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And this is just some thoughts I scribbled as I am writing another long long editorial.
Why are these dramas popular now?
|The developmental history of Korean TV dramas
|Korean dramas, scooped from the sea of myths
|The TV home drama world
|Predictable drama, touching love: How to enjoy the melodrama in drama


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By: Richard Hargis
Disclosure of prior property inspection reports is always unsettling for sellers and brokers. A bad prior report (typically from a busted contract) can adversely affect the continued marketing and sale of a property. A favorable or neutral inspection report would have the opposite effect.
Regardless of how much it might hurt to disclose a prior inspection report, sellers and brokers should always weigh on the side of disclosure. It is well-settled that prior inspection reports constitute material facts concerning the property which a purchaser is entitled to know in making the decision whether to buy the property.
Recent court decisions make it clear that sellers have the duty to disclose information from prior inspection reports obtained by buyers. In one such case from Texas, the jury held sellers liable for failing to disclose the existence of foundation defects revealed in a prior prospective buyer's engineering report, where the prospective buyer terminated the earnest money contract based on the report.
The facts of the case are illustrative of the court's ruling. The prior buyer discussed the unfavorable engineering report with the sellers and offered to sell them a copy of it. The sellers refused, stating that they did not intend to put any additional money into the house before selling it. Thereafter, the sellers listed the property with another broker, but did not disclose knowledge of foundation defects on the state-mandated Seller Disclosure Notice. After the listing terminated without a sale, the sellers advertised the house for sale on their own.
A new buyer prospect responded to one of the advertisements, which described the house as being in "excellent" condition. Although the new buyer noticed minor cracks and a slight slope to the floor, the sellers assured them that the cracks were superficial and routine for a house in that area. The engineer hired by the buyer to do a "walk through" inspection failed to detect defects in the foundation, and the sellers never disclosed the results of the prior inspection.
The buyer purchased the house, and lived there for approximately 3 years before attempting to sell the house. An interested buyer was provided copies of the inspection reports prepared for the current owners when they purchased the home. The buyer decided to hire his own inspector and, by chance, contacted the same engineer who prepared the report withheld from the current owners by the previous sellers, which showed the foundation was defective. The buyer demanded to be released from the contract, and the owners complied. Thereafter, the current owners brought suit against the previous seller.
The jury found that the previous seller knowingly engaged in a false, misleading, or deceptive act or practice and an unconscionable action or course of action, and assessed punitive damages.
In addition to pointing out the need for sellers to reveal information in prior inspection reports, the court's ruling also says, very clearly, that a seller is not absolved from liability just because a buyer had the opportunity to have his own inspections performed. The seller cannot use the failure of the buyer's inspector to find defects as a defense.
The message is clear: Prior reports constitute material facts. A seller's duty to disclose his knowledge of defects does not end when he completes a written Seller Disclosure Notice during the listing appointment. Rather, the seller's knowledge encompasses prior inspection reports obtained by prospective buyers. Sellers have the ongoing duty to disclose any defects revealed in those reports.
J. Richard Hargis is a practicing Houston attorney, Hargis & Harpold, L.L.P. He is a frequent speaker on real estate topics for continuing education programs and before businesses and trade associatons. His articles are regularly featured in local and state real estate publications.
Copyright© 1998, J. Richard Hargis. All right reserved. For information on Mr. Hargis' keynote presentations, continuing education programs, or consultation, please contact Susie Hale, The Frog Pond Group at 800-704-FROG (3764) or email firstname.lastname@example.com.
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subset: commoncrawl


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While the tragic explosion in Jakarta last week is an obvious setback to those combating terrorism in Indonesia, it does not erase the very substantial progress in fighting terrorism – progress that's been ongoing since the 2002 bombings in Bali that killed over 200.
Progress on the prayer front plainly needs to be ongoing as well. Prayer, by individuals anywhere on the planet, can reach out to the Almighty as the source of protection and security. Could prayer, filled with a conviction that the presence of the Divine makes a practical difference, have the potential to enhance counter-terrorism efforts? Absolutely.
Prayer has the capacity to spiritually enhance the safety and security of each individual in a terrorist-prone region. The book of Job in the Bible gives a powerful starting point for such prayer when it says, "Thou shalt be secure, because there is hope" (11:18). Think of that. The Bible not only promises security, but suggests why one can expect security. "Because there is hope." That's why security is realistic, is obtainable.
But if there is security because there is hope, one might ask, then why is there hope? The answer is both simple and profound. There is hope because there is God. The God of promise and of fulfillment who the author of Job glimpsed, the God who is both the divine Life and immortal Love that Christ Jesus so magnificently knew and proved, the God who meets humanity's needs today for comfort and protection as people learn how to turn more completely to Him in prayer – that God is why there is hope. And that hope is why there is security.
The key for progress on the prayer front is not simply to agree that prayer is a good thing, and then go on one's way. The key is to actually do it. To actually pray. To pray relentlessly. To understandingly and unflinchingly turn one's thought and one's heart to divine Life and Love in the same way that the master Christian did.
If you're a Bible reader, you may recall a terrorist episode that crashed in on Jesus. At one point in his ministry, a hostile mob encircled him with the intent of throwing him from the brow of a hill. Was he without hope? One can only assume from the outcome that his hope and confidence remained unshakable. His prayer-based security unfolded in the most unexpected of ways. He apparently simply became invisible to them and went on his way (see Luke 4:14-31).
Monitor founder Mary Baker Eddy discovered that the safety and security emanating from divine Life and Love are so certain, so reliable, so consistent, as to be scientific. She wrote in her primary work, "Science and Health with Key to the Scriptures," "Security for the claims of harmonious and eternal being is found only in divine Science" (p. 232). As people commit themselves to a prayer-based response to terrorist acts, they'll find those prayers serve as a spiritual underpinning to people on the front lines of the campaign to thwart terrorism. They may even glimpse the scientific certainty that because there is God, there is hope. And because there is hope, there is security. We can at least begin to prove this today.


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text: Home > Member Resources > News Service News Service Page ContentNCManagers.org is pleased to offer a news aggregate service for its readers. Sign up today and receive these links straight to your email inbox. These news links are culled from a variety of newspapers and websites from across the state and nation using key words pertinent to our profession. However, please note that NCCCMA is not responsible for the content or tone of the subject matter in these articles. Sign up now.


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Posted 8 years ago
(60 items)
Some nice find's out in the flea market,s! The #5 Fantasy Masterpieces comic is in very nice shape.I would of loved to have found the # 19 Marvel double feature but not to be.The two came from a three for $ 1.00 box and the other was for a great price. Keep Read'm and collect'm there fun.! Frosty21


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Stanford post and former Cy-Fair standout Chiney Ogwumike debuted the second installment of the "Nerd Nation" videos that were made popular two years ago when the women's basketball team wrote, filmed and produced a music video called "Nerd City Kids."
This year, Ogwumike – the Pac 12 Player of the Year – stars in the music video called "N-E-R-D-S." The video includes several student-athletes from Stanford and a few alumni, like Ogwumike's older sister Nneka (Los Angeles Sparks) and Richard Sherman (Seattle Seahawks).
The first video went viral a couple of years ago and the nerd nation label has stuck with Stanford. This year, the video was timed to come out on Selection Monday, when Stanford will find out what seed and where it will play in this year's NCAA women's basketball Tournament.
"We never knew it would get that big," Ogwumike said. "But we wanted to do something that showed that we were student-athletes, that we care about our studies and our sports and that it's ok to care about both. We are proud to be somewhere where academics matter. It's been so much fun."
The new video:
The 2012 video:


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Having problems with your Acer Aspire One PC Notebook ?
Not reading DVD discs
\015 Computer doesn t reading DVD discs\015
Answers :
\015\012has it been reading DVD discs before? Also, does it read other discs apart from DVD's?
\015\012It is possible that your DVD drive's lens (laser) is weak. You can try to use a lens cleaner or cutton board to clean the lens.
\015\012Take care.
\015\012has it been reading DVD discs before? Also, does it read other discs apart from DVD's?
\015\012It is possible that your DVD drive's lens (laser) is weak. You can try to use a lens cleaner or cutton board to clean the lens.
\015\012Take care.
Repair Help & Product Troubleshooting for Acer Aspire One PC Notebook
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text:
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Posted on October 14, 2012


