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EAST PROVIDENCE – And so, Alex Butler and his East Providence High squad was left with this: With nothing left to play for other than pride and principle, the Townies played free and easy Wednesday night while at the same time providing defending state champion St. Raphael Academy with all sorts of fits.
By no means did E.P. run away from the challenge at hand. The Townies stood toe-to-toe with the defending champs until the very end of an eventual 58-55 Saints win that demonstrated to Butler that his team was not ready to enjoy the rest of its February vacation.
PAWTUCKET — The City Council at its meeting of Feb. 8 approved several appointments, including that of renewing the terms of the municipal judge and associate municipal judge.
By a unanimous vote, the council elected former state Senator John M. McBurney to a three-year term as municipal judge and current Sen. Donna M. Nesslebush to a three-year term as associate municipal judge.
John T. Barry
LINCOLN- John T. Barry, 77, of Williams Street, Lincoln passed away peacefully at home on Tuesday, February 21, 2012. He was born in Pawtucket, the son of James and Alice (Knowles) Barry, Sr.
Mr. Barry grew up in Pawtucket, graduating from St. Edwards School and Pawtucket West High School. He was a lifelong Communicant at St. Edward's Church and a volunteer at LaSalette Shrine.
Paula Tierney
CENTRAL FALLS- Paula Tierney, 44, passed on February 20, 2012.
Born in Providence, a daughter of the late Francis and Joan (Hennesey)
She had been employed as a Telemarketer for many years.
Paula is survived by a daughter, Melissa Brooks of Central Falls, a sister, Diane Mayer of Riverside, two brothers, Kenneth Smith of Johnston and Steven Smith of East Providence and several nieces and nephews. She was the former wife of Dennis Brooks.
Steven J. Phillips
PAWTUCKET- Steven J. Phillips, 45, passed away unexpectedly at home on Tuesday, February 21, 2012. He was the beloved husband Elizabeth M. (Allan) Phillips for twenty-three years.
A lifelong resident of Pawtucket, he was a son of Joyce (Renell) Phillips of Pawtucket and the late Richard Phillips.
Steve had been employed by the Sun Chemical Company, Mansfield, for many years. He was a handyman who enjoyed helping others, and was an avid sports fan, especially of the Patriots.
Linda A. Santos
PAWTUCKET- Linda A. Santos, 92, of Pawtucket, died Tuesday, February 21, 2012. She was the wife of the late Anthony Santos.
Mrs. Santos was born in Ludlow MA, the daughter of the late Firmino and Guilheminia (Conceicao) Ferreira.
She was employed at the former Central Braid and Rug in Central Falls.
Mrs. Santos is survived by her daughter Sandra Wesley and her husband Fred of Seekonk MA. She also leaves three grandchildren and four great grandchildren. Mrs. Santos was the mother of the late Lynda Fernandes and the sister of the late James Ferreira.
PAWTUCKET — How much of a pounding from trailer trucks can a 135-year-old bridge take? That's what City Councilors have been wanting to know. Yet the most recent assessment done by the state rates the span as safe and maintains the additional truck traffic is having little to no impact.
Heralding the 1663 Royal Charter for the English Colony of Rhode Island and Providence Plantations as "one of the great human rights documents of the world," Gov. Lincoln Chafee set the stage Tuesday for the celebration next year of the 350th anniversary of King Charles II's grant to John Clarke and Roger Williams.
A history buff who frequently references Roger Williams, the state's founder, and the charter in his speeches and Statehouse presentations, Chafee appointed a 34-member commission to organize a celebration for the sesquarcentennial, which will culminate on July 8, 2013.


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"I'm happy," said Doris Young, mother of 17-year-old Sterling Horton, who was fatally shot in July 2006. "God made him confess, because he could have walked away from my son's murder."
Chief Deputy Hennepin County Attorney David Brown said Tarius Gresham was facing a rape charge from 2012 when he confessed last month to the Horton murder, according to Minnesota Public Radio News ( http://bit.ly/1iJkgh7)
According to the charges, Tarius Gresham admitted to fighting with Horton on the night of July 25, 2006. Gresham told police he pulled out a gun, shot Horton and then ran.
"It's not unusual to have defendants want to offer information about other crimes," Brown said. "What's unusual about this case is that he wanted to offer information that he had committed another crime."
Before accepting his confession, Brown said authorities verified his claim and determined he was responsible for Horton's death.
Gresham was sentenced to 15 years in prison Thursday, which includes time for both the Horton murder and the rape conviction.
"I saw the face that killed my son," Young said. "I saw the young man's face."
Information from: Minnesota Public Radio News,

subset: commoncrawl


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This grammar & punctuation unit for grade 6+ is based on rules for making plural nouns by adding 's/es', changing letters, and identifying nouns that do not change or have irregular forms. The unit includes a copy of the rules plus three activity pages (with answer key). (Find other units by searching 'Grammar 6')
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La endarterectomía es un procedimiento quirúrgico que remueve del revestimiento de una arteria el material de la placa que se ha formado.
Last reviewed 5/21/2012 by Luc Jasmin, MD, PhD, Department of Neurosurgery at Cedars-Sinai Medical Center, Los Angeles, and Department of Anatomy at UCSF, San Francisco, CA. Review provided by VeriMed Healthcare Network. Also reviewed by David Zieve, MD, MHA, Medical Director, A.D.A.M. Health Solutions, Ebix, Inc.
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Shady Side Academy falls just short in PIAA tennis match
TribLIVE Sports Videos
After cruising to a 6-0 first set win in the PIAA Class AAA doubles championships on May 25 at Hershey Racquet Club, it looked like Shady Side Academy's Alok Nimgaonkar and Chris Grubbs would end a five-year WPIAL drought and bring home the gold.
Their hopes were quickly dashed, however, as they fell to West Chester Rustin's John Lim and Derek Summers, 0-6, 6-2, 6-4.
Nimgaonkar and Grubbs jumped out to a 3-0 lead in the third set but couldn't close it out.
For awhile, it looked like it might be an all-WPIAL final as Fox Chapel's Ben Short and Sid Rajupet nearly knocked off Lim and Summers in the semifinals.
They lost in a three-set heartbreaker by scores of 6-4, 3-6, 7-6 (11-9).
Shady Side Academy coach Tom Mercer, despite the loss, was upbeat about the season.
"They made it to the finals," he said. "As a team, we made it to the finals, so it was a very rewarding season. We nearly reached all of our goals but came up short. A lot of teams would love to have the season we had.
"The doubles match was frustrating, though. We started out playing very well. I think their guys were a little bit off of their game after that tense semifinal match but they rebounded nicely and we started making some errors.
"Give them credit, though. We have a strong nucleus coming back and we hope to be in the same position next year."
Marty Stewart is a sports editor for Trib Total Media. He can be reached at 412-782-2123 or email@example.com.
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By MADALENE WILSONGuest writer
All four of Louis and Louisa Durand's children gathered for a family reunion during the middle of May. When asked about their longevity and health, they claim good genes, good food and good old pasteurized water (you know, the water that ran through the pasture before it hit the cistern).
Marie Lott is the oldest at 91 and lives in Price, Emery is 86, Fred is 85 and Isabelle Oveson is the baby at 80. The brothers traveled from Alabama for a two week stay during which time they were able to visit with many family members.
They spent most of their time at Isabelle's home in Huntington which is the centralized gathering place for food and rest, her home emulates the feel of their mother's home. Kandice and Foster, Marie's children; Emery's son Jim; Jackie, Madelene, Kurt, Kelly and Kris - Isabelle's children were able to make it to the reunion and of course, some of their spouses, many grand and great grand children were able to visit too.
Emery and Fred moved to Alabama in 1965 where they worked for Boeing, a subcontractor for NASA and were instrumental in getting the Saturn V (the Apollo missions) in to space and landing on the moon in 1969. They each married "southern gals" and were "adopted" by their wives families. As much as they would like to come back to Utah to live, especially with Emery's son and family living here in Emery County, the number of years spent in Alabama and the large extended families have planted roots that make it difficult to pull out of that area.
All four of the siblings are widowed. They have survived six spouses amongst the four of them. They know the importance of family and have instilled that quality in each of their own families. They stay in touch with each other every weekend via phone calls.
I am very proud to be a member of this particular family. All four of these people have had great influence in raising their next generation; they encouraged integrity, character, hard work, free thinking and also passed on those great genes. And it must also be mentioned that as children, we too drank that "pasture - ized" water.


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Portland Public Schools Science Expo
Date: Thursday, March 10, 2016
Location: OMSI
(High School only)
Contact: Nancy Lapotin at firstname.lastname@example.com
Phone: 503-916-2000 ext. 71327
* Paperwork is due February 19th.
STUDENTS-Please make sure to clear your schedule for the whole day as you must be present to receive awards!!!!!
Here are the schedules for fair day for the students and for the judges.
See you all at OMSI!!!!!
March 10, 2016
Schedule for Students
8:00-8:30 Exhibit Set-up-OMSI Auditorium
Students register, turn in any missing paperwork, get programs and badges and set-up projects.
8:30-8:45 Orientation-Nancy Lapotin (Fair Director)
8:45-9:45 Judges only at projects-students tour OMSI exhibits
9:45-10:00 Return to posters
10:00-12:00 Judges interview students at their projects
12:00-12:30 Lunch break
12:00-2:00 Exhibit Area open to the public. Students must be at their exhibits 12:30-2 additional interviews may take place.
2:00 Take down exhibits and set up chairs in auditorium for Awards Ceremony
2:15-3:00 Awards Ceremony-OMSI Auditorium-Friends and Family Invited. Students must be present to receive awards.
Schedule for Judges
8:30-8:45 Breakfast and orientation
Badges and instructions from Linda Mantel
8:45-9:45 View projects without students present
9:45-10:00 Meet with judging team.
10:00-12:00 Judging with Students
12:00-12:45 Lunch and deliberations (category and special award results due by 1pm)
12:45-1:45 Finalist judging
2:00 Data entry complete, judges excused (Thanks x 106)
Please feel free to pass my email address (email@example.com)on to anyone you think might be interested in this.
Thanks you so much in advance.
We look forward to a fantastic celebration of PPS Student Research Projects this spring!


