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Diflucan In Us
Boston Herald
October 28th, diflucan in us 2012 at 12:43 pm
Some observations as the Patriots and Rams are tied, diflucan in us 7-7, diflucan in us going into the second quarter.
The Patriots' defensive starters were Vince Wilfork, diflucan in us Kyle Love, diflucan in us Chandler Jones, diflucan in us Rob Ninkovich, diflucan in us Jerod Mayo, diflucan in us Brandon Spikes and Dont'a Hightower in a 4-3 base with Kyle Arrington (right) and Alfonzo Dennard (left) at corner and Devin McCourty and Tavon Wilson at safety.
Cornerback Sterling Moore subbed for Spikes in the nickel on the first drive of the game.
Hightower was lined up in the slot in man coverage on a receiver at one point.
Chris Givens beat Wilson on the touchdown because Wilson was too stiff and flat-footed while Givens ran full speed in his direction. Diflucan in us Poor technique on Wilson's part to surrender the deep ball so easiy. Diflucan in us It looked like trouble when Givens was still 10 yards in front of him.
Givens has 11 catches this season, diflucan in us and that's his sixth 50-plus yarder, diflucan in us including his second touchdown. Diflucan in us Givens set a record with five straight games with a 50-yard reception. Diflucan in us The Patriots knew that would be coming.
The Patriots' starting offensive line, diflucan in us from left to right, diflucan in us was Nate Solder, diflucan in us Donald Thomas, diflucan in us Ryan Wendell, diflucan in us Dan Connolly and Sebastian Vollmer.
Brandon Lloyd broke free from an aggressive look from cornerback Janoris Jenkins and scored an easy 19-yard touchdown on the Patriots' third third-and-short conversion on the drive, diflucan in us including a penalty. Diflucan in us But Lloyd was asked this week if the Pats could use the Rams' defensive aggressiveness against them, diflucan in us and he simply smiled and nodded. Diflucan in us He knew.
The Patriots used a big set on second-and-1 before the touchdown, diflucan in us with Michael Hoomanawanui at fullback and Marcus Cannon as a second left tackle. Diflucan in us And surprise, diflucan in us Stevan Ridley got stuffed for no gain by the hardest, diflucan in us cleanest hit of the game at that point. Diflucan in us Not only did the Patriots lose that battle, diflucan in us but they got smoked. Diflucan in us I've thought all along that the Patriot should run when it's given, diflucan in us or at least stay out of the packages that force them to run. Diflucan in us They're at their best when they can take the matchup to the defense, diflucan in us whether it's called with personnel or audibled at the line, diflucan in us and these jumbo run sets take that opportunity away.
Danny Woodhead was open after a defender fell down in the flat, diflucan in us and he turned it into a 24-yard gain. Diflucan in us Wes Welker busted his tail to get across the field to try to make the block that would have sprung him down the sideline, diflucan in us but he was a split second too late.
Arrington was replaced by Moore on the second drive. Diflucan in us He has a head injury and is questionable to return, diflucan in us but he was standing on the sideline without a helmet, diflucan in us which isn't a good sign.
Julian Edelman is back to returning punts, diflucan in us so it looks like the hand injury is just about in the past. Diflucan in us He couldn't do it last week in his return from the ailment.
Great catch by Rob Gronkowski toward the end of the quarter. Diflucan in us That was thrown directly over his head, diflucan in us and those have given receivers like Lloyd and Welker trouble this season. Diflucan in us Not an easy one to haul in, diflucan in us especially when you know you're exposed to a potential hit.
Ridley has caught some criticism for negative plays, diflucan in us but so many, diflucan in us including the one-yard loss on the goal line, diflucan in us are the result of the line or blockers losing the push up front. Diflucan in us That's been the biggest issue in those big sets.
Source: http://www.bostonherald.com/blogs/sports/blitz/index.php/2012/10/28/first-quarter-observations-brandon-lloyds-touchdown-has-pats-rams-tied-7-7/


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Hi everyone. I am Margaret, mom to an almost 7 year old (birthday on tuesday) boy named Robbie (bet you couldn't guess that from my user name :D). I have severe PCOS (had it since I was 16) and AF has been coming about every 34-38 days lately, never had one that was shorter then 21 days between. I am on day 22 (and according to the last few af I am supposed to be most fertile tomorrow). Woke up this morning to spotting. Have never spotted before, first is was red but now pink. Did not dtd last night so not sure what the spotting is about. Maybe it means I will be joining you ladies here :confused:
Anyone had this?

subset: commoncrawl


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EHS Compliance and Regulations Companies in Bulgaria
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Watson-Marlow Fluid Technology Group
Distributor in BULGARIA
Watson-Marlow`s heavy-duty hose pump can reliably handle many harsh materials, including grit-filled sludge, dependably for long periods of time. Peristaltic hose pumps are self-priming, dry running and virtually maintenance-free, with no expensive seals to replace, no check valves to clog, and no rotors and stators to wear out. Unlike other pump types such as gear pumps, ...


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Canon Rebel T3 / EOS 1100D Review
|11 Previous
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Canon EOS 1100D Dynamic Range (JPEG)
Our Dynamic Range measurement system involves shooting a calibrated Stouffer Step Wedge (13 stops total range) which is backlit using a daylight balanced lamp (98 CRI). A single shot of this produces a gray scale wedge from the camera's clipped white point down to black (example below). Each step of the scale is equivalent to 1/3 EV (a third of a stop), we select one step as 'middle gray' (defined as 50% luminance) and measure outwards to define the dynamic range. Hence there are 'two sides' to our results, the amount of shadow range (below middle gray) and the amount of highlight range (above middle gray).
To most people highlight range is the first thing they think about when talking about dynamic range, that is the amount of highlight detail above middle gray the camera can capture before it clips to white. Shadow range is more complicated; in our test the line on the graph stops as soon as the luminance value drops below our defined 'black point' (about 2% luminance) or the signal-to-noise ratio drops below a predefined value (where shadow detail would be swamped by noise), whichever comes first.
Note: this page features our new interactive dynamic range comparison widget. The wedges below the graph are created by our measurement system from the values read from the step wedge, the red lines indicate approximate shadow and highlight range (the dotted line indicating middle gray).
The EOS 1100D's is displaying the tone curve we've seen many times before from Canon, giving good dynamic range (around 8.3EV) and a gentle roll-off as you move towards the highlights. The Neutral Picture Style is a little different from the others, employing slightly lower contrast in the upper mid-tones but clipping a bit more abruptly to highlights, but at the same point above middle gray. Overall this is exactly what we're used to seeing from Canon DSLRs, meaning a little less highlight range than is typically obtained from Nikon and Sony competitors (but much the same as other brands).
The Highlight Tone Priority option (Custom Function II.5) is a method for capturing more information in the brightest parts of the scene. It does this by applying less amplification to the signal coming from the sensor, then compensating for it by using a different tone curve to ensure the correct brightness in the final image. Turn this on and you get just over one stop additional highlight range, matching competing models from Nikon and Sony.
When used at ISO 200, the effect is the same as underexposing a 'normal' ISO 100 shot by one stop, then pulling up the mid tones and shadows to compensate. The result is an image that looks right but doesn't lose the extra highlights you've captured. It means you have to use ISO 200 and upwards, because you're using the sensor at its lowest amplification setting but with one stop less exposure than in normal mode. The penalty is a touch more noise in shadow regions (as shown by the bottom limit of the HTP ON strip being very slightly higher).