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All fans of the UFC are in for a treat when former bantamweight champ Ronda Rousey makes her much anticipated return to the octagon against the current UFC Women's Bantamweight Champion, Amanda Nunes.
Ronda Rousey
After becoming the first American to win an Olympic medal in women's judo – bronze in 2008, Rousey burst onto the pro MMA fight scene and could seemingly do no wrong early in her UFC career. Rousey (aka Rowdy, The Arm Collector) burst onto the pro MMA scene winning 12 consecutive Mixed Martial Arts fights including 6 in the UFC, nine of these wins came by way of her signature armbar submission. She became the poster girl of UFC and life seemed perfect for the American athlete.
Rousey's career made a U-turn after a convincing loss to Holly Holm by way of a spectacular knockout. Rousey suffered from severe depression as a result and she took some time away from MMA to recover. Now she's back to prove that she still has what it takes to be named the best bantamweight female fighter in the UFC, but it won't be a walk in the park.
Amanda Nunes
Someone who would love to stop her in her comeback fight is the current UFC Women's Bantamweight Champion Amanda Nunes.
Fighting out of Salvador, Brazil – a country known for producing great UFC fighters over the years, Nunes is known as "The Lioness of the Ring". Nunes started training Karate from a young age and at age 16 also trained in boxing. Brazilian jiu-jitsu was what she pursued next, earning herself a black belt before competing in MMA. Following brief stings in MMA competitions Strikeforce and Invicta Fighting Championships, Nunes began her UFC career in 2013 and has risen to the top of the ranks, most recently defeating Miesha Tate at UFC 200, gaining the UFC Women's Bantamweight Championship.
My Free UFC 207 Betting Tips
Rousey has spent a long time out of the octagon and all fight fans know her strengths, on the flip-side so do her opponents. Her weaknesses were on full display against Holly Holm, a fight in which Rousey was dominated from beginning to end. I don't think that Rousey has the mental toughness to win this one, her last loss would have put a lot of doubt in her mind. I believe Nunes should be starting as short favourite to win.
My tip: Nunes by knockout. Nunes has proven she has KO power and I think she'll find an opening in Rousey's defenses within the first few rounds.
If you do choose to bet on either fighter to win via the method of victory betting market, you will qualify for a unique bonus offer from CrownBet.com.au listed below.
CrownBet's Unique UFC 207 Bonus Offer
Place a bet on the "Method of Victory" market for our nominated fight.
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Method of Victory Betting Odds
My tip is listed in bold text.
Bet on the method of victory to qualify for CrownBet's unique special offer listed above.
|Ronda Rousey to win by KO
|Ronda Rousey to win by Submission
|Ronda Rousey to win by Points
|Amanda Nunes to win by KO
|Amanda Nunes to win by Submission
|Amanda Nunes to win by Points
Other Top Markets and Betting Odds
The following markets don't qualify for CrownBet's special offer.
Round/Method of Victory Double
Round Betting
All odds were correct at the time of posting and are subject to change.
Good Luck and Happy Punting!
Aussie Punter


text: At Salon 131 in Seminole we come together as a team and do our best to satisfy all of your hair care needs, right in your NEIGHBORHOOD! We offer all styles, from short hair cuts to longer cuts. By keeping up with the current trends and continuously educating ourselves we are able to help you achieve the look that your favorite actors and pop stars wear. You can also create your own new and cool hair style complete with coloring to go with it. Our beauticians are here to help you create the look of your dreams or the season, whichever.


text:
I wish Nigella Lawson were the last straw. Like Rihanna and Halle Berry and Brooke Muelller, this week the seductive British TV chef—beloved for her deep-fried chocolate recipes and now a host on The Taste—became domestic violence's latest poster girl. Only this time, that poster is more like a mirror.
Because we have to ask: What we would have done had we been sitting at the next table on June 6, the day she was eating outside at a posh London restaurant, and suddenly her husband, Charles Saatchi, is reportedly grabbing her throat, and, no, he's not putting a diamond necklace around it; he's choking her, and she's making a terrible, awful "Stop!" face? For the most part, no one did anything. Well, that's not quite true: Someone snapped photos—which rocks because it seems to have allegedly shamed Saatchi to come forward to the police and cop to assault, and Lawson to leave their home with her children. Perhaps he noticed the passerparazzi and that's why he loosened his grip. But had he held it, who knows? It's possible it would have only taken 10 seconds for Nigella to lose consciousness and four to five minutes for her to die. Like what happened to Sylvie Cachay, 33, the up-and-coming swimsuit designer found strangled—BTW, that's the word for a neck grab (choking is what you do on a sandwich)—in a Soho hotel bathtub and whose boyfriend, Nicholas Brooks, is on trial right now for her murder.
Believe me, I understand paralysis. You're hanging out with a mimosa, nudging your girlfriend Hey, isn't that Nigella Lawson? And then something catches your eye—Like what? Is her husband really doing that? Nooooyessss. And you freeze because you wish you hadn't seen it, but damn it, you did. And what does an ordinary person drinking a mimosa do now? Get in a celebrity's face? What if it's not what you think? Really. And what do you ever do when you see a guy getting violent with a girl?
I've written a lot about domestic abuse and heard chilling stories from victims about being beaten to the ground in public and witnesses just walking by. And other stories where a stranger stepped in to say something—and saved the woman's life. Bell Bajao is a campaign that suggests when you see a violent couple, make some kind of noise to cause a distraction, including going up to a neighbor's house where you hear screams and ringing the bell. The idea is to let the abuser know you've noticed and possibly intercept an escalating rage. In the same way, taking photos can out the person. Other advocates suggest that, where possible, you ask the victim if she needs help.
In general, experts stop short of advising that you jump into the middle of the fray—it could trigger the abuser to become more dangerous to either his partner or to you. But they do say to call the police, or 911. And if you aren't sure how to assess the situation, get help at the national domestic violence hotline: 1-800-799-7233
The main thing is to do something. Because doing nothing is one more vote that as a culture we accept that it's OK to strangle your partner a little, even in public, and maybe hit her, punch her, break a bone. A couple of years ago, I spoke to Nancy Glass, Ph.D., at the Johns Hopkins University School of Nursing who has researched domestic violence homicides. When a man strangles his partner, she told me, it increases the odds that he will go on to kill her about seven-fold.