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Viewing convention Program information from 2005
Session Details
Thursday, 29 December
575. Public or Postcolonial? South Asian Writers as Intellectuals
7:15–8:30 p.m., Georgetown West, Washington Hilton
Program arranged by the Discussion Group on South Asian Languages and Literatures
Presiding: Pramod K. Mishra, Augustana Coll., IL
1. "Kureishi's The Black Album and Postcolonial Novel as Public Intervention," Lopamudra B. Basu, Graduate Center, City Univ. of New York
2. "Public and Postcolonial: C. S. Laxmi and Rukhsana Ahmad's Transnational Projects," Josna E. Rege, Mount Holyoke Coll.
3. "Transnationalism and the Politics of Representation in Indian Diaspora Films," Jaspal Kaur Singh, Northern Michigan Univ.
Respondent: Bed Prasad Giri, Dartmouth Coll.
No comments have been posted for this session.
|© 2015 Modern Language Association.


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- Sunday, February 17, 2008
Lucy McKenzie: Hats Off
Date + TimeSunday, February 17, 2008
11:00 a.m. - 2:00 p.m.
LocationKoret Visitor Education Center
Inspired by the work of McKenzie, this program explores environmentally conscious fashion - come ready to make your own hat! The afternoon includes a reading of Aliens Love Underpants, by Claire Freedman and Ben Cart. The family tour begins at 1:00 p.m. in the Koret Center Studio; screenings and readings take place at 2:00 p.m. in the Koret Center Learning Lounge.
Admission + Ticketing
Museum and program admission are free for families.
Major support for family programs is provided by The Wallace Foundation.


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Anna Sui gives her hippie-chic aesthetic a glamorous update with this beaded and fringed silk-georgette dress. The eye-catching embellishments and romantically cut hem take this style into standout territory - perfect the look with a silver clutch for a flash of metallics. Large to size. See Size & Fit tab.
Anna Sui dress: black silk-georgette, sequin, bead, ribbon and fringe embellishment, square neck, gathering from ruffled dropped waist, elongated back hem, fully lined. Concealed hook and zip fastening at side. 100% silk; lining: 100% polyester. Dry clean.
Product code: 177860 - Need help? Contact us


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FIFA World Cup 2014 goal-line technology
In October 2013, FIFA awarded the contract for goal-line technology to German company GoalControl, and its GoalControl-4D system. The Tivoli stadium in Achen was the first to be fitted with a FIFA-certified goal-line technology system. Note that the rival Hawk-Eye technology has already been in use in English Premier League fixtures since the 2013-145 season. Derbyshire-based company Labosport will carry out the final installation testing of goal-line technology systems. It also tested the Hawk-Eye systems for the Premier League.
The World Cup football tournament starts in Rio de Janeiro in Brazil next month and organisers FIFA will be hoping goal-line technology will not be dominating all the headlines. We take a closer look at the technology.
See also: FIFA World Cup tests goal-line technology


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Video: Avalanche risks in Colorado
KUSA - With all the snow that fell last week in the high country the risk for avalanches is very high right now some experts say the worst in 30 years.
Before anyone heads out into the backcountry - whether to ski, snowshoe or just hike - there are some precautions to take.
Brian Lazar with the Colorado Avalanche Information Center stopped by the 9NEWS studio Saturday morning with more information.
(KUSA-TV © 2013 Multimedia Holdings Corporation)


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Is programming...analysis done backward?
(This is another in my emptying of my drafts box of proto-blogs - this one from four or more years ago. Some of it is dated, and I gave it an edit or two, but I think there might be a thought or two in here worth reading).
Software is what architecture would be if building-materials were free. Everybody would have a beautiful, twisting crystal palace in the air, and nobody would be able to find the bathroom - Jesse Glick
Ever stop to think how we do software? When analyzing the real world, people start with existing phenomena, then backtrack to one or more models that describe it - each is an abstraction of the physical phenomena. Then, applying Occam's razor, the simpler explanation wins. That is why you can explain the entire solar system as everything orbiting the earth, and the math will work - but you don't do that in practice, because it's much more complex that way. We call that science.
Then there's this thing called computer science. Interestingly, it doesn't work the same way.
This work is done by people, and human beings, in the sciences, humanities, or in economies, build status hierarchies and compete for places in them. We're primates. It's just what we do. So that complicates things.
On fuzzier subjects (say, Shakespeare), the Occam's razor approach doesn't offer much benefit. You're starting with an artifact of culture and attempting to analyze it in the context of other, well, artifacts of culture (Edward O. Wilson crystallizes the problem brilliantly in Consilience But there is a desire to understand the subject, and to analyze it. Thus we have the field of study of literary theory (a subject I wasted too much of my youth on). Because you can never get Shakespeare down to something as simple as observations of the planets, you can never know if your analytical approach is useful or not (if you could, we would have IT thesis projects like "Write a program that will write Shakespeare's next play, after all the ones he really wrote" - and they would all produce the same play).
Any study of fuzzy subjects tend to have a lot more fads, politics and schools of thought that come and go, simply because it can and there's no place else to go - nothing's provable. There is a discipline called computer science, but it falls far, far short of the things people need to get done some way or other in today's IT world (say the phrase "halting problem" in a room full of people trying to write the next YouTube - you will be an annoyance and they are right, you aren't). If you studied literature any time between 1978 and 1997, think structuralism, semiotics, post-structurialism, post-modernism (if you don't know these terms, count yourself lucky and don't waste your time). A school of thought becomes popular; people compete for status by either using it, or carrying it to further extremes; this provokes others to carry it to still further extremes to try to gain status; eventually it collapses under the weight of its own absurdity and the next fad catches on, and the process repeats. The ebb and flow of religious fundamentalism throughout history has similar patterns.
All of this is possible because in the humanities, nobody is ever going to prove they're right anyway - what better environment to compete for status in, than one where nobody will ever prove you don't have status!
(Note that the study of business is a soft, not a hard science. There is just as much unresolvable complexity in an economy as in a Shakespeare play - it's just as fuzzy, maybe more so. But it just does involve some numbers, so it's easier to pass it off as a hard science, and parts of it, when isolated to the point of not being able to predict anything useful, are hard sciences - math is math).
The proven best practice for figuring something out is to look around, seeing what's happening, and start putting things in boxes. The boxes are abstractions. Eventually you've got a pile of abstractions in some kind of organization, and you can either try to predict the future with it, or try it out on similar things and see if the results map to reality. If it works, you earn some money or status or something along those lines (and if your lucky, it happens within your lifetime). If you really do a good job, it might just raise the quality of life for more people than just you, because everybody's got a new tool that will work consistently.
Then there's software. Yes, there are cases where you write software that is a 1:1 mapping between the world and the software, and its totally testable. But that's usually the exception, not the rule. And usually a 1:1 mapping between the problem and the software that solves the problem is completely unusable - some of the most avid users of the NetBeans Platform are people who adopted the Naked Objects approach to design with entirely predictable and disasterous results - the sane description of a problem to a computer is very different than the sane description of a problem to a person - and confusing the two is a seductive trap.
Very, very often in software, we start with the abstraction! We aren't observing the universe and discovering principles, we are god, creating the universe. Before you have an implementation, define your interfaces. This is the polar opposite of scientific analysis, where you start with a complex world, and find abstractions that consistently describe it. In software, you start with abstractions, and then fill in the complexities. You may derive the abstractions from a mental model based on observation, but you're still starting with the abstractions. And if the complexities - what the software really has to do to fulfill its purpose - don't fit the abstractions well, you get performance problems and other nastiness - because in the end, there is a reality, and it's what work the computer is actually going to do when your software is run. But you're not starting from what the computer is going to do - unless you can afford a lifetime to cogitate on one problem so that you really can think it all through and work backwards from the root problem - and outside of well funded foundations, most of us don't have that luxury.
But isn't it all a little weird? In software, often you're not starting with the world - you're creating a world for other people to live in. People building frameworks (like Java) or operating systems compete on whose world is better (never mind that better for the programmer and better for the customer can be two different things). And that competition is more often defined by religiously held views than by sober analysis. So everybody goes out and tries to get people to religiously hold views favorable to them. If you've created a world, you've got to get people to go live in it, particularly if you hope to make them pay for the privilege, which is the typical way you manage to create a world and feed your family at the same time.
The reason for all the weirdness is, it's not a thing that has a template. Human beings haven't been in the business of creating worlds for very long. As the last vestige of the Roman empire, the Catholic church is the closest thing we have to a reliable template for effective abstraction creation (please don't think me a conspiracy theory believer or anything similar because of that metaphor - sometimes a cigar really just is a cigar!). We're not good at it yet. We don't know the best practices for world creation, if there even are any (and anybody who knows them is either long dead or not about to share them if they aren't). Witness the huge number of web application frameworks out there. Which one is the best? Well, the best at what? It's not quite as unsolvable as proving Shakespeare is better than Hawthorne. But it's close.
So it's a bit like every computer programmer has been given the task "Create the Catholic church, from scratch, and prove it will still be around in a thousand years before you start, if you want to be funded." Fertile ground for charlatans, but somewhere in there, some folks are probably going to do some stuff that does work, and will be observed to work and copied. But it doesn't happen fast and we started from zero within the last 50 years.
The real situation is, we're finding the rules as we go (with businesses that bet wrong as the casualties - but this is how market economies build infrastructure - through failure - consider the U.S railroads, most of which were built by companies that went bankrupt [but left behind railroads] or ATM [bankomat] machines built by competing banks that consolidated).
The closest analogy might really be competition between various religions - and in fact, you get identifiably religious behavior - things like Microsoft bashing, obsession with not breaking object encapsulation, Free Software or Extreme Programming are the faiths. That still isn't a situation human beings have traditionally been in - Extreme Programming doesn't tell you if you should use Windows or something else; object- or aspect- oriented programming doesn't say do or do not use Extreme Programming here. There is no One True Way. So the complexity of the available worlds to live in approaches the complexity of the real world, except they don't map 1:1. Is it any wonder this industry is a bit chaotic? Is it a wonder this creates a market for freakish "software methodologies" (I believe there is one software methodology and it has three steps - hire smart people, give them a problem, and get out of their way while they solve it - but try to sell that to a $7/hr HR recruiting agency...).
The result of all of it is that software has all the benefits of science, and all the handicaps of the humanities. There is an inevitable time delay between creating an abstraction and having it prove itself effective or ineffective - you have to ship the software, maybe several times, before you have real evidence. In that gap, people will behave as they do in the humanities - nothing is provably good or bad, and people will be faddish, place their bets and behave accordingly. Creating a thing and proving a thing are not the same. Right now, the software business emphasizes creating over proving, because there are not enough things that really are proven, and it's a business - you have to place your bets somewhere.
There never was any other way to go.
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text: Movies & Television Not a montage of slightly condescending geek speeches, Ted is something far worse. It wasn't screened for critics by press deadlines. It should not be screened by anyone.TedWW Critic's Score: D-Contempt fuels the comedy of Seth MacFarlane. He's disdainful toward his own meal ticket, the Simpsons rip-off Family Guy. He sneers at other performers: His new talking-bear movie Ted has the gall to shit-talk ... More