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- Canon EOS M58.8%
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- Panasonic FZ2500/FZ20001.9%
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- Olympus E-PL80.1%
- Olympus 25mm F1.2 Pro1.5%
- Olympus 12-100mm F4 IS Pro1.9%
- Olympus 30mm F3.5 Macro0.1%
- Sigma 85mm F1.4 Art3.6%
- Sigma 12-24mm F4 Art2.6%
- Sigma 500mm F4 DG OS HSM Sport2.4%
- GoPro Hero50.8%
- GoPro Karma drone2.2%
|Flower in flower by atdigit
from Random Items Challenge 26
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|A Normal Dat at Thomas's Clap 2016-9379 by Andrew Maltzoff
from Show us SCHOOL!


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Best Buy will return as the anchor sponsor of the No. 17 Ford at Roush Fenway Racing next season, but with a different driver behind the wheel, the organization announced Friday at Chicagoland Speedway.
The electronics and appliance retailer will sponsor the 2011 NASCAR Nationwide Series champion in 12 Cup races, as Stenhouse competes in his first full season in NASCAR's foremost series. Best Buy also will sponsor the No. 99 Ford of Carl Edwards in four Cup races.
Fifth Third Bank and Zest also will return as primary sponsors for the No. 17 car, which has approximately 70 percent of its inventory filled for 2013. Veteran Jimmy Fennig is expected to remain with the 17 car as Stenhouse's crew chief, though team owner Jack Roush reserves the right to change personnel.
"Ricky is a very lucky young driver, because he is able now to step into a mature team," Roush said Friday at Chicagoland. "Regardless of how the personnel winds up being distributed next year, he winds up taking one of the three seats at our table -- which have been very good seats based on the work that (general manager) Robbie Reiser has done on the manufacturing side, and all the crew chiefs at the race track.
"Unlike a lot of rookies that have to wait their time to either grow a team that can give them what they need, or to find a senior driver that backs away (and) leaves a spot open, he is very lucky to have that. Roush Fenway and Best Buy and Zest and Fifth Third are very lucky to have Ricky -- Ricky is going to make a statement in this business."
Stenhouse is fighting for his second straight Nationwide title in Roush Fenway's No. 6 Ford. He trails series leader Elliott Sadler by one point with eight races left.


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This is a lesson about the electromagnetic spectrum. Learners will read two pages of information about the electromagnetic spectrum and answer questions in an accompanying worksheet. This activity is from the Stanford Solar Center's All About the... (View More) Sun: Sun and Stars activity guide for Grades 5-8 and can also accompany the Stanford Solar Center's Build Your Own Spectroscope activity. (View Less)
Materials Cost: 1 cent - $1 per group of students
This is a lesson about the formation of plasma bubbles in Earth's ionosphere. Plasma bubbles cause stars to twinkle and radio signals from satellites to distort. Learners will build a model ionosphere in order to demonstrate and understand this... (View More) process. This activity requires special materials including a laser pointer and silicon-based glue. (View Less)
Materials Cost: 1 cent - $1 per group of students
This is an activity about determining the distance of a solar flare from the center of the Sun's disk. Learners will use transparency grids overlaid on images of the Sun in order to calculate the distance of a solar flare, similar to a signal... (View More) detection method used by scientists. This is the second activity in the lesson "How Does HESSI Take a Picture?" NOTE: The HESSI mission was renamed RRHESSI (Reuven Ramaty High Energy Solar Spectroscope Imager) after Dr. Reuven Ramaty, who was the original Co-Investigator for this NASA mission, and was a pioneer in the fields of solar physics, gamma-ray astronomy, nuclear astrophysics, and cosmic rays. (View Less)
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This is an activity about lunar magmatic differentiation, in which learners simulate the process of geological differentiation. The activity ends with an extension question that asks about the makeup of the highlands of the Moon. This activity is in... (View More) Unit 2 of the Exploring the Moon teacher's guide and is designed for use especially, but not exclusively, with the Lunar Sample Disk program. (View Less)
Materials Cost: 1 cent - $1 per group of students


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Just a couple of days after his 20th birthday, the young Czech climbing phenomenon Adam Ondra succeeded on what is most likely the world's hardest sport climb, making the first ascent of the well renowned La Dura Dura project at Oliana, Spain.
Both Ondra and Chris Sharma had been attempting the line, and it was Ondra, after 9 weeks of effort, who clipped the chains to make the first successful redpoint.
The route, now graded 9b+ is the same grade as the last 'hardest route in the world' (The Change at Flatanger in Norway) that Adam put up last year. They may be the same grade, but Adam thinks La Dura Dura is the harder of the two...
In a recent interview on Planet Mountain, Adam explained how La Dura Dura breaks down:
"The first section is comprised of 15 moves, about 10 metres high, a 9b/9b+ in its own right which leads to a rest, a bad kneebar where I could recover for about a minute before moving up into the second section, 10 metres of 8c+ climbing which leads to two tiny crimps and then the jug and good rest. From here... a further 20 meters of 8b climbing lead to the top."
And Adam also explained why it is harder than The Change:
"If I compare it to Change I think La Dura Dura is harder, but still 9b+. Change really suits my style of climbing, the crux has some moves where you need to be really flexible and it seems to have been naturally made specifically for me. I know it might sound strange, but La Dura Dura has more straightforward climbing, but you really need to get everything wired 100%. I needed about 5 weeks for Change, and 9 weeks for La Dura Dura. I'd say that for me La Dura Dura is a better achievement because it fits my style less."
Oliana has been a forcing ground for world sport climbing after coming in to vogue around eight years ago. Some notable ascents have been Chris Sharma's Papichulo (9a+) in May 2008 and Pachamama (9a+) in May 2009 and of course Adam Ondra's Chaxi Raxi (9b) in April 2011.
Chris Sharma, who was the vision behind La Dura Dura and bolted the route, spoke of it and how it stacks up against other hard routes in this 2012 UKClimbing Interview:
"Well, first of all I definitely see a lot of potential still to push the standards in sportclimbing. One of the biggest challenges is finding the perfect route that is just beyond our limits but that is also something much cooler and more inspiring to warrant trying harder and longer than ever before. In my time here living in Spain and dedicating myself to finding, developing and climbing the hardest routes 've been trying to find that next step. It seems instead of one giant leap it's a lot of baby steps instead. Although there are still no 9b+ routes, within the grade of 9b there are some easier and some harder. For example Neanderthal in Santa Linya might be on the lower end and First Round First Minute is probably nearer the higher end.
But there are two projects that stand out as being a solid step beyond. Perfecto Mundo in Margalef and La Dura Dura in Oliana.
I've been working on both of these lines on and off and have been quite close to redpointing several times. This last season I had the pleasure to work on La Dura Dura with Adam Ondra and it was an amazing experience to try such a hard line with such a good climber. We both had some really good goes but in the end the season came and went without a send. Although it may seem sometimes we walk away empty handed when we fail on our projects, I think redpointing isn't the only marker for progression. With La Dura Dura, I had thought that the route was totally beyond me and that it was a route for future generations. I had previously done all of the moves but was unable to link any of them. Teaming up with Adam, I think some of his motivation and determination rubbed off on me and I realised that maybe it was possible, and by the end of the season we were close to doing it. So even though I have nothing to show for all this time spent on that route I still feel like I had a huge progression in my climbing this year."