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Local baseball fans were aflutter last Friday with the announcement by the St. Louis Cardinals that the major-league club had agreed to purchase their longtime Triple-A affiliate, the Memphis Redbirds. As stated in a release by the Cardinals:
"In an effort to rescue their financially troubled Triple-A affiliate, the St. Louis Cardinals will acquire the Memphis Redbirds. The club's home ballpark, AutoZone Park, will be acquired by the City of Memphis."
Hold on, though, said Memphis mayor A C Wharton later that same day. When it comes to the city's purchase of AutoZone Park, it appears a deal has yet be struck. Said Wharton, as quoted by Daniel Connolly in The Commercial Appeal:
"I do not have a contract. I do not have an agreement or even a draft of agreement to review. . . . What we have, I guess, is sort of a clash of business cultures in which the private sector is moving at a pace that is not exactly in sync with what I, serving in the public entity, have to honor . . . ."
And so the drama continues, a story of "business cultures" now more than 15 years old, since Dean and Kristi Jernigan first secured the $72 million in bonds to build "the finest baseball stadium below the major-league level." That glorious ballpark at Third and Union remains very much a standard to be matched by other minor-league operations. But with more than $20 million of debt giving its brick exterior an extra shade of red, the stadium's value as an asset — not to baseball fans, really, but the city of Memphis — remains vague at best.
The fact is, AutoZone Park was a feat of financial wizardry on the part of the Jernigans. Consider that no other minor-league ballpark had cost as much as $50 million to complete. (Victory Field — home to the Triple-A Indianapolis Indians — opened in 1996 and cost all of $20 million.) Its 44 luxury suites — on two levels — are 44 more than can be found in many, if not most, minor-league parks across the country.
The stadium is the realization of a dream-like vision, but with an economic foundation no more healthy than a heaping dish of barbecue nachos. It's one thing to own and operate the baseball team that plays in the stadium (the Cardinals seem ready and willing to play this role). It's quite another to turn the facility itself into a source of measurable profit, particularly if the new owner is a city government weighted with declining population and stagnant, if not shrinking, business development. At the very least, the city cannot afford to lose money on AutoZone Park. Thus, Mayor Wharton's pregnant pause last Friday.
The Redbirds are hosting an invitation-only event Tuesday evening, one where Cardinals owner Bill DeWitt and general manager John Mozeliak are expected to appear. You have to presume John Pontius and Ray Pohlman — the faces of the nonprofit Redbirds Foundation — will be there too. It will be a gathering of "players" with deep interest in making the business of Redbirds baseball a long-term success story.
Keep your eye on those 44 suites. The park's most distinguishing luxury features were signed to 15-year leases before the park opened in 2000, meaning they are up for grabs after the 2014 season. The Memphis economy was quite different at the turn of the century. (To begin with, there were no Memphis Grizzlies and no FedExForum with its own luxury suites.) For that matter, the global economy was quite different at the turn of the century. Those suites have each brought in an average of $40,000 per season, a revenue stream north of $1.5 million. How many will be renewed, and at what price?
AutoZone Park isn't going anywhere. The Cardinals are flush with the finest minor-league system in baseball, with Memphis the final training ground for rising stars like Michael Wacha. The big righty pitched his first Triple-A game in downtown Memphis last April, then was named National League Championship Series MVP in October. The ultimate dream for AutoZone Park, it turns out, is creating revenue streams as strong and steady as the flow of talent on its way to Busch Stadium.
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I will need to do a few things with my master mold before it's ready for the plaster. The biggest of these is going to be separating the gusset into two pieces. Normally this is done after the rubber copy has been made in order to keep the mold lines on the original to a minimum.
I need to do this because the gusset piece will not release the casting without risking a break to one of the legs. The problem with Imp, however, is that those legs (and the necessary pour hole) occupy a really small space. It's going to be tight working no matter where I cut.
I decide to run my split down the forward tripoded leg. The gusset usually splits to either side of the pour hole. I've already had to make a pour hole that does not bisect the gusset because there wasn't enough room between the two legs on that side. It was tempting to split the gusset along the pour hole and spare that front leg the mold line, but I suspected that design wouldn't really give me much more wiggle to get the piece out with all its legs intact.
The downside was that it would be a complicated cut. I would need to carefully cut the line down that inner front leg, but it would need to fall quite further back if I was going to miss truncating the raised foreleg on the other side. (This will become more clear in the next post with pictures of the resulting piece.)
I should add here that I hate cutting rubber. The rubber I use is really tough. It doesn't cut easily and it will quickly dull an Xacto blade. The important initial slice down the leg, using the point of the blade, isn't hard. After that I spent most of the time thinking, "This is a recipe for lopping off a finger" and "gee, maybe I should have placed the phone next to the workbench in case I have to call for medical help". The split was rough, but I cut it without incident (other than a few new gray hairs).
With the gusset split, all that was left was to fill the sprues. I'm not really picky about getting these just right, especially since the plasteline doesn't really stick well to the rubber. Mostly I just want to cut down on the clean-up I will have to do with each plaster piece.
Now I am ready to start. The basic idea behind a rubber master mold is that, one by one, the pieces will be replaced with a plaster version, until all that is left is a plaster mold encasing the rubber original. The rubber original is then removed and the plaster mold is ready for ceramic slipcasting, and the rubber pieces can be reassembled to make the next plaster mold.
When I began the rubber mold, I started with the big side pieces first, and worked my way inside. The plaster molds go the other way; I start on the inside and work my way out. For this mold, my first piece is going to be the smaller, forward gusset section. That piece gets removed and the rest of the master mold is reassembled.
In the next step, I'm going to be pouring my plaster into the cavity left by the missing piece. The rest of my master mold is going to act as a framework with the rubber original creates the image in the plaster.


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PAVIR is incorporated in the State of California as a 501(c)(3) tax exempt corporation.
Our focus is on Driving Veterans Health Innovation, which we address by supporting research and education activities conducted at the VA Palo Alto Health Care System (VAPAHCS). The range of research activities is described in the research section of this website and represents the vast majority of our activities.
PAVIR also supports various educational instruction and other learning experiences of benefit to employees and veterans. For example, this website's current needs section illustrates our goal to fund professional development for researchers who are in the early stages of their career.
PAVIR is one of 83 active VA affiliated not for profits, based on the legal authority of Title 38 UCS §7361-7366, which authorizes VA medical centers to establish nonprofit research and education corporations to accept and administer private and non-VA federal funds in support of VA's research and education missions. The congressional intent in enabling these corporations is to provide VA facilities with a flexible funding mechanism for the conduct of research as well as staff and patient education.
As of May 2017, PAVIR employs 233 staff, most of whom directly work on research projects aimed at improving health care. PAVIR's core administration offices include 28 employees. The core administration offices provide services including:
- Contracts & Grants administration, both pre- and post-award
- Clinical Trial services, primarily negotiation of budgets and Clinical Research And Development Agreements (CRADAs)
- Finance and Accounting
- Purchasing
- Information Technology
- Human Resources
Prior to July 1, 2014, we operated under the name of Palo Alto Institute for Research and Education (PAIRE).


text:
July 8, 2013
One out of every eight women develops breast cancer at some point of her life. This disease is the second leading cause of cancer death among women, but early detection and screening can save lives.
November 10, 2012
An in-depth and graphical look at some of the more interesting facts and figures surrounding breast surgery in the United States. Source