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Conventional wisdom calls us attention-deprived, constantly seeking the next scrap of info. But Google reveals our true desire: Context. (Wikipedia entries, This American Life's financial crisis explainer, Gizmodo's definitive guide to smartphones, etc.) We'll explore how journalism and media must adapt to meet our insatiable hunger for the bigger picture.
You can also see a great Live blog of this panel by Steve Myers.


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In this chapter, we provide a brief overview of the RNA interference (RNAi) process and discuss technologies and products that can be used for RNAi experiments. A protocol is provided for the enzymatic synthesis of double-stranded RNA in vitro that provides an inexpensive alternative to chemical synthesis of RNAs. We also discuss design and selection of short interfering RNA (siRNA) sequences and describe a vector system, the psiCHECK™ Vectors, that can be used to screen potential siRNA target sequences for effectiveness during RNAi optimization. In addition, the GeneClip™ U1 Hairpin Cloning Systems, a DNA-directed RNAi (ddRNAi) system specifically designed for expression of small hairpin RNAs in mammalian cells, provide a cloning-based approach to allow fast, easy ligation and expression of hairpin oligonucleotides. Various strategies for delivery of siRNA to target cells are discussed, and example protocols for transient and stable transfection of mammalian cells are provided. Finally, methods for quantitating target gene suppression are briefly summarized.
RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA)
suppresses expression of a target protein by stimulating the specific degradation of
the target mRNA (for reviews, see Hannon, 2003; Caplen, 2004; Fuchs et
al. 2004; Betz, 2003a). RNAi has been used to study loss of function for a
variety of genes in several organisms including various plants,
Caenorhabditis elegans and Drosophila,
and permits loss-of-function genetic screens and rapid tests for genetic interactions
in mammalian cells (Hannon, 2002; Williams et al. 2003).
RNAi involves a multistep process (Figure 2.1). dsRNA is recognized by an RNase
III family member (e.g., Dicer in Drosophila) and cleaved into
siRNAs of 21–23 nucleotides (Agrawal et al. 2003; Elbashir
et al. 2001b; Bernstein et al. 2001;
Hammond et al. 2000). These siRNAs are incorporated into an RNAi targeting complex
known as RISC (RNA-induced silencing complex), which destroys mRNAs homologous to the
integral siRNA (Hammond et al. 2000; Bernstein et
al. 2001). The target mRNA is cleaved in the center of the region
complementary to the siRNA (Elbashir et al. 2001c), with the net
result being rapid degradation of the target mRNA and decreased protein expression.
RNAi has revolutionized the study of gene function, and is being explored as a
therapeutic tool (for reviews, see Dorsett and Tuschl, 2004; Hannon and Rossi, 2004).
For example, RNAi has been used to identify gene products essential for cell growth
(Harborth et al. 2001), to cause subtype and species-specific
knockdown of various protein kinase C (PKC) isoforms in both human and rat cells
(Irie et al. 2002), and to specifically target degradation of an
oncogene product (Wilda et al. 2002). RNAi has also been used to
specifically target and prevent viral infections by HIV-1 and HCV in cell culture
(Park et al. 2002) and intact animals (McCaffrey et
al. 2002). These observations open the field for further studies toward
novel gene therapy approaches for anti-cancer or anti-viral treatments using siRNAs
or shRNAs.
An animated
presentation illustrating the entire RNAi process is available on the
Nature web site.
The use of long dsRNAs (>400bp) has been successful in generating RNA
interference effects in many organisms including Drosophila
(Misquitta and Paterson, 1999), zebrafish (Wargelius et al.
1999), Planaria (Sanchez-Alvarado et al.
1999) and numerous plants (Jorgensen, 1990, Fukusaki et al.
2004, Jensen et al. 2004). In mammalian systems, siRNA molecules
of 21–22 nucleotides or short hairpin RNAs (shRNAs) are used to avoid endogenous
nonspecific antiviral responses that target longer dsRNAs (Caplen
et al. 2001, Elbashir et al. 2001a). Yu
et al. (2002) and others (Brummelkamp et
al. 2002b; McManus et al. 2002; Sui et
al. 2002; Xia et al. 2002; Barton and Medzhitov,
2002) demonstrated that shRNAs bearing a fold-back, stem-loop structure of
approximately 19 perfectly matched nucleotides connected by various spacer regions
and ending in a 2-nucleotide 3′-overhang can be as efficient as siRNAs at inducing
RNA interference. si/shRNAs can induce specific gene silencing in a wide range of
mammalian cell lines without leading to global inhibition of mRNA translation (Caplen
et al. 2001; Elbashir et al. 2001a;
Paddison et al. 2002).
siRNAs are the main effectors of the RNAi process. These molecules can be
synthesized chemically or enzymatically in vitro (Micura, 2002; Betz, 2003b;
Paddison et al. 2002) or endogenously expressed inside the
cells in the form of shRNAs (Yu et al. 2002; McManus
et al. 2002). Plasmid-based expression systems using RNA
polymerase III U6 or H1, or RNA polymerase II U1, small nuclear RNA promoters,
have been used for endogenous expression of shRNAs (Brummelkamp et
al. 2002b; Sui et al. 2002, Novarino et
al. 2004).
Design of the siRNA sequence is crucial for effective gene silencing. Rational
design strategies for effective siRNAs are being developed based on an
understanding of RNAi biochemistry and of naturally occurring microRNA (miRNA)
function. Several groups have proposed basic empirical guidelines for designing
effective siRNAs that can be applied to the selection of potential target
sequences (Chiu and Rana, 2002; Khvorova et al. 2003;
Schwarz et al. 2003; Hseih et al.
2004; Reynolds et al. 2004; Ui-Tei et
al. 2004). In addition, strategies for experimentally screening
effective siRNAs from pools of potential siRNAs are being developed (Kumar
et al. 2003; Vidugiriene et al. 2004)
and will remain a useful tool until potent siRNAs can be predicted accurately for
each target gene.
The efficient delivery of siRNAs is a vital step in RNAi-based gene silencing
experiments. Synthetic siRNAs can be delivered by electroporation or by using
lipophilic agents (McManus et al. 2002; Kishida et
al. 2004). siRNAs have been used successfully to silence target
genes; however, these approaches are limited by the transient nature of the
response. The use of plasmid systems to express small hairpin RNAs helps overcomes
this limitation by allowing stable suppression of target genes (Dykxhoorn
et al. 2003). Various viral delivery systems have also
been developed to deliver shRNA-expressing cassettes into cells that are difficult
to transfect, creating new possibilities for RNAi usage (Brummelkamp et
al. 2002a; Rubinson et al. 2003). Successful
delivery of siRNAs in live animals has also been reported (Hasuwa et
al. 2002; Carmell et al. 2003; Kobayashi
et al. 2004).
return to top of page
Identifying an optimal target sequence is critical to the success of RNA
interference experiments. Since it is not possible to predict the optimal siRNA
sequence for a given target, multiple siRNAs will usually need to be evaluated.
Recommendations for the design of siRNAs are constantly being improved upon as
knowledge of the RNAi process continues to expand. At the time of writing this
chapter, the recommendations are as follows: siRNA target sequences should be
specific to the gene of interest and have ~20–50% GC content (Henshel et
al. 2004). Ui-Tei
et al. (2004) report that siRNAs satisfying the following
conditions are capable of effective gene silencing in mammalian cells: 1) G/C at the
5′ end of the sense strand; 2) A/U at the 5′ end of the antisense strand; 3)
at least 5 A/U residues in the first 7 bases of the 5′ terminal of the antisense
strand; 4) no runs of more than 9 G/C residues. Additionally, primer design rules
specific to the RNA polymerase used will apply. For example, for RNA polymerase III,
the polymerase that transcribes from the U6 promoter, the preferred target sequence
is 5′-GN18-3′. Runs of 4 or more Ts (or As on the
other strand) serve as terminator sequences for RNA polymerase III and should be
avoided. In addition, regions with a run of any single base should be avoided
(Czauderna et al. 2003). It is generally recommended that the
mRNA target site be at least 50–200 bases downstream of the start codon (Sui
et al. 2002; Elbashir et al. 2002,
Duxbury and Whang, 2004) to avoid regions in which regulatory proteins might bind.
Not all siRNAs directed against a target gene are equally effective in suppressing
expression of that target in mammalian cells. Therefore, it is important to identify
siRNA sequences that are effective inhibitors of target gene expression. Although
rational designs for selection of potential target sequences have been encouraging in
generating effective siRNAs, accurate prediction of the most effective siRNAs still
remains to be achieved. Current screening technologies are based on semiquantitative,
time-consuming methods and are not easily modified to perform rapid, simultaneous
screening of multiple siRNA/shRNA sequences. However, as the field of RNAi advances,
and more high-throughput applications are adopted, there is a growing need for rapid,
quantitative screening to confirm siRNA effectiveness (Kumar et
al. 2003; Mousses et al. 2003).
Recently, several quantifiable procedures that use reporter genes to help rapidly
identify effective siRNAs have been developed. In these approaches, the change in
expression of a reporter gene fused to a target gene is used as an indicator of the
effectiveness of an RNAi methodology. Here, we describe the psiCHECK™ Vector system,
which is based on use of the bioluminescent Renilla luciferase
reporter gene. The psiCHECK™ Vectors offer several advantages compared to other
fusion approaches such as green fluorescent protein (GFP)- or Flag-tag-based methods.
Measurement of net fluorescence from GFP in cell culture can be difficult and, in
most cases, a flow cytometer is required for quantitation. Flag-tag quantitation
requires Western blot analysis, which can be time-consuming. The high sensitivity of
bioluminescence detection can readily tolerate lower expression levels, and
introduction of a second reporter gene, firefly luciferase, allows normalization of
changes in Renilla luciferase expression, making the psiCHECK™
Vector approach more robust and giving greater reproducibility of results.
The psiCHECK™-1 and -2 Vectors allow quantitative selection of optimal siRNA
target sites and can be adapted for use in high-throughput applications. Figure 2.2
provides a basic illustration of how the psiCHECK™ Vectors are used. Both vectors
contain a synthetic version of the Renilla luciferase
(hRluc) reporter gene for monitoring RNAi activity. Several
restriction sites are included 3′ of the luciferase translational stop codon,
allowing creation of transcriptional fusions between the gene of interest and the
Renilla luciferase reporter gene. Because of the presence of
a stop codon in-frame with the Renilla luciferase open reading
frame, no fusion protein is produced. Consequently, there is no need to maintain
frames when inserting the target gene. Also, toxic genes or gene fragments can be
analyzed using this design without the danger of these genes killing the transfected
The psiCHECK™-1 Vector (Cat.# C8011) is recommended
for monitoring RNAi effects in live cells. Changes in Renilla
luciferase activity can be measured with the EnduRen™ Live Cell Substrate
(Cat.# E6481). This approach permits continuous
monitoring of intracellular luminescence. Renilla luciferase
expression can be monitored continuously for 2 days without interfering with
normal cell physiology.
The psiCHECK™-2 Vector (Cat.# C8021) contains an
additional reporter gene, a synthetic firefly luciferase gene
(hluc+), and is designed for endpoint lytic assays. Inclusion of
the firefly luciferase gene permits normalization of changes in
Renilla luciferase expression to firefly luciferase expression.
Renilla and firefly luciferase activities can be measured
using either the Dual-Luciferase® Reporter Assay System
(Cat.# E1910) or the
Dual-Glo® Luciferase Assay System (Cat.#
To use the psiCHECK™ Vectors for screening siRNA targets, the gene of interest is
cloned into the multiple cloning region located 3´ to the synthetic
Renilla luciferase gene and its translational stop codon. After
cloning, the vector is transfected into a mammalian cell line, and a fusion of the
Renilla gene and the target gene is transcribed. Functional
Renilla luciferase is translated from the intact transcript.
Depending on your experimental design, vectors expressing shRNA or synthetic siRNA
can be either cotransfected simultaneously or sequentially. If a specific shRNA/siRNA
effectively initiates the RNAi process on the target RNA, the fused
Renilla target gene mRNA sequence will be degraded, resulting in