Adam Ondra is sponsored by: Black Diamond , Montura, Entre Prises , La Sportiva , Beal and Hudy Sports
Tonight's Friday video features 22 year-old, up-and-coming US trad climber Molly Mitchell on her new routes,... Read more
As reported previously, German/Norwegian Thilo Schröter has a good time in Rocklands this summer, with multiple ascents of... Read more
Chris Sharma has made the first ascent of Alasha, a Deep Water Solo (DWS) route which he says is of roughly the same... Read more
Adam Ondra is now back in Flatanger where he intends to work some of his remaining projects. On his way there he made a couple of... Read more
In April this year, Piotrek Schab, 19, repeated Chris Sharma's Papichulo at Oliana in Catalunya for his first of the... Read more
After having done his final university exams, Adam Ondra took good time in Flatanger cave last week with the first ascent of... Read more


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Sky Rocket: The Story of a Little Bay Horseby Margaret Cabell Self
A starved, beaten, terrified little horse, headed for the slaughter house, is temporarily left at the riding stables of a Junior Cavalry unit by his dealer-owner. Impressed by his indomitable spirit and his "look of eagles," Colonel Brewster, Commandant of the riding unit, turns the pitiable animal over to his fifteen-year-old son, Tom, to try to rehabilitate the abused animal. Surprisingly, teen-age Pamela, who is nervous and uncertain, too, takes a significant part in the crucial weeks of patient, understanding effort that follow in trying to make the little bay live up to his name, Sky Rocket. Every bit of action in this moving story "might have happened" because the author has not only trained many thousands of boys and girls and hundreds of horses through her Mounted Troop riding classes, but the real Sky Rocket was her own horse, whose rehabilitation she skillfully and lovingly guided.
Select your format based upon: 1) how you want to read your book, and 2) compatibility with your reading tool. To learn more about using Bookshare with your device, visit the Help Center.
Here is an overview of the specialized formats that Bookshare offers its members with links that go to the Help Center for more information.
- Bookshare Web Reader - a customized reading tool for Bookshare members offering all the features of DAISY with a single click of the "Read Now" link.
- DAISY (Digital Accessible Information System) - a digital book file format. DAISY books from Bookshare are DAISY 3.0 text files that work with just about every type of access technology that reads text. Books that contain images will have the download option of 'DAISY Text with Images'.
- BRF (Braille Refreshable Format) - digital Braille for use with refreshable Braille devices and Braille embossers.
- MP3 (Mpeg audio layer 3) - Provides audio only with no text. These books are created with a text-to-speech engine and spoken by Kendra, a high quality synthetic voice from Ivona. Any device that supports MP3 playback is compatible.
- DAISY Audio - Similar to the Daisy 3.0 option above; however, this option uses MP3 files created with our text-to-speech engine that utilizes Ivonas Kendra voice. This format will work with Daisy Audio compatible players such as Victor Reader Stream and Read2Go.


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Sunday, January 10, 2010
7 comments:
- Betty Townsend said...
Oh, I love this!! When and where did you take it? She has a beautiful smile!!
- January 10, 2010 at 8:12 PM
- Erick said...
Very Nice. Great shot of Les!
- January 10, 2010 at 8:28 PM
- Leslie's pics said...
Uh, whatever happened to LIQUIFY?? or chopping me so you can only see me from the neck up?? Thank god that was the only one you posted!! I was getting worried as I scrolled down the page!
- January 10, 2010 at 9:01 PM
- jalna said...
Betty, I love this shot too!! I went with Les to help her with a photo shoot, and I took this before the family arrived. It's by the water fountain between the Elk's Club and Kapiolani Park.
Thanks Erick.
Les, I wanted to post more pictures of all your different faces, but I was afraid you might seek revenge and do the same.
- January 10, 2010 at 9:44 PM
- Christine said...
Sooo happy! Ha,ha! Nice pic of Les!
- January 11, 2010 at 7:53 AM
- Les said...
smart woman. ;)
- January 11, 2010 at 10:18 AM
- jalna said...
Thanks CQ!
Les, I know.
- January 11, 2010 at 9:22 PM
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Help Design a PlayStation Game From Scratch
Vote on fan-submitted ideas and concepts for a game from The Bearded Ladies.
As gamers, we very often think we have great ideas for videogames. Without any real knowledge of how to actually make one, those ideas typically end up shared on a message board at best or going to waste at worst. Sony is now giving creative gamers the opportunity to help devise a new game from scratch -- no game development know-how needed.
The game is to be actually developed at The Bearded Ladies Consulting, a Sweden-based developer that previously worked on PSN games Landit Bandit and Pipe Madness. The only aspect of the new game that's been decided is that it will be an "action platformer" -- everything else, such as the title, plot, and character design, will be thought up by the public.
Each Friday for the next eight weeks, a new challenge will be shared. The following week, the top submissions will be presented on Facebook where they'll then be voted on. The winner each week will be included in the game with the creator receiving a listing in the game's credits.
The first week's challenge is to create a main character and a mission for he/she/it. It's also preferred that some sort of artwork accompany it so The Bearded Ladies have some idea of what it will be working with.
Upon completion, the game will be released on the PlayStation Network sometime later this year. It'll carry a PEGI-12 rating in Europe, so it won't necessarily need to be made for children. There's currently no word on a North American release.
Although it's a Euro initiative, anyone can participate. More details and an example of what's being looked for this week can be seen over at the Euro PlayStation Blog. Submissions are to be made on the Gamocracy forums, located here.
Source: Gamasutra
See Also
Today in Gaming - 2/18/11: Medal of Honor 2 and Infamous 2 Hero Edition
Comments (2)
That souds pretty cool.Posted: Feb 18, 2011 12:00AM PST by steelerzfan101
I am glad to see developers doing this. They should not do this all the time but they should definitely do it a little bit more!
whoaPosted: Feb 18, 2011 12:00AM PST by smile_kill
really i gonna get sum ideas from ecco the dolphin game or maybe nba 2k11


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Carol Hevey named 2010 BusinessWoman of the Year
As executive vice president for Time Warner Cable's East Region, Hevey is responsible for cable operations serving 5.9 million residential video and commercial, high-speed data, digital phone and wireless customers.
She becomes the 26th woman to be honored with the Charlotte BusinessWoman of the Year award for significant, sustained contributions to business and the community. The award celebrates the achievements of exceptional women professionals who exhibit leadership as defined by the values of The McColl School of Business at Queens - competence, character and commitment to the community.
Previous winners include Cathy Bessant, Jennifer Appleby, Patricia Rodgers, Ruth Shaw and Joan Zimmerman.
"Carol Hevey is known for her visionary leadership at Time Warner Cable, and has been selfless in her devotion to community service," said Pamela Davies, president of Queens.
Hevey's previous civic honors include being named to the 2010 Charlotte USA 20: Celebrating Dynamic Women list. She also is a past winner of the Power Women in Cable award from Cable Fax Magazine and Multichannel News' Wonder Women award. She serves on the board of directors of Charlotte Center City Partners, Charlotte Chamber of Commerce and Central Piedmont Community College Foundation. She is the 2011 chairperson of the American Heart Association's Go Red for Women Campaign in Charlotte. She also is on the board of advisors for the Charlotte Area YWCA and the N.C. Governor's Conference for Women.
The Charlotte BusinessWoman of the Year award will be presented at a March 14 luncheon at Queens featuring keynote speakers Cathy Bessant of Bank of America, and Joan Zimmerman of Southern Shows Inc, both former winners.
To register for the luncheon visit: www.queens.edu/bwoy.
- Queens Rallies Together in Time of Need
- Queens Alerts Students of Sexual Misconduct Cases on Campus
- Queens once again makes U.S. News & World Report's Top 25 List
- MBA and MSOD Programs Recognized in CBJ
- Thesis on Social Media Evolves Into Publication
- MSN Program Named Among Top Southern Colleges in Graduate Healthcare Management
- Queens Students Published on WCNC
- Dr. Paul Nitsch Featured in Charlotte Magazine
- Queens Sends Two Swimmers to Rio Olympics
- Conceptual Era Places Emphasis on Liberal Arts


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Skip to comments.A Statement from Rush (Rush offers apology to Slut!)
