papacy (as we conceive it), the East (by Newman's reasoning) would have likely gone into complete heresy in the first Millennium. As it was, the East was rife with Monophysitism and Monothelitism (and also to a lesser extent, Nestorianism) for many centuries: infiltrating the patriarchates in alarming proportion (and we see a clear example of that in 449).
Some argue that the proliferation of those heresies and also rampant iconoclasm, led directly to so many large regions falling rapidly to Islam. The road was paved with these heresies.
The East was in schism five times before 1054, as I have noted, and in all five instances (in the judgment of both sides) they were on the wrong side. Rome was right every time, with regard to these five schisms. I think that speaks volumes. Rome determines orthodoxy. History plainly reveals this. The five schisms are:
The Arian schisms (343-98)
The controversy over St. John Chrysostom (404-415)
The Acacian schism (484-519)
Concerning Monothelitism (640-681)
Concerning Iconoclasm (726-87 and 815-43)
This adds up to 231 out of 500 years in schism: out of communion with Rome (46% of the time). Orthodox agree that all five of these schisms were in error, according to present Orthodox teaching. The Orthodox eventually rejected Arianism and Monothelitism and Iconoclasm. They think St. John Chrysostom is a good guy (Rome defended him then, just as with St. Athanasius). The Acacian schism had to do with Monophysitism.
Orthodoxy is not under the pope now. That changed in the eleventh century. The very fact that the more ecumenical Orthodox see it as preferable to somehow be back in communion with the pope, itself proves that Orthodoxy has lost a key proponent of historic Catholic Christianity. Otherwise, Orthodoxy would be complete in itself, and in need of no component from another Christian communion, as we view ourselves to be. Orthodoxy wouldn't need "our" pope; it would already have one of its own, or deny that it needs one at all (even in the lesser Orthodox sense). But that is not possible in Orthodoxy because of competing jurisdictions.
People who know much more about the situation than I do tell me there are many Eastern Catholics (as opposed to Eastern Orthodox) who are dissidents on the question of papal supremacy, headship, and infallibility. Whether an Eastern Catholic chooses to abide by this or not is their choice. This is the problem in the Catholic Church as a whole: cafeteria, pick-and-choose Catholics. If a Catholic doesn't like some Catholic dogmas, then to be honest, he should not be a Catholic, because he or she is not in full obedience on that point. That doesn't change the fact of what we teach. They would then simply be "Catholics" who are really Orthodox at heart. What else is new? Lots of folks don't consistently follow their own ostensible affiliation. Yet the 1990 Eastern Code of Canons states:
Canon 44 – §1. The Roman Pontiff obtains full and supreme power in the Church by means of legitimate election accepted by him together with episcopal consecration; . . .
Canon 45 – §1. The Roman Pontiff, by virtue of his office (munus), not only has power over the entire Church but also possesses a primacy of ordinary power over all the eparchies and groupings of them by which the proper, ordinary and immediate power which bishops possess in the eparchy entrusted to their care is both strengthened and safeguarded.
§2. The Roman Pontiff, in fulfilling the office (munus) of the supreme pastor of the Church is always united in communion with the other bishops and with the entire Church; however, he has the right, according to the needs of the Church, to determine the manner, either personal or collegial, of exercising this function.
§3. There is neither appeal nor recourse against a sentence or decree of the Roman Pontiff.
A lot of the problems in the Catholic Church (even including very much the sex scandal) came about (I dare say) due to spineless bishops who have been lax in their duty (just as in the Arian crisis and other scandals and low points in the history of the Church). Many bishops followed the model of apostate (or at least wimpish, ineffectual) priests that the prophets were constantly complaining about in the Old Testament. Modernism and liturgical mediocrity or corruption flourishes in many parishes because of this.
But (I would contend) papal encyclicals and acts have been the big force for a reform of both liturgy and orthodoxy. It is Rome that disciplines dissidents, and has provided strong leadership (the Catechism; many superb encyclicals). Thus, it seems to me that the "top-down" model works better for preserving orthodoxy and good morals (e.g., Humanae Vitae again).
In Orthodoxy, in large part because of no such central authority and final say, something like contraception now increasingly takes hold, even though the Orthodox traditionally opposed it as a grave sin, just as we continue to do. The difference? We had a pope who spoke out strongly on the matter in 1968, and it is infallible moral teaching that won't change.
Orthodox argue that Pope St. Leo the Great in the Council of Chalcedon in 451 did not "rule" or have the final say, but rather, that his Tome was deliberated upon and then ratified by the assembly. I heard Metr. Kallistos Ware make this argument in a lecture in Detroit.