reduced Renilla luciferase activity.
psiCHECK™ Vectors Technical Bulletin
psiCHECK™ Vectors
The use of bioluminescent reporter genes for RNAi optimization
siRNA synthesis in vitro provides a useful alternative to the potentially expensive
chemical synthesis of RNA (Figure 2.3). The method relies on T7 phage RNA
polymerase to produce individual sense and antisense strands that are annealed in vitro
prior to delivery into the cells of choice (Fire et al. 1998; Donze
and Picard, 2002; Yu et al. 2002, Shim et al.
The T7 RiboMAX™ Express RNAi System (Cat.# P1700) is
an in vitro transcription system designed for rapid production of milligram amounts of
double-stranded RNA (dsRNA). The system can be used to synthesize siRNAs for use in
mammalian systems (Figure 2.4; Betz, 2003b, Hwang et al. 2004) or
longer interfering RNAs for nonmammalian systems (Betz and Worzella, 2003; Betz, 2003c).
The DNA templates for in vitro transcription of siRNAs are a pair of short, duplex
oligonucleotides that contain T7 RNA polymerase promoters upstream of the sense and
antisense RNA sequences. Each oligonucleotide of the duplex is a separate template for
the synthesis of one strand of the siRNA. The separate short RNA strands that are
synthesized are then annealed to form siRNA.
RNAi experiments in nonmammalian systems are typically performed with dsRNA of
400bp or larger (Elbashir et al. 2001b; Yang et
al. 2000, Hammond et al. 2000). The minimum size of
dsRNA recommended for RNAi in these systems is ~200bp. In general, templates for
transcription of dsRNA for use in RNAi experiments correspond to most or all of the
target message sequence. Data suggests that longer dsRNA molecules are more effective
on a molar basis at silencing protein expression, but higher concentrations of
smaller dsRNA molecules may have similar silencing effects. Data generated at Promega
suggests that smaller dsRNAs can be as effective and efficient at inducing RNAi in
nonmammalian systems (Betz, 2003c).
In the T7 RiboMAX™ Express RNAi System, dsRNA production requires a T7 RNA
polymerase promoter at the 5′-ends of both DNA target sequence strands. To achieve
this, separate DNA templates, each containing the target sequence in a different
orientation relative to the T7 promoter, are transcribed in two separate reactions.
The resulting transcripts are mixed and annealed post-transcriptionally. DNA
templates can be created by PCR or by using two linearized plasmid templates, each
containing the T7 polymerase promoter at a different end of the target sequence.
See the T7 RiboMAX™ Express RNAi System Technical Bulletin
#TB316 for a protocol for in vitro synthesis of dsRNA for RNAi in nonmammalian
Figure 2.5 outlines the protocol for synthesis of siRNA using the T7 RiboMAX™
Express RNAi System. The initial step is generating the DNA template, which consists
of two DNA oligonucleotides annealed to form a duplex. Generally 20pmol of duplex
oligonucleotides are required per 20µl in vitro transcription reaction. Using the
RiboMAX™ Express T7 Buffer and Enzyme Mix allows efficient synthesis of RNA in as
little as 30 minutes. The annealed DNA oligonucleotide template is removed by a DNase
digestion step, and the separate small RNA strands (sense and antisense) are annealed
to form siRNA. The siRNA is precipitated using sodium acetate and isopropanol, and
the resuspended product can be analyzed on polyacrylamide gels for size and
integrity. Quantitation of the siRNA can be accomplished by either gel analysis or
RiboGreen® analysis (Molecular Probes).
Materials Required:
The target mRNA sequence selected must be screened for the sequence
5′-GN17C-3′. The generation of 3–5 different siRNA
sequences for a particular target is recommended to allow screening for the
optimal target site. The oligonucleotides consist of the target sequence plus the
T7 RNA polymerase promoter sequence and 6 extra nucleotides upstream of the
minimal promoter sequence to allow for efficient T7 RNA polymerase binding.
Details on design of oligonucleotides for use with this system are found in the
T7 RiboMAX™ Express RNAi System Technical Bulletin #TB316.
The DNA template can be removed by digestion with DNase following the
transcription reaction. RQ1 RNase-Free DNase (Cat.#
M6101) has been tested for its ability to degrade DNA while
maintaining the integrity of RNA. If accurate RNA concentration determination is
desired, the RNA should be DNase-treated and purified to remove potentially
inhibitory or interfering components.
T7 RiboMAX™ Express RNAi System Technical Bulletin
RNAi in Drosophila S2 cells: Effect of dsRNA size,
concentration, and exposure time
The T7 RiboMAX™ Express RNAi System: Efficient synthesis of dsRNA for
RNA interference
Produce functional siRNAs and hairpin siRNAs using the T7 RiboMAX™
Express RNAi System
In this article, siRNA was used to reduce the level of the PU.1
transcription factor. The siRNA was generated using the T7 RiboMAX™
Express RNAi System. Annealed siRNA was purified by isopropanol
precipitation. Forty-eight hours after transfecting 2.5µg of siRNA into
RAW264.7 cells, RNA and protein were isolated from the cells, and the
siRNA effect was analyzed by RT-PCR and Western blot.
In this article, siRNA was used to silence endogenous mouse and human
NRSF expression in NS20Y and HeLa cells. siRNAs were generated using the
T7 RiboMAX™ Express RNAi System.
DNA-directed RNA interference (ddRNAi) involves the use of DNA templates to
synthesize si/shRNA in vivo. ddRNAi relies on U6 or H1 [RNA polymerase III], or U1
[RNA polymerase II]) promoters for the expression of siRNA target sequences that have
been transfected into mammalian cells (Miyagishi and Taira, 2002; Brummelkamp
et al. 2002b; Novarino et al. 2004).
si/shRNA target sequences can be generated by PCR, creating "expression cassettes"
that can be transfected directly into cells (Csiszar et al.
2004; Castanotto et al. 2002) or cloned into expression vectors
(Sui et al. 2002; Paul et al. 2002; Gou
et al. 2003; Yu et al. 2002). PCR
generation is recommended when rapid screening of numerous siRNAs is desired.
Cloning-based approaches that allow direct ligation of hairpin oligonucleotides into
a ddRNAi vector provide another method for quickly and easily screening various
targets (Yeager et al. 2005). Screening can also be performed
using synthetic RNAs, but this can become expensive for numerous targets.
Vector-based approaches also offer the potential of stable, long-term inhibition
of gene expression by providing siRNAs on plasmids that allow selection of
transfected cells. Vectors with markers, such as puromycin, neomycin or hygromycin,
can be used for suppression of target genes for several weeks or longer. Transfection
with synthetic siRNAs allows for only a transient measurement (usually 48–72 hours)
of the RNAi effect.
The success of ddRNAi depends on several parameters including generation of
vectors containing full-length sh/siRNA sequences, delivery of those vectors into
cells, and expression of the si/shRNA constructs. Most strategies for cloning siRNA
target sequences into expression vectors utilize the design of a hairpin structure.
This design consists of two inverted repeats separated by a short spacer sequence
(loop sequence). After transcription by RNA polymerase, the inverted repeats anneal
and form a hairpin, which is then cleaved by Dicer to form an siRNA.
The GeneClip™ U1 Hairpin Cloning Systems facilitate easy expression of shRNAs in
vivo by ddRNAi-based methods. This system provides a simple, cloning-based approach,
allowing ligation of potential shRNA target sequences into vectors that allow
transient or stable expression in mammalian cells.
The GeneClip™ U1 Hairpin Cloning Systems (Cat.# C8750, C8760,
C8770, C8780, C8790) are designed for rapid and efficient cloning
of hairpin sequences for expression of shRNAs in vivo. The U1 promoter provides the
benefit of production of shRNA with a defined termination site, mimicking siRNA
produced in vivo when the Dicer complex cleaves the loop off the hairpin. Using the
GeneClip™ Systems, complementary oligonucleotides supplied by the user are annealed
and cloned into the predigested vector downstream of the U1 promoter. The linearized
vectors contain overhangs for increased cloning efficiency. The GeneClip™ Systems are
provided in five formats. The GeneClip™ U1 Hairpin Cloning System—Basic is designed
for transient suppression of the gene of interest. The GeneClip™ U1 Hairpin Cloning
System—hMGFP contains the green fluorescent protein gene from Montastrea
cavernosa and can therefore be used to determine transfection
efficiency; it also allows separation of transfected cells by fluorescent-activated
cell sorting (FACS®). The three remaining GeneClip™ U1
Hairpin Cloning Systems provide the ability to stably select transfected cells using
either neomycin, hygromycin or puromycin, so that experimental results do not depend
on transfection efficiency.
An overview of the GeneClip™ U1 Hairpin Cloning System protocol is given in Figure
Once target sites have been selected, the hairpin oligonucleotides are
annealed and ligated into the pGeneClip™ Vector, which is then screened for the
proper insert (Figure 2.7). These two oligonucleotides form a DNA insert that
contains a hairpin siRNA target sequence. Standard desalting of the
oligonucleotides is necessary, but gel purification and 5′ phosphorylation are not
required. Because the oligonucleotides inserted into the vector are short (~60nt),
detection by standard agarose gel electrophoresis can be difficult. To make
detection of inserts more convenient, a PstI site is engineered into the vector
backbone (Figure 2.7). Upon successful ligation of the annealed
oligonucleotides into the vector, a second PstI site is created. Digestion with
PstI therefore results in two easily separated bands of approximately 3kb and 1kb.
If no insert is present, PstI digestion will result in a single band. In our
experiments, ligation of the insert typically results in over 50-fold more
colonies than control ligations containing vector alone. In addition, the PstI
digestion showed over 90% of the colonies contained insert.
The introduction of long strands of dsRNA into mammalian cells induces a strong
interferon response that can lead to an overall shutdown of protein synthesis and
cell death. Short dsRNA molecules (<30nt) can bypass the interferon
response and still function in the RNAi pathway (Elbashir et
al. 2001a). Since the entire mRNA sequence cannot be used in mammalian
cells, various target sites must be selected for use in RNAi experiments.
Unfortunately not all target sequences in an mRNA will work equally well to
suppress target gene expression. Experimental testing of the optimal sequences is
required to confirm a high level of inhibition.
The pGeneClip™ Basic and pGeneClip™ hMGFP Vectors are recommended for use in
transient transfection assays. For many target sequences, transient transfection
can quickly yield cells that can be assessed for the effects of gene suppression,
including changes in phenotype, protein expression levels or other effects.
One consideration in evaluating transient transfection experiments is
transfection efficiency. For example, if only 30% of the cells are transfected, it
may be difficult to detect inhibition of expression, since 70% of the cells were
not successfully transfected and still express the target mRNA. The GeneClip™ U1
Hairpin Cloning System—hMGFP (Cat.# C8790) provides
an shRNA expression vector that contains an internal fluorescent marker for
monitoring delivery efficiency of shRNA-expressing constructs. In addition to
allowing transcription of hairpin target sequences and generation of siRNAs in
vivo, the vector contains an improved, synthetic version of the green fluorescent
protein (hMGFP) gene. The hMGFP gene encodes a 26kDa protein that gives improved
fluorescence and reduced cytotoxicity compared with other GFP proteins. The
presence of GFP allows easy determination of transfection efficiency and allows
selection of transfected cells by FACS® (Cormack
et al. 1996; Sorensen et al. 1999;
Galbraith et al. 1999). Importantly, the expression of hMGFP
does not affect gene silencing by shRNA molecules expressed from the same vector.
The pGeneClip™ Puromycin, pGeneClip™ Hygromycin and pGeneClip™ Neomycin Vectors
allow selection of stably transfected cells. Thus, the results are no longer
dependent on transfection efficiency. If necessary, clonal lines of transfected
cells can be generated if the population of selected cells does not show the
expected inhibition levels. Because integration of the vector into different
positions in the genome can affect expression of the RNAi hairpin, a population of
cells may not show suppression. A clonal cell line that sufficiently expresses the
RNAi hairpin may be required to demonstrate suppression with the pGeneClip™
To test the effectiveness of the GeneClip™ U1 promoter, we monitored reduction
in p53 protein levels after targeting p53 mRNA. p53 expression was targeted using