Posted on 03/03/2012 2:15:11 PM PST by TSgt
For over 20 years, I have illustrated the absurd with absurdity, three hours a day, five days a week. In this instance, I chose the wrong words in my analogy of the situation. I did not mean a personal attack on Ms. Fluke.
I think it is absolutely absurd that during these very serious political times, we are discussing personal sexual recreational activities before members of Congress. I personally do not agree that American citizens should pay for these social activities. What happened to personal responsibility and accountability? Where do we draw the line? If this is accepted as the norm, what will follow? Will we be debating if taxpayers should pay for new sneakers for all students that are interested in running to keep fit?In my monologue, I posited that it is not our business whatsoever to know what is going on in anyone's bedroom nor do I think it is a topic that should reach a Presidential level.
My choice of words was not the best, and in the attempt to be humorous, I created a national stir. I sincerely apologize to Ms. Fluke for the insulting word choices.
Your choice of words set off a little warning bell in my mind.
Maybe Rush was persuaded by his little head? (That is, does Mrs. Limbaugh have any strong feelings on this issue?)
I'd love to see Sarah Palin tweet about and eviscerate this...obvious Dem plant.
As Palin, on every aspect of this subject, holds not four aces, but fifty-two of them.
Your choice of words set off a little warning bell in my mind.
Maybe Rush was persuaded by his little head? (That is, does Mrs. Limbaugh have any strong feelings on this issue?)
I'd love to see Sarah Palin tweet about and eviscerate this...obvious Dem plant.
As Palin, on every aspect of this subject, holds not four aces, but fifty-two of them.
I personally wouldn't enjoy that much confrontation, but if she deserves it so be it. Make sure she knows the truth....
Why isn't her *boyfriend* paying for the contraceptive?
He's the one c**ing inside her, not me.
Oh, she's sleeping around? #OccupySandra
Since you love the diabolical CT stuff, pictures of him and an under-aged girl during his South America vacation trip alone together doing.... would be appropriate for the sort of blackmail you are indicating.
Limbaugh went beyond merely using the S-word. He said that she should sell sex tapes to pay for birth control.
I think that he was trying to be funny, not being literal, and I don't want to pay for birth control. But aren't we hypocrites if we defend Limbaugh on this issue and attack such "comedians" as Letterman ("slutty flight attendant look") and Maher for making similar preposterous remarks about Palin?
And how about the Playboy article about "hate rape" of conservative women in 2009?
Although all of them have rights under the 1st Amendment, I think Limbaugh handed Obama a gift. And I have a 1st Amendment right to say it wasn't funny, and it supports the false notion that conservatives are vindictive yahoos.
If you did your own research instead of counting on what Rush tells you you would know that Dems do not call Rush an entertainer, They call him the leader of the Republican party.
You're wrong. I've seen the left call Rush an "entertainer" on more than a few occasions. Numerous other Freepers recall it too.
I have done it for a while now
I stand corrected. I should have said:
Rush's business is political commentating. He does it in an entertaining way. It's the left that minimizes Rush's political commentary by calling him an entertainer. You've been doing it for a while too.
Now why don't you go back to the ' bubble' and listen to Rush's past skits lecturing on how to NEVER give in to pressure from the left, just like he just did now.
I haven't defended Rush on his apology. Nothing that I posted would even indicate that. That makes your comment stupid.
I have no use for light banter, insider jokes interspersed with discussion of politics as though it's a horse race.
Rush took the Dem's bait and paid for it.
It was open line Friday. He should have let the listeners talk about Sheriff Joe and possible WH involvement in Breitbart death.
If they were claiming he was just an entertainer then they would have NO basis for demanding that Republican congressional leaders repudiate him. They could only do this by claiming her is a leader which is what they been doing for 2+ years.
Break out of the Rush bubble. He caved just like he lectures others to never do.
I was one of the few that never got upset for those 'attacks' on Palin because I believe it is the price one accepts to pay for being a confrontation public figure. As you noticed it brought much hand wringing here. Well guess what? I thought Rush's comments were funny. Not great taste but entertaining. If I was asked to defend Rush I would reply that he is an entertainer.
Now the reaction to such 'attacks' is part of the political game of war. Obviously Dems were much more effective with this with Sandra Fluke than any relating to Palin, getting Rush on the networks. I just don't understand why it matters.
No, the problem Rush has is he endlessly harped on all the personal 'attacks' on Palin and many times claimed they were proof she was a 'threat' as a potential Republican candidate. Well does this mean Rush thinks that Sandra Fluke is a serious threat? No, once again Rush trapped himself.
This was a gift to really go to war with for the good of the country and take down the congressional Rats, Obama, liberal academia and MSM another peg or two the way they were pushing it, Rush ran away.
Tells me something about his character when it's time to toughen up and press the attack.........He ain't really here with the tea party *Little Folk*.
>Im trying to recall when a vile leftist spewing on TV or radio made an apology. I cant recall one, ever. Anyone?<
Letterman (the pig) apologized to Sarah Palin and her family for making a sexual joke about her daughter and Alex Rodriguez at a Yankees game. Willow, the youngest daughter, was with Governor Palin at the game and the pig Letterman got her mixed up with Bristol.
....I haven't defended Rush on his apology. Nothing that I posted would even indicate that.
If they were claiming he was just an entertainer then they would have NO basis for demanding that Republican congressional leaders repudiate him. They could only do this by claiming her is a leader which is what they been doing for 2+ years.
Are you claiming that the left is logical and consistant, because they aren't. They will call Rush a leader when it suits their propaganda and they will call Rush an entertainer when it suits their propaganda. As I said, the left has called Rush "just an entertainer" numerous times. They do it when they want to dimmish his political commentary.
Break out of the Rush bubble. He caved just like he lectures others to never do.
My comment wasn't about Rush caving. It was about yours and others description of Rush being an entertainer. This is the second time I'm pointing this out to you. Yet you persist.
What part of the following quote from my previous post are you having trouble understanding?
She's having sex outside of marriage and that is fornication. Such immoral behavior is a trait of a slut. I'll hasten to add that among the Communist Goals is the following item:
26. Present homosexuality, degeneracy and promiscuity as "normal, natural, healthy."
Right after that cited goal of Communism is this:
27. Infiltrate the churches and replace revealed religion with "social" religion. Discredit the Bible and emphasize the need for intellectual maturity which does not need a "religious crutch."
If Sandra Fluke wishes to engage those long-term sexual relations with that one man you cited, it needs to be within the bonds of marriage. Yes, I'm old fashioned and my views, which I believe are Bible-based, are narrow and intolerant of what passes for morality nowadays. I date back to 1943 and the "New Morality" is the "Old Immorality" in my opinion.
But you asked a question in all sincerity and I gave you my unvarnished honest answer. Even here on Free Republic, where we Christians need not apologize for our convictions, there will be probably some snickers headed my way. So be it. I'll go ahead and take my lumps but will stand by what I just wrote here and cling to my so-called "religious crutch".
In any case, thanks for asking and I hope you'll find my response worthwhile even if you disagree.
What you are claiming is ridiculous. House libs led by Pelosi sent Boehner a letter DEMANDING that he repudiate Rush's comments. For your claims to make sense the letter would have to say:
" Dear Mr House Speaker. A well know entertainer Rush Limbaugh recently insulted a private citizen who is lobbying for woman's issues. We demand that you as House Speaker repudiate this 'entertainer's' comments because it is the job of House speaker to review every popular entertainers comments and comment on them "
The letter NEVER said that. Dems are saying that Rush is our leader not just an entertainer, Now they got OUR LEADER (as you seem to think he is) to cave, Great logic,
Maybe my sense of humor is biased by the likelihood that this episode will help destroy religious freedom and re-elect Obama.