text:
Hello and welcome to ExamPundit. Here are the Important Current Affairs of 1st September, 2015. These are Important for the upcoming IBPS PO V Exam. We are also providing 5 Stock GK Expected Questions.
RTGS will not operate
on 2nd, 4th saturdays: RBI
on 2nd, 4th saturdays: RBI
- The RTGS system, which allows real time transfer of large
funds, will not be operated on second and fourth Saturdays with banks observing
public holiday on that days, the Reserve Bank said on Tuesday.
- However, the system will operate for full days on other
Saturdays of a month, RBI said in a notification.
- Banks are observing public holiday on every second and
fourth Saturdays of a month.
- RBI has also effected changes in RTGS time window.
- As per the new time schedule for regular days, including
Saturdays except second and fourth, business opening would be at 0800 hours
while initial cut off (customer transactions) would be 1630 hours. The final
cut-off (inter-bank transactions) will be at 1945 hours and IDL reversal
between 1945-2000 hours.
- The close of business is at 2000 hours.
- As per earlier timings for Saturdays or short days, the
initial cut-off was at 1400 hrs and final cut off at 1500 hours.
- The close of business was at 1530 hours.
- RTGS means Real Time Gross Settlement which refers to
continuous or real time settlement of funds.
- It is primarily meant for large value transactions. The
minimum amount to be remitted through RTGS is Rs 2 lakh. There is no upper
ceiling for RTGS transactions.
At 7 per cent, India
remains fastest growing major economy
remains fastest growing major economy
- India remained the fastest growing major economy of the
world for a second straight quarter, with GDP growing at 7 per cent during
April-June 2015 against 6.7 per cent in April-June 2014. Growth in the previous
quarter, January-March 2015, was, however, higher at 7.5 per cent.
- Data released the Central Statistics Office (CSO) on Monday
showed marginal improvement in private consumption expenditure but gross
capital formation rate — a barometer for investments — continued to decline. It
was down to 27.8 per cent from 29.2 per cent in April–June 2014.
- A source at the CSO explained that growth would have been
better had it not been for the poor rabi crop.
- Growth during the quarter was driven primarily by the
services sector. The manufacturing and construction industries recorded
favourable growth too. The agriculture sector saw a decline, growing at a mere
0.6 per cent. Growth also slowed in the sectors 'mining and quarrying' and
'electricity, gas, water supply and utility services'.
- In terms of the gross value added, (GVA) growth was 7.1 per
cent against 7.4 per cent in April-July 2014. The revised methodology for GDP
(Gross Domestic Product) calculation subtracts subsidy and adds taxes to the
GVA to arrive at the GDP.
- The Modi Government's budget announcement of higher public
spending this financial year did not reflect in the data released as at growth
was low at 2.7 per cent in 'public administration, defence and other services'.
Union Government
approved Smart National Common Mobility Card model for seamless travel across
approved Smart National Common Mobility Card model for seamless travel across
- The Union Ministry of Urban Development on 1 September 2015
gave approval for Smart National Common Mobility Card (NCMC) model for seamless
travel across India.
- The model will enable commuters to travel by different
metros and other transport systems across the country besides retail shopping
and purchases.
Features of Smart
National Common Mobility Card (NCMC)
National Common Mobility Card (NCMC)
- It is based on EMV Open Loop Card system with stored value
- It meets travel needs based on stored value of money and can
be used for travelling by any means of transport and also enables account based
retail applications.
- The model is an improved version of nationwide common card
prevalent in Singapore.
- The National Payments Corporation of India (NPCI) will be
involved in indigenous development and management of Clearing and Settlement of
payments, Simulating Cards, Terminals and network, a support base of vendors
for providing certified tools, cards, terminals and other services.
- The Centre for
Development of Advanced Computing (C-DAC) will develop the standards and
hardware for Metro gates/validators along with related ecosystems.
- Funding of 4.47 crore rupees approved by the ministry for
developing the model.
Entertainment ties up with French studio Pathe
Entertainment ties up with French studio Pathe
- Anil Ambani's film production and distribution outfit
Reliance Entertainment has tied up with French film production and distribution
house Pathe to distribute director Gurinder Chaddha's next movie titled
Viceroy's House.
- This is the first time that Pathe has collaborated with an
Indian outfit for the distribution of a film.
- Pathé will distribute the film in the UK and France and will
handle sales throughout the rest of the world while Reliance Entertainment will
distribute the film in India.
- The company holds leadership position in the art-house and
indie film distribution space in the UK and France.
- Apart from producing and distributing films (in cinema and
home video), the company also owns cinema halls in France, Netherlands and
Switzerland (total of 1,000 screens).
Anjali Duggal takes
charge as Financial Services Secretary
charge as Financial Services Secretary
- Anjuly Chib Duggal, a Punjab cadre IAS officer, has assumed
charge as Secretary in the Department of Financial Services in the Finance
- She has replaced Hasmukh Adhia, who has taken over as
Revenue Secretary in the same Ministry.
- Prior to taking over as Financial Services Secretary, Anjuly
was holding the charge of Corporate Affairs Secretary.
- She had also earlier worked in various capacities including
Joint Secretary, Additional Secretary and Special Secretary in the Department
of Expenditure in Finance Ministry.
A K Jha takes charge
as NTPC interim CMD
as NTPC interim CMD
- NTPC's Director (Technical) A K Jha today took additional
charge of Chairman & Managing Director of the company as an interim
- After refusing to give extension to Arup Roy Choudhury as
CMD of NTPC, the government has decided to give additional charge to Jha for
three months from September 1 as an interim arrangement.
- A search committee headed by Power Secretary P K Pujari is
currently looking for a full-time CMD for NTPC.
- The committee will send the list of short-listed candidates
to the Appointments Committee of the Cabinet for a final call.
- Jha, 58, is a graduate in Mechanical Engineering from BIT
Sindri, Ranchi University, and holds LLB from the Delhi University.
- He joined NTPC in 1977 as an Executive Trainee and was
directly associated with its flagship project — Singrauli (5×200 MW).
India beat Sri Lanka
by 117 runs to win series 2-1 in Sri Lanka after 22 years
by 117 runs to win series 2-1 in Sri Lanka after 22 years
- India today clinched their first Test series victory on Sri
Lankan soil in 22 years with an emphatic 117-run win over the hosts in the
third and final cricket Test despite a valiant century by rival skipper Angelo
- After setting a daunting victory target of 386, India
managed to dismiss the Sri Lankans for 268 on a wicket which slowed down
considerably to wrap up the series 2-1, their first overseas series victory
since June, 2011 against the West Indies.
- It was new captain Virat Kohli's first series win ever since
he took over reins of the team from Mahendra Singh Dhoni.
- The last time an Indian team won a series in Sri Lanka was
way back in 1993 under Mohammed Azharuddin's leadership when they sealed the
series with 1-0 scoreline.
- The fifth day's action was not devoid of drama as more
on-field altercation took place with Kusal Perera being involved in a heated
exchange with Indian players.
- Ashwin took the last wicket of Nuwan Pradep to bring about
India's moment of glory, triggering scenes of wild celebrations in the dressing
room as the players hugged each other and picked up the stumps as mementos.
Ishant Sharma, three
Sri Lankans charged for misconduct
Sri Lankans charged for misconduct
- India paceman Ishant Sharma and three Sri Lankan players
have been charged for misconduct during the ongoing third test between the
neighbours, the International Cricket Council (ICC) said on Tuesday.
- Sharma, who was fined 65 per cent of his match fees for
giving send-offs to Lahiru Thirimanne and Dinesh Chandimal in the second test,
landed in trouble with the Sri Lankans in the final contest as well.
- Thirimanne, Chandimal and paceman Dhammika Prasad, who had
an animated altercation with Ishant on Monday, have also been charged with code
of conduct breaches, the ICC said.
Highest mountain of
North America Mt McKinley renamed as Denali
North America Mt McKinley renamed as Denali
- The highest mountain of North America was on 28 August 2015
renamed as Denali instead of its existing name Mt McKinley by US President
Barack Obama. This designation will now recognize the sacred status of Denali
to generations of Alaska Natives.
- The mountain, which sits in the 6 million-acre Denali
national park, has been known as Denali in Alaska since 1975. Now officially,
the Denali name will be used by all and it will also take effect for all
federal usage and on all official maps.
- The change comes after a debate between lawmakers from
Alaska and Ohio. In January 2015, Republican Senate Lisa Murkowski of Alaska
introduced legislation that sought to officially call the mountain what
Alaskans and indigenous people called it Denali or The High One.
- The historic change came during Obama's three-day
presidential trip to Alaska. It is the latest bid by the President to fulfill
his 2008 campaign promise to improve relations between the federal government
and the nation's Native American tribes.
Tejinder Pal Singh
named as Australian of the Day by Commonwealth Bank
named as Australian of the Day by Commonwealth Bank
- An Indian-origin driver in Australia Tejinder Pal Singh was
on 31 August 2015 named as Australian of the Day by Commonwealth Bank of
Australia for feeding the homeless in Darwin for the past three years.
- He dedicated the last Sunday of every month to feed the poor
and homeless in northern Darwin after his night shift as a cabbie. He cooked
around 30 kg of Indian food to feed the homeless. The free lunch comprises
chickpeas, rice and vegetarian curry.
- His van itself carries the sign Free Indian food for hungry
and needy people, Provide Sikh family.
- The Commonwealth Bank has been sponsoring the Australian of
the Day campaign for over 35 years. The campaign recognises the extraordinary
Australians who have made a big difference to the country.
& Authors –
& Authors –
30 Women in Power:
Their Voices, Their Stories by Naina Lal Kidwai
Their Voices, Their Stories by Naina Lal Kidwai
- The book 30 Women in Power: Their Voices, Their Stories was
in news in August 2015. It was written and edited by Naina Lal Kidwai.
- It is a collection of narratives on modern Indian women who
have been pioneers and led large organizations – in banking, law, the media,
advertising, government services, health care, consulting, the fast-moving
consumer goods sector and the not-for-profit space.
- She is an executive director on the board of HSBC
Asia-Pacific and the chairman of HSBC India.
- She is also a non-executive director of Nestle SA and was
the first woman president of the Federation of Indian Chambers of Commerce and
Industry (FICCI).
Expected Questions on Stock GK:
Expected Questions on Stock GK:
1. Dada Dhuniwale
Thermal Power Plant is located in which state?
Thermal Power Plant is located in which state?
– Madhya Pradesh
2. Sri Penusila
Narasimha Wildlife Sanctuary is located in which state?
Narasimha Wildlife Sanctuary is located in which state?
– Andhra Pradesh
3. Kanaka Durga Temple
is located in which state?
is located in which state?
– Andhra Pradesh
4. Captain Roop Singh
Stadium is located in which city/state?
Stadium is located in which city/state?
– Gowalior, Madhya Pradesh
5. Which of the
following is the capital city of Paraguay
following is the capital city of Paraguay
– Asuncion
Team ExamPundit