shRNAs cloned into a pGeneClip™ Basic Vector. More than 90% inhibition was
observed for the protein compared to a nonspecific control (data not shown). To
test p53 reduction in vivo, 293T cells, which contain elevated levels of p53, were
transfected with a pGeneClip™ Basic Vector expressing p53 shRNA. Compared to
nontransfected cells, the cells containing the pGeneClip™ Vector expressing p53
shRNA exhibited greater than 85% reduction in p53 protein levels (Figure 2.8,
Panel A).
GeneClip™ U1 Hairpin Cloning Systems Technical Manual
pGeneClip™ Vectors
GeneClip™ U1 Hairpin Cloning Systems for expression of short hairpin
RNAs in vivo
Successful RNAi experiments are dependent on both siRNA design and effective delivery
of siRNA duplexes into cells. RNAi delivery strategies vary depending on the target
cells or organism. For example, C. elegans may be injected (Fire
et al. 1998; Grishok et al. 2000), soaked
in (Tabara et al. 1998), or fed (Timmons and Fire, 1998; Kamath
et al. 2001; Fraser et al. 2000) dsRNA.
Successful delivery of interfering RNA has also been achieved by microinjection of RNA
into Drosophila embryos (Kennerdell and Carthew, 1998) and mouse
oocytes (Wianny and Zernicka-Goetz, 2000). Delivery to Drosophila
S2 cells in culture can be achieved by incubating the cells with the chosen RNA (Clemens
et al. 2000; Betz and Worzella, 2003). Use of DNA-based
approaches like ddRNAi vectors allows use of standard transfection reagents/methods; for
example, cationic lipids, calcium phosphate, DEAE-dextran, polybrene-DMSO or
electroporation (Caplen et al. 2001; Elbashir et
al. 2001a).
Once annealed, hairpin oligonucleotides are ligated to the appropriate pGeneClip™
Vector, the resulting constructs can be used for transient or stable transfection.
The GeneClip™ U1 Hairpin Cloning Systems provide a choice of vectors containing
various selectable markers (neomycin, hygromycin or puromycin) that can be used for
stable expression of a pool of cells or individual clones. Transfection of the
plasmid DNA into human cells may be mediated by cationic lipids, calcium phosphate,
DEAE-Dextran, polybrene-DMSO or electroporation. Transfection conditions will need to
be optimized for your particular system. Guidelines for transfection of the
pGeneClip™ Vectors are provided in Technical Manual #TM256. General considerations
for transient and stable transfection are given below.
High transfection efficiency is essential for achieving substantial suppression
levels using a transient transfection approach. Prior to testing for suppression
of the target protein, optimize the transfection conditions for maximum efficiency
in the system to be tested. The optimal conditions will vary with cell type,
transfection method used and the amount of DNA. When using the pGeneClip™ Basic,
Puromycin, Hygromycin or Neomycin Vectors, optimization can be performed using a
GFP reporter such as the Monster Green® Fluorescent
Protein phMGFP Vector (Cat.# E6421). The pGeneClip™
hMGFP Vector already contains the GFP reporter. The GFP reporter can also be used
to determine transfection efficiency for the assay. To test the effectiveness of
the pGeneClip™ Vector constructs (screening various sequences for levels of
inhibition), the use of a reporter, such as GFP, is highly recommended. This
control can be performed as a separate transfection to determine the percentage of
the cell population transfected or as a cotransfection where flow cytometry can be
used to sort GFP-positive cells. The level of target RNA suppression in
transfected cells can then be determined by taking the transfection efficiency
into account.
Obtaining maximum suppression requires optimizing specific assay conditions. We
have observed variations in suppression efficiency as a result of the cell line,
cell culture conditions, target sequence and transfection conditions. Varying the
amount of transfection reagent, amount of DNA and cell density can influence
transfection efficiency. Obtaining the highest transfection efficiency with low
toxicity is essential for maximizing the siRNA interference (suppression) effect
in a transient assay. Additionally, maintaining healthy cell cultures is essential
for this application. The key considerations are discussed more fully below.
Cell Density (Confluence) at Transfection: The recommended cell
density for most cell types at transfection is approximately 30–50%; this level is
lower than standard transfection experiments where cells are plated at 50–70%
confluency. The optimal cell density should be determined for each cell type.
Continued proliferation and the need to passage cells should be considered when
determining the number of cells to plate.
Cell Proliferation: The successful suppression of gene expression
requires actively proliferating and dividing cells, so it is essential to maintain
healthy cell cultures. It also is important to minimize the decrease in cell
growth associated with nonspecific transfection effects and to maintain cell
culture under subconfluent conditions to assure rapid cell division. We recommend
using the CellTiter-Glo® Luminescent Cell Viability
Assay (Cat.# G7570) to monitor cell viability and
Time: The optimal time after transfection for analyzing interference
effects must be determined empirically by testing a range of incubation times.
Typically little inhibition is seen after 24 hours, but the maximal suppression
time can vary from 48–96 hours, depending on the cells used and the experimental
targets tested.
For stable expression, antibiotic selection must be applied following
transfection. Cell lines vary in the level of resistance to antibiotics, so the
level of resistance of a particular cell line must be tested before attempting
stable selection of the cells. A "kill curve" will determine the minimum
concentration of the antibiotic needed to kill nontransfected cells. The
antibiotic concentration for selection will vary depending on the cell type and
the growth rate. In addition, cells that are confluent are more resistant to
antibiotics, so it is important to keep the cells subconfluent. The typical
effective ranges and lengths of time needed for selection are given in Table 2.1.
For example, to generate a kill curve for G-418 selection, test G-418
concentrations of 0, 100, 200, 400, 600, 800 and 1,000µg/ml to determine the
concentration that is toxic to nontransfected cells. Once the effective
concentration of antibiotic has been determined, transfected cells can be selected
for resistance.
Once the effective concentration of antibiotic has been determined, transfected
cells can be selected for resistance, as outlined in the protocol below.
See Figure 2.8, Panels B and C for stably transfected cells that suppressed
expression of p53.
The protocol outlined below was used to successfully deliver PCR products of
various sizes (180bp or 505bp) generated either from the 778bp ERK-A target or from a
control plasmid containing the Renilla luciferase gene
(phRL-null Vector; 500bp or 1,000bp) to Drosophila S2 cells in
culture (Figure 2.9; Betz and Worzella, 2003). Purified, in vitro-synthesized ERK-A
dsRNA was introduced into Drosophila S2 cells using the method
described by Clemens et al. (2000) following the protocol
described below.
Reduction of the targeted gene expression can be measured by 1) monitoring phenotypic
changes of the cell, 2) measuring changes in mRNA levels (e.g., using RT-PCR), or 3)
detecting changes in protein levels by immunocytochemistry or Western blot analysis
(Huang et al. 2003; Kullmann et al. 2002; Lang
et al. 2003). The suppression effect will vary depending on the
target, cell line and experimental conditions.
Controlling for nonspecific effects on other targets is very important. As a negative
control, cells can be transfected with either a nonspecific or scrambled target
sequence. This will show that suppression of gene expression is specific to the
expression of the hairpin siRNA target sequences. When suppression is determined by
Western analysis, positive controls for other genes (e.g., tubulin or actin) should be
included (Huang et al. 2003). Additional controls may also be
desirable (Editorial (2003) Nat. Cell Biol. 5,
Several summary articles are available that suggest various options for controls
that should be incorporated into RNAi experimental design to ensure accuracy and
correct identification of an RNAi effect (Editorial (2003) Nat. Cell
Biol. 5, 489–90; Duxbury and Whang, 2004). The preferred
control is to show restoration of functionality of a gene through artificial
overexpression of the target gene in a form that is not affected by RNAi. For
example, the target gene can be engineered to contain silent mutations that render
the mRNA invulnerable to the RNAi effect and introduced into the cell on a plasmid
vector. If such constructs "rescue" the original function of the gene, this is a good
indication that the observed suppression is mediated by RNAi. Use of siRNAs targeting
several different areas of the same gene to suppress expression may also be used to
provide evidence that an effect is mediated by RNAi. The observation of the same
suppression effect using more that one target RNA can confirm that the observed
effect is indeed RNAi.
For experiments using in vitro-synthesized siRNAs, the minimum concentration of
RNAi showing an effect should be used to avoid nonspecific effects due to the
introduction of large quantities of RNA into the cell. Ideally, any observed
suppression should be confirmed at both the mRNA and protein levels. Northern
blotting and quantitative, real-time RT-PCR can be used to demonstrate reduction of
expression at the RNA level. Quantitative Western blotting, phenotypic and functional
assays are some of the options available to show protein knockdown.
Scrambled siRNAs and siRNAs containing a single mismatch can be used as negative
controls. However, the latter are regarded as more informative. Positive controls
with RNAs known to exhibit an RNAi effect may also be useful.
Products may be covered by pending or issued patents. Please visit our Web site
for more information. For additional patent and licensing information on the
psiCHECK™ Vectors, see statements (a) through (c), below.
(a)For research use only. This product and/or its use is
subject to one or more of the following Promega patents: U.S. Pat. Appln. Ser. Nos.
09/645,706, 10/664,341, 10/943,508, U.S. Pat. Nos. 5,670,356, 6,387,675 and
6,552,179, Australian Pat. No. 698424 and various corresponding patent applications
and issued patents. The terms of the limited license conveyed with the purchase of
this product are as follows: Researchers may use this product in their own research
and they may transfer derivatives to others for such research use provided that at
the time of transfer a copy of this label license is given to the recipients and the
recipients agree to be bound by the conditions of this label license. Researchers
shall have no right to modify or otherwise create variations of the nucleotide
sequence of the luciferase gene except that Researchers may: (1) clone heterologous
DNA sequences at either or both ends of said luciferase gene so as to create fused
gene sequences provided that the coding sequence of the resulting luciferase gene has
no more than four deoxynucleotides missing at the affected terminus when compared to
the intact luciferase gene sequence, and (2) insert and remove nucleic acid sequences
in furtherance of splicing research predicated on the inactivation or reconstitution
of the luminescent activity of the encoded luciferase. In addition, Researchers must
do one of the following: (1) use luminescent assay reagents purchased from Promega
Corporation for all determinations of luminescence activity resulting from the
research use of this product and its derivatives; or (2) contact Promega to obtain a
license for the use of the product and its derivatives. No other use or transfer of
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(b)Licensed from University of Georgia Research
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2,105,984 and related patents.
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William Anders
From Wikipedia, the free encyclopedia
(Redirected from Bill Anders)
|William Alison Anders
|NASA Astronaut
|United States
|October 17, 1933
Hong Kong
|Other occupation
|Major General, USAF
|Time in space
|6d 03h 00m
|1963 NASA Group
|Apollo 8
|Mission insignia
William Alison Anders (born October 17, 1933 in Hong Kong) is a former United States Air Force officer, NASA astronaut, businessman, and engineer. He is was a member of the Apollo 8 space mission. Anders, Frank Borman and Jim Lovell were some of the to leave the orbit of the Earth and go to the Moon.
The crew took the first pictures taken of the earth from the Moon.[1][2]
The Anders crater on the Moon is named in his honor.
References[change | change source]
- ↑ Remarks by the President at the National Academy of Sciences Annual Meeting (2009)
- ↑ International Space Hall of Fame - New Mexico Museum of Space History