Granting that description, this would be, in my view, an instance of the papacy being at an earlier stage of development. It was still developing at this relatively early date. Christianity had only been legal for 138 years. This accounted in large part for a slow earlier development of the papacy, as Cardinal Newman argued.
Even Christology had much more development to go through at that time. The canon of the Bible had only been established for two generations. Mariology was still developing quite a bit, and also the doctrine of the saints. The iconoclasm issue (tying into communion of saints and the incarnation) was not resolved till several centuries after that.
So it is not unusual to hold, as I do, that the doctrine (and operation and function) of the papacy was also developing, which is why infallibility was not declared till 1870. Orthodox will rail against Vatican I but they can't deny that it was the result of a very slow development that took 18 centuries. Hence, if that developed slowly, all the more would we expect a much more primitive papacy in 451, which is very early. At that point things were relatively more "conciliarist" — though, I suspect, not as much as what Orthodox think, judging from very strong papal statements I have read in Pope St. Leo the Great (some of which seem to go even further than Pope St. Gregory the Great 150 years later).
Now, some Orthodox I have debated will argue that it is development of doctrine that is the bugaboo and where Rome went so wrong. I think that is nonsense (sorry for my overly subtle terminology). In my book on Orthodoxy, I argued that Orthodoxy has developments, too, just as Catholicism does. Here is a portion of what I wrote:
Orthodox often seem to disparage development of doctrine, or argue that Orthodox development (insofar as it occurs at all) is essentially different from Catholic development. As an example, let us consider hesychasm. One can trace it back in kernel form to St. Gregory Nyssa, St. Basil the Great, or Origen, yet it was not fully developed until St. Gregory Palamas (1296-1359). So this seems to me to also be a "development of a category" (a type of prayer and devotion, and — especially — the corresponding theology), since it has to do with the nature and essence of God, and a distinction between essence and energy. As such, it is hardly distinguishable (philosophically speaking) from similar refinements of category such as the homoousios, Theotokos, transubstantiation, or procession within the Godhead.
It was not formally adopted (relating it to the Divine Energies and Uncreated Light), until the Councils of Constantinople in 1341, 1347, and 1351, when it then became, according to the Oxford Dictionary of the Christian Church, "an accepted part of Orthodox tradition." I don't see how this is any different (chronologically or essentially) from, for example, Catholic developments of transubstantiation or the Immaculate Conception (both fully developed one or two hundred years before hesychasm was) [or the papacy]. Can Orthodox claim that hesychasm and related concepts existed in their developed form all the way back to the early centuries of the Church? I think not.
Papal infallibility cannot be rolled back. It is a de fide dogma. By its very nature it is permanent. How the inherent power is exercised, however, can be discussed, and has been in high-level talks. And this is how Vatican II complemented Vatican I. Once papal infallibility was established once and for all and "out of the way," then the road was set for a fuller development of a still-supreme pope working more directly in concert with councils and bishops.
Vatican II gave the strongest statement of conciliar infallibility in the history of he Church: but it is conciliar infallibility in cooperation (always) with the pope, and with his approval, which is what sets it apart from orthodox conciliarism. That's why the papal decree came first, then more development on conciliar ecclesiology.
I believe in papal ecclesiology because I think it is also modeled in the Bible, on Petrine primacy. Peter had prerogatives that no one else had, or had only in concert: exactly as in our ecclesiology. Peter was the leader, whereas in Orthodoxy, Peter, John, James, and Paul or some such would all be leaders, with no one having final say in cases of disagreement, leading to separate jurisdictions (and sometimes, historically, heresy as a result).
The Jerusalem Council worked the same way. St. Peter's words seem to have been decisive, and he acted as Leo the Great did in 451. St. James also played a key role, being the resident bishop. It had final authority. St. Paul went around declaring its binding decrees (Acts 16:4).
We are operating the same way the early Church did: just more developed. We still have popes; we still have councils. Orthodoxy has no popes and rarely holds councils; certainly not ecumenical ones, as they were in the first millennium and after, in terms of drawing from bishops all over the world.
But there were several mostly Eastern ecumenical councils then, too, so even if some of ours in the last 1000 years were mostly Western (that would be part of the Orthodox critique), that would be no different in essence from the early mostly Eastern ones: just "balancing the score a bit."


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Here is the article I wrote for the January Issue of RSVP MN Magazine. You can find it here.
The rise of the "Phonetographer" generation is upon us. Even though Facebook was the No. 1 social media app overall in 2013, its photo sharing subsidiary Instagram was the fastest growing app among the top 10.