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text:
- Methodology
- Open Access
A streamlined method for systematic, high resolution in situ analysis of mRNA distribution in plants
© Drea et al; licensee BioMed Central Ltd. 2005
- Received: 31 August 2005
- Accepted: 06 October 2005
- Published: 06 October 2005
In situ hybridisation can provide cellular, and in some cases sub-cellular, resolution of mRNA levels within multicellular organisms and is widely used to provide spatial and temporal information on gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time. Whole-mount and reverse transcriptase-PCR (RT-PCR) based protocols increase throughput, but can compromise both specificity and resolution. With the advent of genome-wide analysis of gene expression, there is an urgent need to develop high-throughput in situ methods that also provide high resolution.
Here we describe the development of a method for performing high-throughput in situ hybridisations that retains both the high resolution and the specificity of the best manual versions. This refined semi-automated protocol has the potential for determining the spatial and temporal expression patterns of hundreds of genes in parallel on a variety of tissues. We show how tissue sections can be organized on microscope slides in a manner that allows the screening of multiple probes on each slide. Slide handling, hybridisation and processing steps have been streamlined providing a capacity of at least 200 probes per week (depending on the tissue type). The technique can be applied easily to different species and tissue types, and we illustrate this with wheat seed and Arabidopsis floral meristems, siliques and seedlings.
The approach has the high specificity and high resolution of previous in situ methods while allowing for the analysis of several genes expression patterns in parallel. This method has the potential to provide an analysis of gene expression patterns at the genome level.
- Floral Meristem
- Cellular Resolution
- Automate Protocol
- Uridine Triphosphate
- Nucellar Projection
In situ hybridisation (ISH) is the method of choice for describing the spatial expression pattern of a given gene. High resolution protocols provide cellular and even subcellular resolution. In multicellular organisms, ISH complements northern blotting, RT-PCR and microarrays, where the extraction of RNA from whole tissues invariably results in the loss of spatial information. Microarrays allow many genes to be studied in parallel and are currently one of the most powerful tools to study gene expression. However, microarray outputs often need to be verified by independent methods, such as ISH [1, 2], and because these downstream methods have a much lower capacity, verification is usually limited to one or a few genes. ISH must therefore be made more efficient and less time-consuming.
A number of variations on the traditional in situ protocols have been reported, including whole-mount ISH (WISH) [3], in situ PCR [4, 5] and the use of vibratome sectioned tissues [6]. The main shortcoming of ISH is undoubtedly the low-throughput nature of the technique. In situ PCR (ISPCR) and RT-ISPCR are elegant techniques that can increase both sensitivity and throughput but they are at best only semi-quantitative [5] and it is desirable first to ascertain the expression pattern by conventional means in order to establish suitable conditions for each probe.
Efforts to make the ISH technique into a highly parallel, systematic process have been successful in flies and primitive chordates [7–9]. Attempts have been made to address this issue in plants using WISH and in situ PCR techniques [10, 11] although actual throughput remains undetermined.
High-throughput protocols used for animal embryos normally involve whole-mount methods [7, 8, 12], thus avoiding the need to section material. The challenges in applying similar techniques to plants include the large size of the tissues and the variable nature of the cell wall. These factors can variably compromise the penetration of probe and make microscopic examination more difficult and time-consuming. WISH is a possibility for Arabidopsis roots and seedlings [11], at least for low- and medium-throughput. However, when performed on other larger tissues, such as seeds, WISH may require embedding and sectioning after the in situ has been performed to evaluate the results [13]. Therefore, the high-throughput advantages gained in the early stages of such procedures are effectively cancelled out.
Promoter fusions with reporter genes are another option for cellular localisation of transcripts but this approach has recognised shortcomings [14]. Elements controlling gene expression are known to be located not only in the traditional promoter region upstream of the coding region, but intergenically and, potentially, a considerable distance from the gene [15, 16]. The resources required for mass transformation and the fact that not all plant species are amenable limits the application of this approach to well-studied model species.
As well as providing an independent means of screening genes for the desired expression profiles (differential expression, domain specific expression etc.) in gene discovery efforts, we envisage that high-throughput mRNA ISH is entirely feasible and will complement the ever-growing microarray data resources available [2, 29]https://www.genevestigator.ethz.ch/; http://www.weigelworld.org/resources/microarray/AtGenExpress/. Recently real-time RT-PCR has been adapted for high-throughput processing [30]. While these approaches provide a wealth of expression data for functional genomics, they are unable to provide the spatial resolution that often directly reflects functional involvement in developmental processes. This inherent drawback in microarray technology has been elegantly addressed using cell sorting to isolate pure populations of a given cell type from the Arabidopsis root. However, this innovative approach is limited to species where suitable and diverse cell line markers are available [31]. It is further limited to tissues whose cells can be separated and sorted: roots are susceptible to protoplasting enzymes but shoots and many other tissues are not.
With these considerations in mind, we have deconstructed the "traditional" ISH protocol and developed a protocol for ISH that retains high resolution and specificity but integrates a degree of automation to a standardised and streamlined protocol. We have used wheat grain and Arabidopsis floral meristems as tests for this new high-throughput protocol and show that it is capable of highly parallel processing.
Results and discussion
Generating an integrated protocol
(i) Plant tissue preparation
Plants grown under desired conditions were harvested, trimmed to allow penetration of solutions, and immediately fixed. These steps were carried out manually in both the standard and new protocols but, in order to automate the new protocol, the samples were placed in a Tissue-Tek Vacuum infiltration processor for further processing.
The Tissue-Tek machine permits use of a combination of vacuum and pressure to exchange solutions at defined times and temperatures, relieving the operator of a large number of tedious steps as well as standardising the process. The initial fixation, dehydration and wax infiltration steps can take over a week to complete using the manual protocol, whereas the automated protocol reduces this to 24 hours. We evaluated various plant tissues prepared by both methods for tissue integrity and preservation of mRNA (signal strength). Arabidopsis tissues are equally well preserved and stained by both protocols but the automated procedure provided enhanced preservation in developing wheat seeds (data not shown). Many plant tissues, such as mature leaves, are very difficult to embed in wax using the manual protocol but even these recalcitrant tissues can be accommodated by altering the timing or pressure of processing.
When the sections had adhered to the slide, and the silicone isolators were removed, the slides were treated to remove wax and to make the sections receptive to the hapten-labelled probes. These down-stream treatments are universal to all tissues and appropriate for automation. However, these treatments are complex and we evaluated which ones were functional (i.e. produced an enhanced, yet specific signal) and which were redundant. Using a training set of probes and the traditional manual protocol, we systematically eliminated or reduced each step in the protocol and visually evaluated the final result. Slides were processed in parallel but with a proportion subjected to a protocol that omitted one or more steps normally used. This led to a reduction in the number of ethanol dehydration steps and elimination of the 're-fixation" step after proteinase K treatment. Some steps, although not absolutely essential, appeared to enhance the reliability of the process and these were retained: for example, acetic anhydride treatment was found to reduce background (especially on poly-lysine coated slides) and we increased the time allowed for de-waxing in xylene while applying agitation using the VP2000 slide processor (see below).
Finally, using a basic open-plan slide processor (VP2000), common in medical cytology labs, a reduced section-processing protocol with essential steps only was automated and made completely hands-off. This also eliminates much of variation possible in a multi-step, closely timed procedure and led to more reproducible signals.
(ii) Probe-making
We also assessed the yield of different RNA polymerases. Although in vitro transcription is possible with any one of T7, T3 or SP6 RNAPs, T7 was undoubtedly the most efficient providing high yields of almost every cDNA. SP6 was found to be the least consistent, but could produce reasonable yields for a low proportion of clones. We did not investigate this further and routinely used T7 RNAP.
Next, we minimised the protocol for the in vitro labelling of the PCR products to the following essential steps only: transcription with dig-UTP (digoxigenin uridine triphosphate) for two hours, immediate hydrolysis for 30 minutes for all probes and immediate precipitation with ammonium acetate (which also neutralises the carbonate hydrolysis buffer) and ethanol for 30–60 minutes. The labelling procedure was performed in 96-well plate format and took a total of four hours. To monitor transcription efficiency, a small aliquot from each batch of transcription products was run on an agarose gel to ensure that transcription is working and we repeated this test after the hydrolysis step.
The manual protocol describes individual hydrolysis times for each probe depending on the length of the DNA but we have found that once probes are hydrolysed to below a certain length, more detailed definition of an optimal probe size is not required. Hydrolysis fragments the probe and, in theory, the small fragments can access the RNA within tissue sections. However, subjecting the same transcript to several various hydrolysis incubations had little effect on signal strength. As template lengths ranged from 0.5 to 1.5 kb, we subjected each labelled transcript to the same hydrolysis treatment. When assessed on an agarose gel, all probes produced small fragments within a comparable range. Labelled transcription products were resuspended in TE and could be stored at -70°C where they remain usable for at least several months.
Accurate quantification of probes by dot-blot requires serial dilution but a simple qualitative test using a 1:100 dilution of each probe is usually sufficient to assess probe quality. If labelling is detected at this dilution, the same dilution in Hybridisation Solution (HS) can be denatured briefly and added to the slide for hybridisation. Since the probes are single stranded RNA, they are very prone to degradation during storage. Stability is much enhanced by diluting probes directly into the HS and storing them at -20°C: the HS contains 50% formamide which inactivates RNases.
(iii) Hybridisation and signal detection