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To link to this article, copy this persistent link:
(Apr 05, 2012) On January 2, 2012, the President of Gabon promulgated Decree No. 0001 on the Finance Law for 2012. Tax incentives under the new Law apply to cement production and to the wood industry. (Albert Atangana, Gabon: Finance Law 2012 – Details, TAX NEWS SERVICE (Apr. 2, 2012), International Bureau of Fiscal Documentation online subscription database.)
Companies in the cement production sector will enjoy:
- a seven-year period of exemption from corporate income tax;
- reduction of the withholding tax rate from the standard 15% rate to 10% on distribution of dividends and interest payments;
- full deduction of interest incurred for business purposes;
- a seven-year period of exemption from VAT on certain operating expenses; and
- possible refunds of input VAT on equipment used for business purposes. (Id.)
Benefits for companies engaged in the wood industry include:
- a five-year period of exemption from corporate income tax and from the minimum lump-sum tax;
- a five-year period of exemption from withholding tax on distribution of dividends and interest payments;
- application of the declining balance depreciation method for certain equipment; and
- creation of a special renewal reserve for certain equipment. (Id.)
In addition, the Law applies a reduced 5% customs duty rate on imports of personal computers and a registration duty of 1% on the contribution in kind upon a company's formation or increase of share capital. The Law also requires that oil subcontractors keep their local accounts under the OHADA Simplified Accounting Standards (Systéme Comptable Allégé). The above provisions apply from January 1, 2012. (Id.; for an overview of taxation in Gabon, see, for example, Gabon Highlights 2012, DELOITTE (last visited Apr. 3, 2012).)
OHADA is the Organization for the Harmonization of Business Legislation in Africa (L'Organisation pour l'Harmonisation en Afrique du Droit des Affaires). (OHADA (last visited Apr. 2, 2012); Accueil, OHADA website (last visited Apr. 2, 2012); OHADA, under "Glossary," in Action Plan Developed by Ordre des Experts Comptables et Comptables Agréés de Côte d'Ivoire, at 2 (Dec. 2010), IFAC [International Federation of Accountants].)
|Wendy Zeldin More by this author
|Taxation More on this topic
|Gabon More about this jurisdiction
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The Global Legal Monitor is an online publication from the Law Library of Congress covering legal news and developments worldwide. It is updated frequently and draws on information from the Global Legal Information Network, official national legal publications, and reliable press sources. You can find previous news by searching the GLM.
Last updated: 04/05/2012