I certainly don't want to include myself among those who chant the mantra "Anything that demeans women is not art!"
I think this apology is an admission that the "slut" affair was not Rush's best work. Remember when Rush introduced the term "feminazi?" I think he was better in those days. Read the comments of one woman who was kicked out of the "women's movement" over a disagreement about whether a song was "art":
And these feminists of the New Haven Women's Liberation Rock Band went into a rage, surrounded me, practically spat in my face, literally my back was to the wall. They're screaming in my face, "Art? Art? Nothing that demeans women can be art!" There it is. There it is! Right from the start. The fascism of the contemporary women's movement.
Well the libs at MSNBC certainly seemed to think it would.
My thoughts were that this was a funny distraction from the sad fact that congressional Republicans were being slaughtered by Democrats. Republicans have much bigger problems than Rush. That comment by the Santorum supporter about aspirin struck me as more problematic than this. Why is Rush relevant?
I would say that Rush's "joke" and subsequent surrender is demoralizing for some on the Right, and very encouraging for those on the Left who are paying attention.
Why is Rush relevant?
One answer is that we are debating it on FR. "There is nothing either good or bad, but thinking makes it so." - from Hamlet, Act 2, scene 2.
Well those on 'the right' that think that Rush was some superhuman savior-prophet better wake up because he is a very flawed human with some some unique entertainment abilities who does not have all the answers as they seem to think he does.
Dems were encouraged by Obama beating up on Republicans before Rush said anything about this.
"When are we going to go after Bill Maher, Ed Schultz and Keith Olberman? Why do they get off the hook?"
Bill Maher doesn't have sponsors and is therefore untouchable. As long as he keeps his ratings over 1 million viewers, HBO won't drop him. The only thing that can sink him is if he lets up on conservatives. His viewers expect his material.
The other two do have sponsors though, so that are fair game.
Indeed. The question is whether he can inadvertently help the Left.
Dems were encouraged by Obama beating up on Republicans before Rush said anything about this.
You may be right that, in the long run, this may not make much of a difference. Or maybe Rush threw gas on the fire. We'll see what happens, although I think an exact cause-and-effect analysis in November will be impossible.
I forgot about Levin such that Rush was such an overpowering figure.
"But aren't we hypocrites if we defend Limbaugh on this issue and attack such "comedians" as Letterman ("slutty flight attendant look") and Maher for making similar preposterous remarks about Palin?"
Calling that slut a slut is not a preposterous remark, but a simple truth. A single woman who wants $3,000 for contraception is a slut.
Remarks about Governor Palin by such low-life scumbags as Letterman and Maher were malicious lies, and therefore deserving of condemnation.
One is allowed to deplore the deplorable, including sluts and malicious liars, while defending the good, which includes Governor Palin. No hypocrisy is involved.
BTW, you are an agent provocateur from the sleazy byways of the left, right?
I'm fine with Rush protecting his own personal interests, don't we all?
When the shooting begins and American lives are on the line for liberty get back to me we can review this issue.
For now, our part is to ensure the sponsors who bailed on him never make a profit again.
If you read her testimony carefully, you will discern that she was acting as an advocate for sluts. She did not refer to herself or her habits once.
If she does not meet the Sullivan definition of a public figure (and she may not), then calling her a slut (unless Limbaugh knows her personally) was arguably libel, and definitely slander, and once called out he really had to back down.
"If you read her testimony carefully, you will discern that she was acting as an advocate for sluts. She did not refer to herself or her habits once."
Let's see, would that be better described as "a transparent ruse" or "a distinction without a difference?"
I doubt that Limbaugh would have much difficulty proving the truth of his assertion if the matter went to trial.
>She siad $3000 during law school. I imagine that is based on a monthly cost, which is how contraceptives are typically purchased, not a per act cost.<
Do the math. You can get a prescription for birth control that will set you back $30.00
So the strumpet is so flippin' dumb it will take her 100 years to get through law school?
Go back and read her transcript. She was relaying stories from other women at Georgetown, and speaking on their behalf. Nowhere in her transcript does she say SHE is the one needing free contraception. Her sex life is never mentioned.
Everybody is assuming she's sleeping around. She may be, but that's not what the transcript shows.
Rush was correct to apologize, because by calling her a slut he (and many people on this board) accused her specifically of being the one with the morals of an alley cat. There's no evidence of that.
This seems peculiar in that Rush's listeners wouldn't be expected to espouse Fluke's "values." The sponsors didn't want to appear to support his values? Seems like just being a sponsor would alienate Fluke's fellow believers.
If we all cancelled our subscriptions to HBO and told them it was because of Bill Maher, I think they'd take notice.
And this is my point: Conservatives aren't willing to fight at all. :(
You know, you may be on to something there. Maybe she's so outraged because she doesn't engage. Or, maybe she has been involved with LGBT for reasons she's not discussing.
By all means, Rush should apologize to her. Making herself a public figure, through the help of Pelosi and the Democrats, she has certainly warranted no disrespect or condemnation of her opinions. Dissembling about her past, she deserves no circumspection about her motives. Going on national TV, with the help of 'friends' in the media, her 'outrage' at the mere suggestion—expressed in public by Rush, but thought by many—that she has epressed and opinion for personal reasons, which she couched in carefully crafted public statements.
Or, maybe she just has imaginary friends or is hypothesizing for the sake of having chosen Georgetown admininistration as an opponent in her quest for a right of some kind for the last 3 years; after all, we all have a 'right' to engage in sexual activities, no matter our proclivities or perversity, and expect others to pick up the tab in the short or long run.
I am being sarcastic, of course. Frankly, I'd like to see her on a set, in person, with Rush, to defend her actions, opinions, and motives. After all, 0bama has said everyone should speak and listen to 'the other side' in considering their own beliefs. What should she have to fear from a conservative who 1) upholds religious freedom as a guarantee of US law, 2) adherance to a moral code (however outdated in her mind—or arrived at late in life as Rush apparently has), and 3) sense of personal responsibility?
To wit, I give you, by way of background on Fluke:
(It's best laid out/formatted at this link—http://www.jammiewf.com/2012/sandra-flukes-appearance-is-no-fluke/—, but the text of it is here)
"Sandra Flukes Appearance Is No Fluke
Posted by Just a Grunt on Mar 02, 2012 at 10:49 am
For me the interesting part of the story is the ever-evolving coed. I put that in quotes because in the beginning she was described as a Georgetown law student. It was then revealed that prior to attending Georgetown she was an active womens right advocate. In one of her first interviews she is quoted as talking about how she reviewed Georgetowns insurance policy prior to committing to attend, and seeing that it didnt cover contraceptive services, she decided to attend with the express purpose of battling this policy. During this time, she was described as a 23-year-old coed. Magically, at the same time Congress is debating the forced coverage of contraception, she appears and is even brought to Capitol Hill to testify. This morning, in an interview with Matt Lauer on the Today show, it was revealed that she is 30 years old, NOT the 23 that had been reported all along.
In other words, folks, you are being played. She has been an activist all along and the Dems were just waiting for the appropriate time to play her.
While she is described as a third year law student they always fail to mention that she is also the past president of Law Students for Reproductive Justice.