At the end of the pre-treatment in the VP2000, the slides were dried and the silicone incubation chambers were carefully applied (Figure 2D). 40 μl of HS/probe mix was applied to each chamber in a standardised order from 96 well plate to slide (i.e., plate A1-A8 to slide 1 etc.,) such that 96 probes (including controls) can be applied to 12 slides (Figure 2F). Hybridisation was overnight at a standard 50°C in a conventional incubator on horizontal slide racks.
Use of hybridisation chambers with the same dimensions as the isolator allowed at least 8 different probes to be applied to a single slide. A range of sizes of isolator and hybridisation chambers are available and different sizes may be better suited to different tissue types (Figure 2G). Use of glass coverslips for incubation steps in the manual protocol normally requires prolonged, but gentle, washing to remove coverslips without damaging the underlying sections. However, the chambers can be quickly removed and the slides were loaded immediately for a short washing step in the VP2000. In addition, we also evaluated the various post-hybridisation steps and found that the RNase treatment step could be omitted completely. The RNAse step is thought to decrease background staining in ISH but slides prepared with or without RNase are essentially identical.
After standard incubations in blocking solution and Anti-DIG-AP (alkaline phosphatase) antibody, the slides were developed colorimetrically and a sample of the output is seen in Figure 2E. Using the colorimetric system rather than fluorescence means the progress of development can be monitored under a dissecting microscope and the problem of plant tissue autofluorescence is avoided. All reactions were stopped simultaneously, and all slides mounted permanently and could be stored for image-capture on the microscope.
(iv) Processing of results
Previous methods using one probe per slide meant that microscopic analysis was time-consuming and involved positioning many individual sections for optimal picture quality. In the automated protocol, with uniformly arranged sections representing multiple probes on every slide, image capture can be streamlined and has the potential for further automation.
(v) Data collection and storage
We describe a semi-automated system for highly parallel processing of ISH. We have introduced a substantial degree of automation to produce a system for performing high-throughput RNA-ISH on hundreds of plant tissue sections simultaneously without loss of resolution, specificity or sensitivity. This slide processing system has a capacity of at least 96 probes per week/per person for multiple developmental stages or experimental treatments and, with the exception of imaging, is scalable. Therefore, it is now feasible to contemplate genome-wide spatial analyses of gene expression at cellular resolution in both crop and model plant species.
To achieve genome-wide coverage in any species, certain prerequisites are necessary. First, sequences representing the expressed genes must be suitable for making probes and be available in an organized format. Second, the remaining manual steps must be further streamlined and, ideally, automated. Finally, automated image collection, analysis and quantification methods need to be developed.
Several projects are currently underway whose collective aim is to provide the expressed Arabidopsis genome as organized libraries of clones. Thus, a large proportion of Arabidopsis genes are currently available as trimmed ORFs (open reading frames) from SALK http://signal.salk.edu/cdnastatus.html and ESTs (expressed sequence tags) from ABRC (Arabidopsis Biological Resource Centre) http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm, or as 3' UTRs (untranslated regions) or as groups of structurally related genes from special projects such as REGIA (Re gulatory G ene I nitiative in A rabidopsis) http://www.epsoweb.org/catalog/EU/fp5/REGIA.htm. These collections tend to present genes in a consistent format that is amenable to automation. In most crop species, where the genomes are as yet incomplete, large EST collections are available. However, even these can provide useful information when used in conjunction with microarrays, where gene expression data can be confirmed and resolved to individual cell types and tissue layers [19].
Throughput is somewhat lower in Arabidopsis than in wheat, largely due to the time required for sectioning the smaller and more heterogeneous tissues. The proportion of usable sections is less and the tissue complexity requires greater imaging times. However, the smaller size of Arabidopsis facilitates whole mount approaches [3, 11] and, used with confocal microscopy and fluorescent imaging, this could completely eliminate manual sectioning without compromising either resolution or throughput.
Further automation is therefore required. Recent developments in slide processing have provided improved and more cost-effective slide processors that automate all the steps from de-waxing to mounting, including hybridisation (unpublished results) but imaging and analysis remain significant rate-limiting factors. Automated imaging and analysis involving machine-learning, are essential to extend this approach to the analysis of whole genomes. Such approaches have been developed for the analysis of tissue microarrays in the analysis of protein expression in various cancers, though these technologies still involve protein immunohistochemisty more than mRNA in situ hybridization [26–28]. However, as they employ a similar colour-based detection system, the technology should be transferable.
Preparation of plant material
Wheat plants (variety Savannah) were grown under controlled environment conditions (16°C, 16 h light) and ears tagged daily at anthesis. Arabidopsis Col-0 was grown in glasshouse under a 16-hour light regime. Wheat grains harvested at 3, 6 and 9 DAA were trimmed and Arabidopsis floral meristems were removed just after bolting. All tissues were fixed in 4% paraformaldehyde, then transferred to the Tissue Tek VIP (Vacuum Infiltration Processor, Sakura) for an automated fixation/dehydration/infiltration process as follows: fixative 6 h 35°C; 70% ethanol 1 h 35°C, 80% ethanol 1.5 h 35°C; 90% ethanol 2 h 35°C; 100% ethanol 1 h 35°C; 100% ethanol 1.5 h 35°C (repeat for 2 h); xylene 0.5 h 35°C (repeat for 1 h and again for 1.5 h); wax 1 h 60°C (repeat once, then again twice for 2 h). All steps were performed under vacuum. Samples were then transferred to the Tissue Tek Embedding Console for embedding in paraffin wax.
Section preparation
Sections (14 μm) from wheat samples at the required stages and 8 μm sections from floral meristems were cut on a Leica Microtome (RM2125RT) and ordered on polysine slides containing the silicone isolators (Grace Biolabs). After drying down at 42°C overnight suitable sections were selected for pretreatment.
Pretreatment steps were performed using the VP2000 Slide Processor (Vysis) using the following program: xylene 20 min (twice); 100% ethanol 10 min, then through a 95%, 85%, 50%, 30% ethanol series (2 min each), PBS (3 mM NaH2PO4, 7 mM Na2HPO4, 130 mM NaCl) 3–4 min; proteinase K (2–3 μg/ml in 100 mM Tris, 10 mM EDTA pH7.5) 30 min at 37°C; glycine (0.2%) 2 min; PBS 3–4 min; acetic anhydride (0.5% in 0.1 M triethanolamine pH 8) 10 min; PBS 3–4 min, then back through the ethanol series. Slides were completely dry at this stage and could be stored at 4°C until hybridisation.
Generating templates and labeling probes
Wheat cDNAs for screening are supplied as inserts in a vector derived from pSPORT1. Primers were designed in order to append a T7 RNAP site to the 3' end of the insert with the other primer nested inside the native vector T7 RNAP site; T7.2 5' GAATTGTAATACGACTCACTATAGGG CCAGTGAATTGAATTTAGG 3' and R7.2 5'AGGGAAAGCTGGTACGCCTGC 3' (T7 RNAP promoter binding site underlined). Arabidopsis histone H4 was amplified from pBluescript and AP3, AG, REM from pGEM vectors with universal forward and reverse primers for subsequent transcription with T7 RNAP. PCR reactions were performed with the following cycle: 94°C 3 min, then 30 cycles of 94°C 45 s, 63°C 45 s and 72°C 1.5 min, final extension of 72°C for 6 min. For 96-well plates PCR-product purification was done using the Montage Clean-up Kit (Millipore).
In vitro transcription was performed in 10 μl reactions for 2 h at 37°C in the presence of digoxigenin-UTP (Dig-UTP)-nucleotides (0.35 mM). Hydrolysis was carried out immediately in 100 mM carbonate buffer pH10.2 at 60°C for 30 min, and products precipitated in 2.5 M ammonium acetate and 3 vol absolute ethanol for 1 h at 4°C. Plates were centrifuged at 4000 rpm for 30 min and pellets resuspended in 30 μl TE (100 mM Tris, 10 mM EDTA) buffer. Dilutions (100 x) were made in water and 1 μl of each spotted on nitrocellulose for dot-blot: 30 min in blocking solution (Sigma), 30 min in anti-DIG-alkaline phosphatase (Roche); 5 min wash in TBS (10 mM Tris, 250 mM NaCl); 5 min in AP-buffer (100 mM Tris, 100 mM NaCl pH 9.5; 50 mM MgCl2) and developed as described above until signal was sufficient. All probes were then diluted 100-fold in hybridisation solution (300 mM NaCl, 10 mM Tris pH 6.8, 10 mM NaPO4, 5 mM EDTA, 50% formamide, 5% dextran sulphate, 0.5 mg/ml tRNA, 1 × Denhardt's, 0.1 mg/ml salmon testis DNA) and maintained stably at -20°C until hybridization.
Hybridisation and washing
Chambers (Grace Biolabs) were applied securely to the slides (after pre-treatment) and probes (diluted in hybridisation solution) were applied to one well (2 sections) for the 3 stages individually. Coverslips were placed on the chambers to prevent evaporation and hybridisation was performed overnight in a 50°C incubator.
Chambers were removed and slides arranged in the VP2000 for washing program: 15 min in 2 × SSC (0.3 M NaCl, 0.03 M Na citrate), 50% (v/v) formamide at 40°C; 40 m in the same at 50°C; 20 min in 1 × SSC, 50% (v/v) formamide at 50°C (all steps with constant agitation); 5 min in 1 × SSC at room temperature; 5 min in 1 × TBS at room temperature. Then slides were transferred into trays for staining: 1% blocking solution (Roche) in TBS 1 h, 1 × TBS containing 1/3000 dilution of Anti-DIG AP and 0.05% (v/v) Tween-20 1 h; 4 × 10 min washes in 1 × TBS; 5 min in AP-Buffer (0.1 M Tris, 0.1 M NaCl, 50 mM MgCl2); developed in AP-Buffer containing NBT (0.1 mg/ml) and BCIP (0.075 mg/ml) for a maximum of 24 h. Slides were then washed several times in water to stop the reaction followed by sequential washes in 70% and 100% ethanol to remove excess stain (the duration of the ethanol washes depends on the level of colour development which was monitored by eye). Slides were then allowed to dry and permanently mounted in Entellan (Merck).
Image capture and analysis
One section for each stage for each probe screened was photographed on a Nikon E800 microscope using a digital camera under brightfield conditions for wheat sections and with UV filter for the calcofluor-counterstained Arabidopsis sections. Images were recorded sequentially as ordered on the slides. Magnifications and camera settings remained unchanged for all images through all stages for wheat section and likewise for floral meristems.
The authors wish to thank Sarah Collier and Mary Byrne (John Innes Centre) for expertise working with Arabidopsis; Cathie Martin, Jan Traas, and Robert Sablowski for probes; Steve Evans and Gawain Bennett (Syngenta) for help with the wheat probe preparation; David Leader, Wolfgang Schuch and Simon Bright for facilitating the collaboration with Syngenta and the BBSRC, Syngenta and the John Innes Centre for funding.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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The Garfield County 4-H Shooting Sports Council will host a 3-D Archery Shoot on Saturday, March 18th, 2017. Registration will begin at 8:00 am, and the shoot will start at 9:00 am.
It will be held at the Garfield County Fairgrounds Indoor Arena – 1001 Railroad Ave, Rifle. Fees are $10 for 8-12yrs, $20 for 13-18yrs, and $25 for ages 19 and over. Prizes will be awarded to the top three places in each age group. Yardages will be marked over 30 yards and binoculars are welcome. Concessions will be available. Proceeds go to help send Garfield County 4-H Shooting Sports Contestants to contests at State Fair. Open to the public.