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Animals (Animalia. The Kingdom Animalia (=Metazoa) is one of a handful of lineages rooted far back in the branching "tree" that represents the history of life on Earth.
This lineage that is composed of those organisms we know as "animals" represents one of the three major origins of multicellularity (the other two large and diverse groups of multicellular organisms are the fungi and the green plants). It is difficult to list characteristics that apply to all animals, since various branches of the animal tree have undergone a range of significant modifications. However, most animals obtain energy from other organisms. They generally feed on them as predators (killing and eating a prey item); parasites, including herbivores feeding on plants (feeding on their "prey" without killing it, at least not immediately); or detritivores (ingesting tiny bits of decomposing organic material such as fallen leaves).
The diversity of animals is impressive. Discover the world's most endangered species. Photographic Dictionary. Warm-blooded Cold-blooded Birds Domestic animals Invertebrates.
Animal Diversity Web. Wild Animals Online - An online encyclopedia of wild animals - facts, photos, pictures, information. A-Z Animals - Animal Facts, Information, Pictures, Videos, Resources and Links. Animal-World, Dr. Jungles Exotic Pets, Animals, Aquariums. Extensive encyclopedia of animal information. Animal Facts Encyclopedia. Animal Pedia - The Free Online Animal Encyclopedia.


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New York Knicks v Boston Celtics
Kevin Garnett of the Boston Celtics grabs a rebound against the New... NyhetsfotoAmerikanska proffsligan i basket,Basketboll,Boston - Massachusetts,Boston Celtics,Gripa,Helkroppsbild,Horisontell,Kevin Garnett,Massachusetts,New York Knicks,Sport,Studsa tillbaka,TD Garden,USAPhotographer Brian BabineauCollection: National Basketball Association 2013 NBAEBOSTON, MA - JANUARY 24: Kevin Garnett #5 of the Boston Celtics grabs a rebound against the New York Knicks on January 24, 2013 at the TD Garden in Boston, Massachusetts. NOTE TO USER: User expressly acknowledges and agrees that, by downloading and or using this photograph, user is consenting to the terms and conditions of the Getty Images License Agreement. Mandatory Copyright Notice: Copyright 2013 NBAE (Photo by Brian Babineau/NBAE via Getty Images)


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I was always told that you should never meet your heroes, lest you be disappointed. It's better to have them live larger than life in your head, rather than realize the grounded reality. Provisionally throwing conventional wisdom to the wolves, I accepted an assignment in 2006 that required I interview a hero. We were to meet at the Rock and Roll Hall of Fame and Museum. The Cleveland appearance was to coincide with a new exhibit debuting at the Rock Hall: Revolution Rock: The Story of the Clash. I was to pen a feature for a national music magazine. It was to focus on my interview with Mick Jones.
Like many, my first exposure to The Clash came courtesy of Combat Rock, specifically "Rock the Casbah". I thought it was kosher. It opened an engaging if initially enigmatic Pandora's box, from which came "Janie Jones", "Career Opportunities", "Complete Control", "Tommy Gun", "Safe European Home", "The Guns of Brixton" (with lyrics You see, he feels like Ivan/born under the Brixton sun), "Clampdown", "The Call Up", "Ivan Meets GI Joe", "Train in Vain". I knew neither the songs nor the lyrics were about me – far from it – but they literally spoke my name at times. Two decades later, they still do. I still want to play in Garageland, with Mick Jones as my guitar hero.
The day of the interview I decided to forgo breakfast and lunch, fearful the butterflies in my stomach would revolt. I'd never been nervous for an interview before, and I've never been since. This was a hero, though. My enthusiasm meant I arrived at the Rock Hall nearly an hour ahead of schedule. I checked in as media, briefly wandered through the hall, and took a seat in the waiting area, reviewing my notes and checking my tape recorders. Other journalists from around the country trickled in, and we exchanged pleasantries. The three journalists next to me sat guessing whether Jones had recently "done anything besides Big Audio Dynamite". He had, and they were curiously unprepared. After a half hour, Jones and a small entourage brushed by the waiting media, not so much as glancing at us, leaving the faint trail of recently smoked cigarettes. I wondered whether this cold reception would be echoed in the one-on-one interview. A Rock Hall marketing exec explained that we would each have a strictly enforced 10 minutes with Jones. I was first.


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Spike the Dinosaur Battery
Click on photo for more information and prices!
Features and Benefits * Compact Battery Pack Charger with LED * Input: AC 110V, Wall Plug * Output: DC 12V, 300mA charge. * Built in Standard Female Tamiya connector * Reliable 12V DC Power Supply * Charges 6.0-9.6V NiMH and NiCd Battery Pack * Individual white box packed.


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Barnstable Municipal Airport is beginning a $1 million project to fix up the cement "pillow" that stops planes from overshooting Runway 6/24 and crossing Route 28.
It's serious stuff, as that's just what a wayward plane did a decade ago. Miraculously, no one was injured as the aircraft came to a halt in the nearby shopping plaza.
The three-week project may cause intermittent flight delays and short-term closures of the runway.
Click HERE to subscribe to e-mail updates from barnstablepatriot.com.
CONTACT US | The Barnstable Patriot | 4 Ocean Street | P.O. Box 1208 | Hyannis, MA 02601 | 508-771-1427 | Copyright © 2015 Local Media Group, All Rights Reserved.


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Top-ranked recipe named "Grilled Pepper and Black Olive Relish (Gc)"
Try this Grilled Pepper and Black Olive Relish (Gc) recipe, or contribute your own. "Bell pepper" and "Relish-sc" are two tags used to describe Grilled Pepper and Black Olive Relish (Gc).
In a mixing bowl combine all ingredients and season to taste with salt and freshly ground pepper. Serve with grilled chicken or fish. Copyright, 1997, TV FOOD NETWORK, G.P., All Rights Reserved Busted by Gail Shermeyer <firstname.lastname@example.com> on May 24, 1997 Recipe by: TVFN: GRILLIN & CHILLIN SHOW #GR3610 Posted to MC-Recipe Digest V1 #637 by owner-mc-recipe on Jun 7, 1997
|My private notes
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text: Stephen Edwards | IBDB: The official source for Broadway Information IBDB: The official source for Broadway Information Official Broadway League sites: - sites - broadway.org broadwayleague.com broadwayfanclub.com kidsnightonbroadway.com stagespecs.org tonyawards.com Quick Search For: In This Category: All Show People/Org. Theatre Season Stephen Edwards Composer Male Productions ProductionsDate of ProductionsFour Baboons Adoring the Sun[Play, Original] Incidental music by Stephen Edwards Mar 18, 1992 - Apr 19, 1992Orpheus Descending[Play, Drama, Revival] Electronic score by Stephen Edwards Sep 24, 1989 - Dec 17, 1989 Find out where Stephen Edwards and are credited together © 2001-2015, The Broadway League, All Rights Reserved. (Copyright and Disclaimer)


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The Sugarland Express
1974109 minutes
Steven Spielberg's debut film stars Goldie Hawn and William Atherton as Lou-Jean and Clovis Poplin, a down-on-their-luck couple who lose their child to the state of Texas and decide to pull out all stops to get him back. Lou-Jean pops Clovis out of jail and the two make off with their son, taking him away from his foster parents. But the long arm of the law isn't too far behind, especially since the couple's holding a cop hostage.


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Keep comfortable while showing some fashionable flair in this Lili knit dress from Element. It features lace sleeves, a scoop neck and a metal brand tag on the back.
- 100% Acrylic
- Imported
- Metal brand tag
- Model is 5' 9'' and measures approximately 34-28-36
- Model is wearing a size S
- Rib-knit collar & cuffs
- Scoop neck
- Size S has a 26" outseam
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text: There's a fair argument that the Simpsons credits gags are now a better reason to watch the show than anything that follows them – particularly in the case of their recent Game Of Thrones homage. But Matt Groening's show set the standard more than 20 years ago and has merely been fine-tuning and tweaking the formula since, adding more characters as the town of Springfield became as important as the Simpsons themselves and endlessly refreshing those blackboard and couch gags. And you know what? It's still a nailed-on classic, mixing physical comedy with character-establishing moments and all scored to that endlessly catchy Elfman theme. And those ever-changing opening and closing gags remain a showcase of comedy genius.


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Main Content:
Discover a beautiful assortment of ladies underwear from Gap.
Gap Collection
In the long ago days you had to choose between bikinis and briefs and had no problem selecting the right color scheme, simply because white, black, gray and nude were your only alternatives. Luckily, times have changed. At this point, quality underpants come in different styles and are available in a rainbow of colors and different fabrics. Want sophisticated lace or softest cotton? You've got it. Choosing the best type of panties is no longer a stressful task. Gap gives you unlimited access to an ample collection of cool, American-styles designed to match your style, personal preferences and silhouette.
Gap ensures a timelessly elegant collection of womens panties with girly, romantic and urban cool accents. When you're feeling sexy and confident, you can always opt for a red, blue, black or burgundy curve-hugging hipster tanga that will automatically boost your power of seduction. The lace-trim V thong is always a safe bet, due to its quality fabric, incredibly gorgeous design and versatility. Last but definitely not least, printed thongs represent the base of a relaxed, sporty outfit that you can wear throughout the entire day. Additional features, like the fantastic lace trim give the otherwise minimalist and sleek design a very feminine touch.
Gap Style Tips
Whether you are a big fan of stripes, monochrome creations or floral and geometrical patterns, the Gap ladies collection has everything it takes to please all tastes. Truth be told, the wrong type of womens under pants can compromise your entire outfit and impact your image. Too-tight can create unaesthetic bulges and draw unnecessary attention to lumps that are not exactly flattering. On the other hand, under wear designed to ensure a comfortable fit will give you the proper license to wear everything your heart desires while accentuating your beautiful curves in a natural, non-ostentatious manner. So much more than an accessory; they are your trusted ally when it comes to flaunting your beautiful body and sporting the clothing items that you love the most.
Regardless of the style of panties that you are looking for, the color and prints that raise your enthusiasm, Gap has everything covered.


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Posted: Apr 19, 2013 1:16 PM by Ashley Colley
BOSTON -- Due to the city of Boston being on lockdown while law enforcement continues its manhunt efforts for a suspect in Monday's marathon bombing, the Boston Red Sox have postponed their game with the Kansas City Royals scheduled for Friday night.
The announcement came from Boston officials just after 2 p.m. CT on Friday afternoon. No word has been made on a make-up date and time. KOMU will bring you updates when they become available.