July 30 2011
Does your campuss LSRJ chapter face opposition in regard to facilitating a comprehensive conversation about reproductive justice? Well mine definitely does! While my campus has a mix of people with different backgrounds, and a rich liberal arts community, the Midwest doesnt exactly scream bleeding liberal. Some LSRJ chapters at conservative campuses face opposition in the form of other, more conservative, student run organizations; some face it from their administrations, and others from their peers, or the community in general. Whatever the opposition is, it can be incredibly frustrating and disheartening.
The question is, how do we combat this conservative opposition and oppression, in order to facilitate a discussion and educate others about the RJ movement? I am obviously not alone in facing these problems, as Sandra Fluke of Georgetown lead a packed room in a discussion on this question at the first Issue Caucus that I attended at the Leadership Institute, LSRJs national conference at Berkeley.
While no solution was definitively reached, and I personally dont begin to have the right answer, I was really charged by the discussion and feel many great ideas were presented. Some campus chapters decided to take an adversarial approach, feeling it important to use those scary words the opposition fears.
Further background research on Ms Fluke reveals that she got her start in government in New York in 2009.
Sandra Flukes professional background in domestic violence and human trafficking began with Sanctuary for Families in New York City. There, she launched the agencys pilot Program Evaluation Initiative. While at Sanctuary, she co-founded the New York Statewide Coalition for Fair Access to Family Court, which after a twenty-year stalemate, successfully advocated for legislation granting access to civil orders of protection for unmarried victims of domestic violence, including LGBTQ victims and teens. Sandra was also a member of the Manhattan Borough Presidents Taskforce on Domestic Violence and numerous other New York City and New York State coalitions that successfully advocated for policy improvements impacting victims of domestic violence.
As the 2010 recipient of the Women Lawyers Association of Los Angeles Fran Kandel Public Interest Grant, she researched, wrote, and produced an instructional film on how to apply for a domestic violence restraining order in pro per. She has also interned with the Coalition to Abolish Slavery & Trafficking; Polaris Project; Neighborhood Legal Services of Los Angeles County; Break the Cycle; the Domestic Violence Legal Empowerment and Appeals Project; NOW Legal Defense and Education Fund; Crime Victim and Sexual Assault Services; and the Human Services Coalition of Tompkins County.
Through Georgetowns clinic programs, Sandra has proposed legislation based on fact-finding in Kenya regarding child trafficking for domestic work, and has represented victims of domestic violence in protection order cases. Sandra is the Development Editor of the Journal of Gender and the Law, and served as the President of Law Students for Reproductive Justice, and the Vice President of the Womens Legal Alliance. In her first year, she also co-founded a campus committee addressing human trafficking. Cornell University awarded her a B. S. in Policy Analysis & Management, as well as Feminist, Gender, & Sexuality Studies in 2003.
My only question is, how does someone go from being a champion of domestic violence issues to an expert of womens reproductive health issues?"
Finally, 0bama called Fluke in a gesture of support; has he been reported as having called or otherwise expressed support for the communities devastated by tornados and families who have lost loved ones and perhaps everything they have? I mean, it is only fitting that Branson, MO, 'does not qualify' for FEMA funds, right? Or that the folks in Marysville, IN, will just have to fend for themselves without any WH attention, right? Fluke and the Democrats' obfuscation of tearing both religious freedoms and freedom of speech (as a two-fer) is the principal initiative of the day, jobs, debt, gas prices, poverty caused by inflation, food prices and the common good of Americans be damned, right?
I am confident that I would not like or approve of that aspect of Fluke's life, but I do not think Limbaugh's choice of that word was wise. But worse than that, he went on to say that she should sell sex tapes to pay for birth control, and in so doing, dragged himself at least temporarily down to the level of Letterman and Maher. If you approve of Limbaugh's "sex tape" comments, and disapprove of Letterman and Maher, that is hypocrisy.
I hope Limbaugh snaps out of it and returns to his old self, which was not helping Obama hold on to power.
At a minimum, ignoring a very important part of my post, you tried to smear me. Either you are a careless reader, or your post was dishonest. That's a shame, because I think you really hate Obama and his gang as much as I do.
What you are claiming is ridiculous. House libs led by Pelosi sent Boehner a letter DEMANDING that he repudiate Rush's comments. For your claims to make sense the letter would have to say:....
Your example doesn't contradict my point. It's ridiculous to think that it does.
Do you need it explained to you why it doesn't?
To claim that Rush is not "just an entertainer" is not logically the same thing as claiming that Rush is "our leader".
Don't try to put words in my mouth. You're not smart enough.
"he was right and his fans stand behind him"
Exactly. I don't think those advertisers are going to get the slut to make up the financial difference they are going to lose, she said she doesn't even have the money to buy condoms. However, maybe she could be the spokesman for Sleep Number beds! Double HA!
I agree with you. Rush should have known better than to wade into this territory. While I totally agree that taxpayers should not have to pay for someone else's birth control, Rush stepped in it and opened himself up to this.
Not a good moment for Rush.
Obviously, we really do not know if this woman is a slut. After finding out what her agenda is, we could safely
qualify her as political "whore" for the left.
My upset the other day is that this has become an issue now. All the media is doing is hitting our candidates and trying to force them to take sides. Either thay have to agree she may be a slut, or denounce Rush.
I like Rush and have listened to him for years, but this was not a better moment for Rush in the climate we have now.
"and once called out he really had to back down."
Not really. Rush by defeinition is an entertainer and just like the liberals such as Bill Maher calling Sarah Palin a cu** he has the same protections under the law.
Why do we always feel the need to run and hide?
After finding out what her agenda is, we could safely qualify her as political whore for the left.
At Georgetown law, she is the former president of Law Students for Reproductive Justice, an editor for the Journal of Gender and the Law, and vice president of the Women's Legal Alliance. She has a bachelor's degree in Feminist, Gender & Sexuality studies from Cornell.
Maybe she has a pleasant personality, but I would not like to be locked in a room with her.
Now you are contradicting on your own argument. Are you claiming you don't consider him a leader ? When your main ridiculous argument is that 'the left' is calling Rush an entertainer instead?
I don't know if you were waiting for your leader, err your 'political commentator', HA_HA, to tell you what happened on the Sunday shows but I will give you a heads up.
Pretty much every MSNBC host demanded that the Republican on the show explain their position on what Rush said about Sandra Fluke and his apology to her. NOT one called Rush an entertainer as you claim they are. If they did then the Republicans could have all replied :'You answered your own question'.
Rush IS an entertainer and a very successful one. He talks about golf, football, his experience at parties, his experiences traveling, etc, etc, and a few jokes about Democrats, Don't do the LEFT's bidding by making him into something more.
Cantor took the liberal bait and gave Rush an especially hard slap-down, loser. Newt's was more measured.
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


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Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss- and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Utilizing this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
While recent technological advances provide the means to efficiently scan the human genome to identify genes associated with diseases1-2, the subsequent functional characterization of these genes is a bottleneck in translating these discoveries into mechanistic insights and ultimately into therapeutics. Genome-scale RNA interference reagents have recently been created to enable systematic loss-of-function mammalian genomics3. To perform complementary gain-of-function gene studies, comparable libraries of arrayed cDNAs or open-reading frames (ORFs) are required along with efficient methods to employ these reagents in cell-based assays.
We4-5 and others6-9 have previously reported the construction of genome-scale ORFeome collections. These collections are useful templates for subcloning9 or recombinational transfer8 between vectors and protein production8, and they enable certain applications including interaction mapping10, but there are limits to the direct applications of these collections. They vary dramatically in terms of gene representation, format, and functionality, as well as quality measures such as the extent of clonality, sequence annotation, and experimental validation (see Supplementary Table 1). Tellingly, gain-of-function screening now lags behind the use of RNAi.