text: Founded in 1935, the Tony Award-winning Oregon Shakespeare Festival (OSF) is among the oldest and largest professional non-profit theatres in the nation. Each year OSF presents an eight-and-a-half-month season (February through early November) of eleven plays in three theatres plus numerous ancillary activities, and undertakes an extensive theatre education program. Operating on a budget exceeding $26 million, OSF presents more than 780 performances annually with attendance of approximately 400,000.


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This article is part of our special report Resource Efficiency.
SPECIAL REPORT / Industrial and environmental groups are lining up to pressure the European Commission on how best to measure resource efficiency. Businesses have warned that a one-size-fits-all approach will hamper economic growth at a time when Europe strives to emerge from its sovereign debt crisis.
The European Commission is to present on 14 December the results of recommendations made by experts on which indicators are the more suitable for monitoring progress under the Resource Efficiency Roadmap set out in September 2011.
Today, each EU citizen consumes 16 tonnes of material annually, of which six tonnes are wasted, with half going to landfills. The aim of the roadmap was to decouple resource use from economic growth.
Resource efficiency means minimising the negative environmental impacts generated by the use of natural resources in a growing economy. To measure it, the EU needs indicators and targets, which will also help to track and guide the progress made up to 2020 and 2050.
"Improved accounting is essential for wise management," Jacqueline McGlade, executive director the European Environment Agency, told a recent World Resources Forum. "You can't manage what you don't measure."
The EU executive suggested measuring progress by introducing a 'lead' "resource productivity" indicator that would measure GDP against material consumption expressed in euros per tonne. This lead indicator will be complemented by the "dashboard" of indicators – such as for carbon, land and water – which was already proposed in the Resource Efficiency Roadmap.
This approach would serve as a basis for resource efficiency targets.
"This will be a critical exercise for the 27 member states but we've seen the model of effort sharing for the climate targets and that works well," German Social-Democrat MEP Jo Leinen said.
But there is broad disagreement on this measure.
'Severe shortcomings'
Anec, an NGO defending consumer interests in standardisation issues, contends the GDP-linked indicator "suffers from severe shortcomings" because the EU's economies differ greatly.
In a response to the Commission's recent call for consultation on resource efficiency indicators, Anec said the indicator is good for measuring de-industrialisation, "but this is not the objective."
The European Steel Association, or Eurofer, said "there is no such thing as a one-size-fits-all indicator" and that "it would be dangerous to work with overall indicators."
Other groups – including Orgalime, the European Engineering Industries Association – say indicators need to be based on robust data.
"Resource efficiency indicators need to make sure decisions are based on a deep analysis," Veronique Steukers of the Nickel Institute agreed.
Euromines, the European association of mining industries, says that not all existing indicators for measuring resource efficiency are applicable to extractive operations and may therefore provide a wrong basis for decision making.
Concepts such as "resource use", "material footprint" and "material intensity" often underweight the importance of stability and durability of materials, according to Euromines.
William Neale, who is responsible for resource efficiency in the European Commission's environmental department, acknowledged that finding the right headline indicator that sets a direction and a goal isn't easy. "It's a case of getting the right indicators. It's a difficult task, intellectually speaking." he said.
The Commission defends the GDP indicator because it is the one for which all member states have solid data.
Neale also thinks that this indicator could help businesses tap funds necessary for new investments.
"if we say this is the way things are going then companies, investors, funds and so on can start seeing that that is the writing on the wall. That gives increased confidence and predictability in the direction we need to go, rather than then hitting the constraints of supply, and the price volatility and hikes and so on," he said in an interview.
Green groups are wary
Environmentalists have criticised the indicator, saying it ignores land, water and carbon footprints. Businesses have also complained, claiming that the indicator should also take into account the environmental benefits of raw materials use, not just the damage.
"This indicator cannot be used to achieve the Commission's vision for 2050, whereby the EU's economy respects constraints and planetary boundaries," Friends of the Earth Europe said in documents presented to the Commission.
The GDP measure does not accurately show whether an economy has improved or worsened its resource use," the green campaigners wrote.
Friends of the Earth Europe suggested the Commission should not set aggregated indicators that combine economic and environmental information. At the moment, the lead indicator does exactly this, since it is the ratio between the environmental impact related to resource use and the overall economic indicator – in this case GDP.
"It is essential that the indicators used are consumption-based, transparent and with a direct link to the statistical system," Friends of the Earth Europe said.
Ernst Ulrich von Weizsäcker, co-chair of the UN's International Panel for Sustainable Resource Management, said there is a need for lawmakers to intervene in regulating resource use, as long they are not "too prescriptive".
"Nothing is moving in the right direction if all is left to the markets," but intervention should not be too bureaucratic," von Weizsäcker said.
"The different data sources might be a challenge. We need to get the process started. We can't wait until we have the perfect data before we set targets and indicators - we would lose too much time and we don't have any time to waste. Setting targets and indicators shall bring the EU on track to become the most resource efficient economy" Leinen added.
Commenting on the use of the lead indicator defined as material resource productivity measured through a consumption-to-GDP, Friends of the Earth Europe said: "By adding different statistical data using weights and proxy converting factors, the result of this indicator is an artificial one that does not reflect Europe's use of resources."
Orgalime, the European Engineering Association, said: "The proposed lead indicator of Domestic Material Consumption (DMC) shows considerable weaknesses in terms of its capacity to reflect all aspects of Resource Efficiency in an appropriate and reliable manner. This risks leading to misinterpretations in environmental and economic terms and should therefore not be applied."
Martin Lehmann, of the World Resources Forum Secretariat, said the GDP indicator is "better than nothing", but the EU should start looking at the bigger picture and try to include social indicators, such as wellbeing, also in its list. "I think the industry, if they go step by step forward to reach resource efficiency and sustainable development, they will in the end not depend on one indicator – because they will end up performing well in all of them if they reduce material imports, energy use and water use," Lehmann said, adding that he expects the Commission to settle on "five to ten main indicators".
"Without indicators we are a bit lost, we do things but we don't know whay we are doing and how well."
The Commission launched in June 2012 a Resource Efficiency Platform in a bid to set new standards for resource efficiency in the EU. The platform has been divided into three working groups:
- Circular economy;
- Setting objectives and measuring progress;
- Framework conditions for investment in resource efficiency.
Indicators and targets are needed in all these three groups and hence the next meeting of the platform, on 14 December, will focus mainly on discussing and possibly drawing up a set of indicators. Stakeholders have already sent the platform around 170 responses to the initially proposed indicators.
- By 2014: Commission asked to revise the 2020 recycling targets set in the Waste Framework Directive
- Summer 2013: European resource-efficiency platform expected to issue a first set of recommendations for short-term measures that can be adopted in about 12 months
- By 2014: Commission to revise 2020 recycling targets set in the Waste Framework Directive
- Mid-2014: Mandate ends, the platform makes recommendations for the longer term
Further Reading
NGOs and Think Tanks
- WWF: Response to the Commission consultation "Options for resource efficiency indicators"
- Friends of the Earth Europe: Indicators for better resource use
- McKinsey: Mobilizing for a resource revolution
Business and Industry
- Euromines: Position on Resource Efficiency
EU official documents
- European Parliament: A resource-efficient Europe (compromise text)
- European Commission:Resource Efficiency
- European Council:Economic aspects of the roadmap to a resource-efficient Europe
- PreLex:Roadmap to a Resource Efficient Europe [FR] [DE]
- Press release on the launch of the Resource Efficiency Platform: Janez Poto?nik European Commissioner for Environment "How can we do more with less" Launch of high-level panel on Resource Efficiency Brussels, 5 June 2012
European Commission
- European Resource Efficiency Platform: Website
- Online Resource Efficiency Platform (OREP): Website
- Resource efficiency in the European Semester ( the 'Promoting growth and competitiveness' section)
- The European Semester
- Innovation partnership to overcome Europe's raw materials shortages
- Directive on the promotion of the use of energy from renewable sources