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Harmony Public Schools are high performing K-12 college preparatory charter schools focusing on math, science, engineering, and computer technologies. For more than a decade, our schools have provided students from under-served communities the opportunity to excel in their studies in a small classroom environment focused on providing the skills they need to succeed in life.
With a college acceptance rate of 100 percent, Harmony Public Schools have earned the reputation of providing a distinct, high-quality education. Our schools also have the honor of being Texas Science, Technology, Engineering and Mathematics (T-STEM) field pioneer schools.
Harmony's vision is to lead our students from the classroom into the world as productive and responsible citizens, and even Nobel laureates.
The Texas-based non-profit organization was established by a group of dedicated educators and university academicians who were passionate about the way children approached math and science.
To fulfill Harmony's goal of educating students across the nation, Harmony Public Schools has developed strategic partnerships outside of the state. Harmony's unique and replicable school model has been proven to work in multiple locations within Texas and beyond.
Based on our experiences with partner schools in Arkansas, Louisiana, Missouri, New Mexico, Oklahoma, and Tennessee, we plan to continue to develop Harmony's partnership strategy with the goal of maximizing our impact outside of Texas.
It is the policy of Harmony Public Schools not to discriminate on the basis of gender, national origin, ethnicity, religion, disability, academic, artistic, athletic ability, or the district the student would otherwise attend.


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As a reseller, you have the ability to leverage the Ontrack PowerControls software suite as well as access the world's leading innovator in the data recovery industry, Ontrack® Data Recovery services.
With nearly 200 engineers worldwide and more than 1,400 years of combined data recovery experience, Ontrack Data Recovery services can offer your customers additional value and add another revenue stream to your organization.
Find out more or call 800.872.2599.
|Info Sheet


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See other Munich properties
Euro Youth Hostel
Real Reviews
Real reviews from fellow travellers
These are trusted reviews from guests who have actually stayed here. We hope you find them helpful.
Note: we only give a property a rating once it has received a minimum of 3 reviews.
- " The hostel was very clean and secure, although located in an area that felt a little sketchy at night (especially as a woman walking home alone). In addition, the common area downstairs was not really conducive to meeting people--I felt like groups mostly stayed on their own and didn't really mix with those they didn't know. That being said, the staff were wonderful, and one of the front desk people was nice enough to switch my room when I felt uncomfortable in my first assignment! "
- 24 Aug 2015 Anonymous USA
- " This is a very social hostel. The bar is always full of people during happy hour and sometimes beyond. But, noise did not travel to our room on the second floor. You need a key card to get into most areas, like the bar and each floor. Although the rooms were clean, the bathrooms could have been cleaner. Staff was very helpful when my I was feeling ill and helped my boyfriend find open pharmacies that were nearby. They offered breakfast, all you can eat, for about 5 euros. It was worth the money. "
- 15 Aug 2015 Anonymous USA
- " the bedroom was alright for a 12 bed only some young fellas it was good, very loudy but I expect that. "
- 02 Aug 2015 Anonymous France
- " 2 minute walk away from the train station which is great, but I found it a bit difficult to find the street as there were road works going on. Overall good hostel. Best bit about it was the social area. Room got very hot at night though which made it difficult to sleep. Staff were also helpful with anything I needed. "
- 21 Jul 2015 Anonymous Scotland
- " Helpful staff, delicious/filling breakfast (5eu extra). HUGE hostel- 5th floor room was hot with no AC. Downstairs bar area great for meeting people- about a 100 people crammed in to watch a soccer game one night. High energy hostel, not a quiet spot! "
- 06 Jun 2015 Anonymous USA
- " The location is excellent, it is super near to the central train station. "
- 01 Jun 2015 Anonymous Hong Kong
- " The location is really good. really close to the station. And has great facilities. It was also clean. And staff was willing to help you a lot. "
- 02 May 2015 Anonymous South Korea
- " Definitely one of the best hostels I have been to. Everything is clean and both wifi and plugs are readily available by bedside. Security and location are amazing as well. "
- 21 Apr 2015 Anonymous Canada
- " During my stay, I experienced horrible electricity problem at daytime. When I was going to use the laundry service to clean my clothes, the hostel went out of electricity all of a sudden. I had to wait so long to get the problem fixed. Luckily all of my clothes were washed but it was already four hours later. Moreover, I appreciate the high security of room since everyone has his own electricity supply to charge own electronic appliances. Staff are nice. "
- 12 Apr 2015 Anonymous Hong Kong
- " An excellent hostel. Much better than Wombats. Nice bar. Great beer. Clean rooms. Top location. Fast Wifi. "
- 04 Mar 2015 Anonymous Ireland


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At the beginning of the week I was at my parents and the Osteen's were on. Joel was speaking of "double for your trouble". That following morning I woke to a dream where I opened a book, believe it was the bbile but not sure, and the Words I clearly saw were, "Double for Your Trouble".
So, I guess one could say that the message just stuck with me and the dream may not be from God, I'm not saying it is, but think so. Either way, throughout the years the Lord has given me consistently the message that what we go through is bringing us to a better place in Him
Those painful trials are bringing you to a better place in Him where the pain will soon be lost in the joy of more of Him.
I know it isn't just a message for me as it is biblical so applies to all his children.


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Trial Set In Wayne Newton Museum Dispute
October 4, 2012 7:26 AM
(Las Vegas, NV) — Wayne Newton and his business partners will square off in a jury trial.
Judge Elizabeth Gonzalez has set aside three weeks in May to hear a dispute over a museum at the entertainer's estate. Plans called for Newton's Casa de Shenandoah east of the Las Vegas Strip to become a tourist destination.
The Texas bankers bankrolling the project say they have spent over $50 million dollars, and now want out.
Newton and his wife claim the partners are trying to squeeze them out of their 20% interest in the museum.


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Most Popular in:
Event Coverage
Record Attendance for CEW's 2013 Beauty Product Demo
By: Nancy Jeffries
Posted: March 15, 2013
Main floor of the 2013 CEW Beauty Product Demo.
page 2 of 4
The winner will also be announced at CEW's Beauty Awards luncheon in May.
Trends and News
Jacobson noted the 2013 Product Demo was the largest in its 60-year history, and several key trends had become apparent over the year. "Nails, for example, are not slowing down," said Jacobson. "We're seeing everyone doing a nail product." The response to the Sally Hansen Manicure station on the exhibition floor gave every indication that nails will continue to express and impress for the coming year.
"For lips," said Jacobson, "there are hybrid lip products, combining color and treatment without irritation, and as far as skin care goes, there are lots of spot treatments." More advances are also being seen in at-office and at-home treatments. "This is why we've added at-home tools this year," said Jacobson, who noted that laser technology was increasingly being targeted for at-home use. She cited Dr. Schultz's progressive peel, a three-month peel you can do at home, as an example. "Skin care is still big," she said, "and there's also a lot of color." She said that the independents have also made an impact. "This year, we've increased the indie submissions by 50%."
In the hair category, the news is color and treatment, and eyes still have it, with new advances in eye treatments, brighteners, serums and treatments for dark circles. "Everyone is looking for the perfect ingredient," said Jacobson, a boon to the supply side contributors, and there's also a great deal of activity in the natural arena. "Consumers are dictating what direction the products will take, with everything from multifunction to natural," she said. "We're also seeing similar products in prestige and mass, so there's a blurring of the lines. The prestige brands are strong, and after women held back for a while, we're seeing a return to prestige spending. They're definitely spending. There are also a lot of prestige brands that are still very affordable."
The product line-up was impressive throughout, with Lip Hybrids, which provide both moisture and color, featuring some of the hottest trends. YSL Beauté offered Rouge Pur Couture Vernis a Levres Glossy Stain—with the color of a lipstick, the shine of a gloss and the endurance of a stain; Giorgio Armani Beauty showcased Lip Maestro, with a velvet matte finish and the intensity of a lip lacquer; Lancôme L'Oréal USA showed Rouge In Love, lip color with a feather-light feel; and Revlon featured Just Bitten Kissable Balm Stain, a lightweight lip stain fused with a pampering balm.


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Royer & Brooks
Campaign Finance$6,000 Given
Figures are based on itemized contributions reported to the Federal Election Commission and state agencies. Please note that:
- contributions under $200 are not reported, and so are not included in totals.
- only contributions from individuals and organizations to candidates are included. Various accounting measures and more exotic contribution types are excluded.
- contributions are matched based on organization and recipient name reported within each election cycle. Contributions using an incorrect or non-standard version of the name may be missed.
- corporate name changes and mergers may cause figures to differ from those of the Center for Responsive Politics.
- organization totals include known subsidiaries of the organization.
For more information, please see our campaign finance methodology page. Lobbyist bundling data is described on our lobbyist bundling methodology page.
Top Recipients
- PAC Color Block
- Organization's PAC(s)
- Employee Color Block
- Associated Individuals
Republicans vs. Democratsin dollars
State vs. Federalin dollars
Top PAC Recipients
- PAC Color Block
- Organization's PAC(s)
- Employee Color Block
- Associated Individuals
Lobbying$1,380,000 Income
Figures are based on lobbying activity reported to the Senate Office of Public Records. Reported dollar amounts are required to be accurate only to the nearest $20,000. For organizations whose primary business is lobbying, we display total income and top clients. For organizations that are not primarily lobbying firms, we display total amount spent on lobbying and top lobbying firms hired.
For more information, please see our lobbying methodology page.
Lobbying Carried Out by Royer & Brooks
Names of Lobbyists
Client Amount Rent-A-Center $710,000 Federal Home Loan Bank of New York $400,000 Renco Group $160,000 Ironwood Global $110,000
Most Frequently Disclosed Lobbying Issues
- Banking,
- Trade,
- Housing
Most Frequently Disclosed Bills
Bill No. Title H.R.1588 Consumer Rental Purchase Agreement Act S.881 Consumer Rental-Purchase Agreement Act of 2011


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|The Talkeetna pattern is for a colorwork Hat & Mitten set. The pattern was published in the Winter 2009 issue of the online knitting magazine Petite Purls and you can view and print the pattern here.
This pattern is FREE!
Pattern Info
Finished Size: Hat – 18 (20)". Mittens – 5 (5.75)". To fit 2-5 (5-8) years.
Yarn: Brown Sheep Company Lanaloft Sport.
Needles: US 3 (3.25 mm) or US 4 (3.5 mm) depending on desired size. The hat requires a 16″ circular and a set of DPNs. The mittens require DPNs.
Gauge: 28 sts and 34 rows [26 sts and 30 rows] = 4″ (10 cm) measured over charted colorwork.
For more pattern details, please visit the Talkeetna Ravelry page.
Additional photos can be found in my Talkeetna Flickr set.