Here, we report the creation and characterization of two publicly available genome-scale human ORFeome collections: the human ORFeome version 8.1 Entry Clone Collection (hORFeome V8.1) and the CCSB-Broad Lentiviral Expression Library. Together, these collections are: (i) extensive, comprising 16,172 distinct ORFs mapping to 13,833 genes, (ii) clonal and sequenced, as each ORF plasmid is derived from a single bacterial colony and nearly all clones are fully sequenced, (iii) versatile, due to use of Gateway recombinational cloning11-12 (iv) enabling of cell-based functional screens, as the Expression Library encodes these clones in a lentiviral expression vector that produces consistent titers and gene expression levels and permits delivery to most cell types, and (v) available via ORFeome Collaboration (Supplementary Note 1).
We assembled these collections in four phases: First we expanded our previous collections to 19,281 ORFs in polyclonal format largely using existing protocols4-5; second, we derived clonal plasmid isolates from single bacterial colonies; third, we sequenced these clonal isolates and used the sequence data to choose clones for inclusion in hORFeome V8.1; and fourth we transferred clones to a lentiviral expression vector to create the CCSB-Broad Lentiviral Expression Library.
We expanded our library by transferring recently available ORFs from Mammalian Gene Collection (MGC)9 cDNAs into the Gateway system using directed PCR4-5 to create Entry vector clones while removing stop codons (Fig. 1a, top). To maximize throughput during this initial phase, clones were represented as a non-clonal pool of bacteria derived from recombinational cloning of PCR products. We next resolved this polyclonal library into clonal isolates using a robust and efficient workflow (Fig. 1a, middle) in which we isolated two colonies per ORF bacterial stock (see online Methods, Supplementary Note 2) from which we prepared ORF templates for sequencing.
We developed an optimized process to leverage next-generation sequencing and efficient alignment algorithms13 to efficiently sequence large numbers of ORF clones at high coverage (Fig. 1a, bottom). Clonal isolates for each ORF were pooled. Using Illumina sequencing technology, we compared efficiencies of sequencing full vectors versus purified ORF inserts only (Supplementary Fig. 1a). While purified ORF inserts yielded higher median sequence coverage, the added clone manipulation led to substantially greater coverage variability (Supplementary Fig. 1b-g, Supplementary Table 2) so we proceeded to sequence pools of full entry-clone plasmids. For some clone pools, we employed an alternative approach in which we PCR-amplified ORF sequences from individual bacterial colonies and sequenced the amplicons in a multiplexed, pooled format previously reported14 using 454 technology. Both methods were effective at sequencing ORF clones (Supplementary Figure 2), but our protocol based on Illumina technology yielded higher yields at lower cost per attempted clone (data not shown), and was therefore used to sequence the majority (84%) of the collection.
To assemble ORF sequences, reads from each clone pool were aligned to MGC reference sequences. Adequate reads were obtained to produce full ORF alignments for > 27,000 clonal isolates from 14,722 polyclonal ORFs, at the fold-coverage required for accurate base-calling (Supplementary Fig. 3). ORF sequences were annotated for mismatches, insertions, and deletions (Supplementary Tables 3,4). To evaluate the sequence accuracy of multiplexed Illumina and 454 sequencing combined with our automated alignment algorithms, we re-sequenced >121,000 nucleotides from 287 ORFs by the Sanger method, and found a confirmation rate of >99.99% of nucleotides. For each original ORF stock, the clonal isolate that most closely matched the MGC reference sequence was selected for inclusion in the hORFeome V8.1 collection. 198 clones with missing start codons were omitted.
Of 14,524 retained sequenced ORFs (Figure 1b), 82% (12,736) were either sequence-identical to the MGC reference or had one synonymous error, and comprise the majority of the hORFeome V8.1 collection (Fig. 1c, Supplementary Fig. 3). Another component of the V8.1 collection, denoted as the hORFeome V8.1 Mutant Subcollection, consists of 1,788 ORFs that had more than one synonymous or any non-synonymous mutations or other errors, and were retained since these plasmids may prove useful in some applications. We supplemented the fully sequenced set of ORFs with 825 clones comprising the hORFeome V8.1 Partially Sequenced Subcollection, including 597 clones from our recently described subcollection of kinases and kinase-related ORFs (clonal isolates, end-read Sanger sequencing in 2 directions)15 (see Supplementary Note 3) and 228 clones that were sequenced using next-generation technology over only part of the intended MGC ORF sequences. Finally, we denote the 823 clonal versions of isoforms that were removed prior to pooled sequencing as the hORFeome V8.1 Unsequenced Subcollection. Overall, hORFeome V8.1 includes 16,172 clonal ORFs, mapping to 13,833 human genes, of which 14,524 clones (90%) for 12,940 genes (94%) are fully sequenced (Supplementary Fig. 3, Supplementary Tables 3,4).
We next determined which currently annotated human transcripts are represented in hORFeome V8.1. The 14,524 fully sequenced library clones were mapped to National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) coding transcripts, and we found that 10,216 ORFs map with > 99% homology and constitute full length coding sequences (Fig. 1d, Supplementary Fig. 4). 1,545 additional ORFs represent partial coding sequences. The remaining 2,763 sequenced ORFs map to non-coding transcripts or to RefSeq transcripts with lower homology or are not currently found in RefSeq. Since the original MGC cDNA source templates for these clones were derived from expressed cellular transcripts, some of these non-full length clones may represent un- or mis-annotated but physiologically relevant transcripts. Indeed, incomplete knowledge of the transcriptome is a major challenge to obtaining a comprehensive ORF resource (Supplementary Note 4).
hORFeome V8.1 enables many applications as it permits rapid ORF shuttling into any Gateway-compatible expression vector. To enable large-scale screening of this collection in mammalian cells, we developed, optimized and validated a series of Gateway-compatible mammalian expression vectors (pLX series, Supplementary Fig. 5) encoding numerous desirable elements (see online Methods). We elected to shuttle the entire hORFeome V8.1 collection into the pLX304-Blast-V5 vector to create the CCSB-Broad Lentiviral Expression Library (Fig. 2a).
We conducted a pilot experiment on 509 ORF clones to assess: (i) protocols to transfer the entry library, (ii) high-throughput production of DNA and virus, and (iii) ORF expression in A549 cells (Fig. 2b-d, Supplementary Figs. 6-9). Plasmid DNA production and viral packaging were achieved in 96-well format with consistent DNA yields and titers averaging 2.1 × 106 infectious units (IU)/ml (Fig. 2c, Supplementary Fig. 7a). Titers were preserved across all ORF sizes (Fig. 2c, Supplementary Fig. 8a). We assessed ORF expression via quantification of V5-epitope tag expression and observed that approximately 90% of ORF lentiviruses induced expression signals greater than 2 standard deviations above the control mean (Fig. 2b, d, Supplementary Figs. 7b, 8b, 9).
Using the optimized protocols, we then produced the CCSB-Broad Lentiviral Expression Library in the pLX304-Blast-V5 vector, successfully isolating a single bacterial colony from 98.5% of reactions (15,935 total clones). To estimate the accuracy of the final collection of expression vectors, we performed end-read sequencing of 325 colonies and confirmed 98.2% accurate transfers (see online Methods). The utility of this resource for systematic functional genomic screens in mammalian cells is illustrated by recent results from a screen of a pilot subset of this collection (597 genes), which identified novel mediators of resistance to RAF inhibition in melanoma15. Additional pilot experiments confirm that this resource enables other readouts including immunofluorescence (Supplementary Figure 10).