Instagram is an online photo and video sharing and social networking service that enables its users to take pictures and videos, apply digital filters to them, and share them on a variety of social networking services such as Facebook, Twitter, Tumblr and Flickr. It first launched in 2010. In 2012 Instagram was acquired by Facebook for $1 billion and made a subsidiary. Instagram saw a growth in audience by 66 percent to 32 million users in 2013 (according to Nielsen Data) and shows no trace of slowing down.
While Instagram is made up of the same demographics as Facebook, it is also still growing in popularity among teens, which are shifting to single purpose or messaging apps, including Instagram, Snapchat, Whatsapp, Whisper and others.
Instagram has many uses in event marketing from several perspectives. On the marketing side, Instagram is being used for everything from shots of the venues to displays, speakers, products, crowds and interaction. On the consumer side, guests, visitors and attendees are participating in contests, recording presentations, products and displays for co-workers that are off-site, and for reviews.
Here are three ways you can successfully integrate Instagram into your events this year:
Promote your event or participation in events:
If you are traveling to a national trade show or hosting your own event, document your location with Instagram to promote traffic to your booth or event. Shoot photos of the outside of the venue to help with directions. Share photos of your booth or event to allow people to see what they will see at the event. Post photos of your booth reps or event managers so that people can more easily recognize your team.
Promote your products, services or offerings:
Highlight any new products, etc. that you will offer at the event. Especially feature a product if it is an exclusive being offered during the run of the event. Photos of this sort are great to share across Twitter, Facebook and your other marketing channels such as newsletters. This can expand your reach even to people that aren't in attendance at the event.
Engage your audience with a photo contest:
Have attendees take photos on Instagram and post them with a message that includes a keyword you are tracking (a hashtag for example) and offer a prize to participants. This extends your reach even further as they share the photos on their channels and to their networks.
The ideas are only limited by your imagination. 2014 is upon us and the trade shows and events are in motion. Smile and get ready for your close-up!


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I know I am early but planning the holidays already =) Want to make cupcakes for gifts this year and was wondering if you would be so kind as to share what cupcake is 'Christmas' to you. Thanks!
I don't usually do cupcakes for Christmas but I want to make these this year: http://www.cbc.ca/holiday/images/xmas_tree_Cupcakes.jpg
I'd love an excuse to make a cupcake wreath, too. http://www.freewebs.com/davebarb_99/apps/photos/photo?photoid=6236622
Those are so cute =) Thanks for sharing!
I want to give cupcakes for Christmas, too! I am thinking of red velvet with cream cheese icing and some festive and easy decoration. I just think the red is pretty for the holiday and here in the South, everyone seems to like it!
I just love this cupcake:
They are so tasty with a light peppermint flavor, and I love the simple look of crushed up peppermint candy canes on top. I made these last Christmas and they were a hit.
hmm to me I think it is chocolate mint yum!
I made an egg nog cake for Christmas and it was yummy. Just sub the egg nog for the liquid and cut back a bit on the sugar. Add a little cinnamon and smidge of nutmeg to the frosting and you're all set.
I love Christmas! I have been thinking about this already too... I think I am going to do a Creme de Menthe cupcake (filled with ganache). I also want to do a Gingerbread Latte cupcake. Haven't quite figured out what the components will be yet though. I just love gingerbread lattes!
hi there i wan't to as well! i think they are a great idea and i can't wait until Christmas! i found these cool pics on blogs and stuff. and i quite like the ones below....
i really like the santa one in this picture...
and also this site has great ideas..
and this is quite clever!
hope this helped!
Hmm... I'm not sure if it would work for a cupcake, specifically, but my favorite cake recipe for Christmas is the White Spice Pound Cake found in the Cake Bible. Love it. Especially with a light, egg-nog flavored buttercream.
For me its the robin cupcake here:
Typical English Christmas card design. Looks too good to eat.
Of all the Christmas cupcakes I've ever done, my most popular request is the Winter White Cupcake I did. Took a special 1 day Wilton Class and they turned out so good. You can search for Maria46 and fine them in my gallery or you can search for Winter White Cupcakes.
That's really great idea to planning the favourite Christmas cupcake. Here are my favourite Christmas cake that i really like
Thank you,
Here are my favorite Christmas cupcakes! Real pieces of art! Enjoy!
I'm planning on making gingerbread with cinnamon cream cheese frosting and a little gingerbread man cutout, egg nog with egg nog buttercream, peppermint cake with white chocolate buttercream with crushed candy canes on top, and sugar cookie cupcakes with vanilla buttercream with sanding sugar or a small cutout cookie topper.
I think you got plenty of ideas on what cupcakes you can make; so would just give you some tips on cupcake boxes. As you just mentioned that you need to gift them, have you thought about the boxes you'll use to pack them up. I think you should buy or make theme-based cupcake boxes, which is Christmas here, of course.