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Fresh Fruit Salad and Sour Cream Crepes
- 1/4 cup Kemps Sour Cream
- 1 Egg, large
- 1/4 cup Kemps milk
- 1 Tbsp Butter, melted
- 6 Tbsp All-purpose flour
- 1 Tbsp Mint, fresh, chopped fine
- 1/4 tsp Salt
Fruit Filling
- 1 cup Fruit, chopped (any combination)
- 1 tsp Granulated sugar
- 3 Tbsp Kemps Sour Cream
Maple sour cream topping
- 1/2 cup Kemps Sour Cream
- 2 Tbsp Orange juice
- 1 Tbsp Maple syrup
- Combine all of the ingredients for the crepe batter in a small mixing bowl.
- Heat a small non-stick skillet over medium-high heat and lightly coat with nonstick cooking spray.
- Add approximately 1/4 cup of batter to the pan and swirl the pan quickly to spread the batter.
- Cook the crepe completely through, flip it gently, and then remove and place on wax paper. Keep warm before serving.
- Toss the fruit, sugar, and sour cream together. Refrigerate.
- Combine the sour cream, orange juice, and maple syrup in a small mixing bowl.
- To assemble, place a crepe on a serving plate, fill with fruit mixture and roll. Top with the maple sour cream and serve.
Makes 3 Crepes.
Serving Suggestion: Sprinkle with confectioners sugar and garnish with fresh fruit.
Per Serving: 280 Calories; 12g Fat; 9g Protein; 33g Carbohydrate; 106mg Cholesterol; 140mg Calcium; 8g Saturated Fat; 363mg Sodium
How Did This Recipe Work Out for You?
Please return to http://www.kemps.com/recipes/fresh-fruit-salad-and-sour-cream-crepes/ to rate and review this recipe. Your comments and suggestions are appreciated by others.