In summary, we report here the construction of the most fully sequenced, flexible and annotated version of the human ORFeome to date. The entire collection, comprising both source (entry) clones and lentivirus vector expression clones, is available without restriction through the ORFeome Collaboration (Supplementary Note 1). We anticipate that these collections will greatly facilitate the systematic functional assessment of human genes that mediate cellular phenotypes.
Supplementary Figure 1 Pilot experiments to optimize pooling strategy for next-generation sequencing of ORF clones.
Supplementary Figure 2 Coverage histograms of sequencing hORFeome V8.1.
Supplementary Figure 3 Flowchart of hORFeome V8.1 creation.
Supplementary Figure 4 Alignment results of 14,524 completely sequenced clones with current NCBI RefSeq transcripts.
Supplementary Figure 5 Plasmid maps of pLX lentiviral expression vectors created as part of this study.
Supplementary Figure 6 Confirmation of viral preparations.
Supplementary Figure 7 Determination of virus titer and ORF expression.
Supplementary Figure 8 Virus titer and ORF expression are maintained across a wide range of ORF lengths.
Supplementary Figure 9 Western blot showing expressed ORFs.
Supplementary Figure 10 Viral preparations enable immunofluoresence high throughout screens.
Supplementary Table 1a Clonal and sequenced ORF Gateway entry clone collections
Supplementary Table 1b Comparison of Nomura and CCSB-Broad ORF collections
Supplementary Table 2 Illumina sequencing pilot data
Supplementary Table 3 Overview of next generation sequencing results
Supplementary Table 4 Annotated list of hORFeome V8.1 and CCSB-Broad Lentiviral Expresson Library.
Supplementary Note 1 Availability of clones and distribution procedures
Supplementary Note 2 Pilot experiments to determine number of colonies to isolate per polyclonal ORF.
Supplementary Note 3 Supplementing hORFeome V8.1 with kinase ORFs.
Supplementary Note 4 Challenges of completing the human ORFeome
Supplementary Note 5 Detailed high-throughput protocol of single colony isolation.
Supplementary Note 6 Details of pooled sequencing protocol optimization experiments.
Supplementary Note 7 Computing virus titers.
Supplementary Note 8 Li-COR in-cell Western and immunoblotting.
We acknowledge B. Piqani, I. Budianto, D. Szeto, T. Hirozane-Kishikawa, V. Swearingen and A. MacWilliams who participated in the generation of various intermediate ORFeome libraries, T. Nieland who offered technical advice, S. Hoang who provided automation support, J. Bochicchio, S. Young, A. Berlin, C. Russ and the Broad Institute Genetic Sequencing Platform who assisted with sequencing and alignment of reads, M. Garber who assisted with ORF sequence annotation, and J. Zhao, T. Roberts and T. Golub who participated in the generation of the kinase sub-collection. This work was supported by Broad Institute Scientific Planning and Allocation of Resources Committee (SPARC) funding, The Ellison Foundation (D.E.H., M.V.), DFCI Institute Sponsored Research funds to CCSB and CCGD and NIH R33 CA128625 (W.C.H., D.E.R., D.E.H). M.V. is a "Chercheur Qualifié Honoraire" from the Fonds de la Recherche Scientifique (FRS-FNRS, French Community of Belgium).
Note: Supplementary information is available on the Nature Methods website.
Author Contributions: J.S.B., Xia.Y. and D.E.R. wrote the manuscript and designed and supervised the process of creating clonal, sequenced ORFs and expression vectors from starting polyclonal ORF pools via Illumina sequencing. D.E.H, K.S.-A. and M.V. designed and supervised the process of cloning ORFs from MGC cDNA templates to generate Gateway entry clones as well as creating clonal, sequenced ORFs via 454 sequencing. Xin.Y., D.B., L.G., and R.R.M. created starting polyclonal ORF pools from MGC cDNA templates. T.H., Y.S., C.F., and C.L. performed bioinformatic analyses. R.L. produced the clonal ORF library and did LR reactions, transformations, colony picking and ORF DNA preparations. J.S.B., S.R.T., H.H., R.R.M., K.S.-A., and C.M.J. created the kinase sub-library and performed pilot experiments. O.A. and J.S.B. created and tested pLX vectors. J.S.B., T.B., T.H., C.F., C.L. and T.M.G analyzed sequencing data. T.B., T.M.G., Xia.Y. and D.E.R. conducted BLAST analysis of ORF sequences. C.N., Xia.Y. and S.S. created virus from ORFs. C.N., S.R.T., C.M.J. and Xia.Y. performed ORF expression assays. M.V., W.C.H., D.E.H. and D.E.R. supervised the project.
Competing Interest Statement: No competing interests to declare
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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124 NW Booth St
(541) 479-1505
Category: Moving & Storage
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825 Northeast F Street
(541) 471-6311
Category: Self Storage - Packaging
Foothill Mini-Storage
2828 Foothill Blvd
(541) 476-0245
Category: Household Goods Moving & Storage
Franklin's Moving & Storage
1900 NW Washington Blvd
(541) 476-7735
Category: Moving & Storage
Grants Pass Equipment Rentals Inc
851 SE Milbank Rd
(541) 476-5219
Category: Moving & Storage
Green's Sewing & Vacuum Distributors
560 Northeast F Street
(541) 476-1231
Category: Containers Industrial
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Google teaming up with British Library to digitize old booksBreaking News
Google is teaming up with the British Library to digitise 250,000 books, published between 1700 and 1870.
The British Library announced the deal that will involve scanning approximately 40 million pages. The out of copyright books will be available free of charge online via Google Books and the British Library website.
The British Library, located near St Pancreas in London, acts as the national library of the United Kingdom, and is one of the world's largest libraries, holding over 150 million items (books, manuscripts, newspapers, maps etc) from nearly every country in the world.
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- David Kennedy recalls his dinners with President Obama
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Remember who called AMSC $3 when it was $43? WPRT is going to collapse as soon as the hucksters are done spinning their fraud stuffing this in every sucker's account it can find. Avoid.
$.30 loss per share??! Company continues to lose money. Stock isn't worth even $10/share.
-Chic N. Little
I doubt it can drop to $10 anytime soon.
They just pulled 5M new shares out of their arse and were able to sell them for $43
sold them for $43 when the price was $48. lol
Regardless, AMSC did an offering at $18. Stock shot to $43+ on nothing but losses...nearly $1 billion of them to-date. Doesn't matter. The ponzi bubble kept inflating and got pumped on hype and speculators driving up to pump and dump. Well, it took 2-years and me shorting that from $18 to $43, but I kept my $3 target on it. :)
So I'm not saying $10 tomorrow. Oh no. This hype is Fraud St's flavor of the time just like all the dotcom stocks when earnings/revenues didn't matter and web visits and puppet socks on TV did.
Stock is strong, no doubt. Wish you guys the best. Just understand this is all speculation and just like FSLR, STP, SPWR, etc. I'm warning you all. Who knows, maybe I'm wrong this time. lol
Well, I'm amazed that anyone would buy this stock with widening losses. You know Fraud St. is going to sell into any good news as this pee-wee company tries to grow into size 40 shoes. Good luck.
amen brother!!!!!
buy puts $$$$$$$$$$$$$$$$$
Big Fatty Broke
stepped in front of a moving train
hey bigfatty_guns = minor threat - in my eye
listen to the lyrics
what the f are you saying?
BFG reiterates Sell on WPRT shares.
FSYS numbers out and not good.
Nat gas is the new solar hype. Long-tern nat gas will do well, like solar, but investors getting hosed now. Pump and dump into hype. Bank on it.
Now that you have made your point why not move on to another stock. I guess there is scam and scum. You are both.
shut up fat boy!