For instance, you may buy a simple yellow colored cardboard box and decorate it using golden sparkle and tie it with a single beautiful ribbon. Be sure to make the knot properly else it will look bad. You can also paste some graphics or logos onto the box like you may write "Happy Christmas" on the box with sparkle or color pens and add a picture of Santa or goodies. It will look amazing.
And if you are going to throw a party, you can also place your delicious cupcakes on stands for your guests to enjoy.
Hello I am a new member here ant I love red velvet cupcakes with creme cheese frosting.But last year I had maked a turrón(it is from Spain in Christmas) cupcakes and were delicious.Bye!
also a nice flavour would be.....
chocolate with a candy cane buttercream
traditional fruit cake with a fruit mince filling
redvelvet with a eggnog buttercream
:) hope this helped!
Original message sent by beantownsara88
I love Christmas! I have been thinking about this already too... I think I am going to do a Creme de Menthe cupcake (filled with ganache). I also want to do a Gingerbread Latte cupcake. Haven't quite figured out what the components will be yet though. I just love gingerbread lattes!
Do you have th recipe for Creme de Menthe cupcakes you wouldn't mind sharing? They sound soo good, ok so I'm a but of a peppermint(ish) addict!


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I adore diplomatic language. I love the insinuations, the backhanded compliment, the veiled insults… it is, I believe, what makes reading countless speeches and articles worth doing.
For this reason, I love Christine Lagarde. I love how she gently slid a shining stiletto into Chancellor George Osbourne's soft gut, a belly bloated by the nourishment he's denying to so many in our society, so desperately in need.


text: Last night at the annual Tony Awards, the soon-to-be-on-Broadway musical Ghost showed off a little scene of supernatural singing. Based on the 1990s movie starring Patrick Swayze, a ghost — represented as a ghost on stage by the blue light that follows him — stalks his non-dead girlfriend. Here's the Broadway cast singing their version of "Unchained Melody" onstage. It's all very dramatic, but we kind of love the blue boyfriend.


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Globally, over 780 million people lack access to safe drinking water and 2.5 billion people lack access to sanitation. A growing population and rising food demands may increase the call for water associated with agriculture, which already constitutes 70 percent of the claim on freshwater resources. Projections are that by 2025, two-thirds of the world's population could be living in severe water stress conditions. Ensuring the availability of safe water to sustain natural systems and human life is integral to the success of the development objectives, foreign policy goals, and national security interests of the United States. To address global water-related development needs, the Water and Development Strategy provides increased focus for USAID's water programs, emphasizing approaches that are sustainable, well-targeted, informed by past effort, work through host country systems, and use emerging science and technology.
Last updated: May 22, 2015


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Jury selection getting back under way after delay in Maine Zumba studio prostitution case
ALFRED, Maine (AP) � Jury selection in the trial of a man accused of helping a Zumba fitness instructor run a prostitution business is resuming in open court, where members of the jury pool could face embarrassing questions about their views on sex, adultery, pornography and prostitution.
The judge in the trial of married insurance businessman Mark Strong Sr. held two days of closed-door questioning of potential jurors before the state Supreme Court ordered the process opened to the public. Jury selection was to resume Wednesday morning.
Jury selection stalled for more than three weeks after prosecutors appealed the dismissal of 46 invasion-of-privacy counts against Strong. Strong, who's from Thomaston, still faces 13 other counts dealing with promotion of prostitution.
Strong and fitness instructor Alexis Wright, who ran the Zumba studio in the seaside community Kennebunk, have pleaded not guilty.
Prosecutors say Wright engaged in prostitution in the dance studio. She'll be tried later.
Justice Nancy Mills has gone to great lengths to seat a jury despite intense news overage generated by the prostitution scandal in Kennebunk, a town of about 10,000 residents known for its ocean beaches and old mansions. More than 140 potential jurors were called for the case, and each of them had to fill out a questionnaire.
The case has generated national and international headlines because of its location in a quiet seaside hamlet next to Kennebunkport, home of the Bush family's Walker's Point summer compound, and the scale of the prostitution alleged by prosecutors.
Police have said more than 150 people are suspected of being clients and many of them were videotaped without their knowledge. A lawyer who has seen a client list says it includes some prominent names. Those who've been charged include a former mayor, a high school hockey coach, a minister, a lawyer and a firefighter.
Strong said he helped Wright launch her Pura Vida dance-fitness studio in Kennebunk by co-signing for her lease and loaning money that was repaid in full. He said he didn't know about allegations of prostitution.