text:
Englewood Cliffs Guitar Lessons
Fort Lee School of Music offers the highest caliber guitar program in Bergen County to students of all ages and levels. The guitar department consists of experienced teachers who have established successful careers as performers and educators. Students are motivated to learn good technique, strong work ethics, chords, scales, rhythms, music reading, analysis and interpretation. These are skills that can be applied both musically and in all walks of life.
Our guitar program focuses on acoustic, classical, jazz or electric guitar.
Private lessons for all ages & levels
Lessons meet once a week.


text:
The 2010 Internet Summit in Raleigh, North Carolina wrapped up yesterday, with Internet professionals and entrepreneurs from across the United States coming together to discuss topics such as mobile marketing and social ecommerce. Our very own Bob Young was a keynote panelist and featured speaker discussing the future of both the web and books – no doubt two very broad and engaging topics.
Bob's featured talk was entitled "There is No Such Thing as a Book" – claiming that "whatever replaces the book on the Internet is not going to look like a book." During Bob's talk and as a fan of René Magritte, I couldn't help but imagine someone somewhere in the world wearing a t-shirt with a picture of a Kindle or an iPad and the sentence "Ceci n'est pas une livre" (I'll wait while you go Google Translate that). Traditionally, we have all come to know and love books in their physical form, but now, "books" are hyper-mobile strings of binary code easily accessible and translatable on multiple devices. A modern-day book's physical properties are seemingly confined only by the Wi-Fi signals that transmit them.
Bob argued that, "the device you are reading on is going to become more pleasurable," as a plethora of textual enhancements like video and hypertext accompany the written word. The question for some, however, is whether all you need is a good story? Things like video, hyperlinks and an Internet connect may actually detract from a book's narrative – diminishing the pleasure derived from an uninterrupted read. Personally, I think that in the future, some readers may intentionally choose to remain on one side of the digital divide, opting to read stories on books – not devices. But of course, that will remain a question of preference and choice, and if there is one thing the future of the web will include – it is choice.
For those of you who were unable to attend the event and enjoy all the interactivity that digital media has to offer, be sure to check out a recap of the lively discussions on Twitter #isum10.
We'd like to thank the organizers of the Internet Summit for putting on such an informative and well-organized event, and we look forward to seeing you again next year.
Gavin Jocius writes for the Lulu Blog – Archived

subset: commoncrawl


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|Camera make and model:
|Canon EOS REBEL T2i
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|2017:01:04 20:35:09
San Francisco, California
San Francisco truly needs no description.
January 4th, 2017
We arrived in San Francisco for a few days vacation and immediately went to the Golden Gate Bridge.This image viewed 1 times, as of last night.


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[Verse 1]
My image is deceiving
Don't start believing, what you see on the surface
My face, a road map
Of gullies and ravines but you don't see my genes
Pumping me up, like a young juvenile
Senile, but not in my dictionary
Girls, yeah, you better be wary
Sneak a peak at my physique, you're gonna shriek
Cause I'm a health freak, palates and yoga
I ginkgo baloba parsley broccoli
Puffing on my magic weed flax seed
500 grams a day of horny goat weed
So I can give the girls all the wood they need
Proud of my stuff, when she says what's up
Don't have clone my testosterone
I got enough left for sex on the cell phone
I can still do it
Yeah, I can still do it
Oh, I can still do it
Yeah, I can still do it
[Verse 2]
All hype, stereo type, a guy over eighty?
Can't cut the mustard, Soft as custard
What's bouncing in his head, ain't bouncing in his bed
It's all a myth, and I get so pissed
I make a fist with they insist
My family jewels can make a Christmas list
I get so pissed, when I see a swinger
It's mind over matter, good God
Suddenly I'm a tripod
I'll pole vault a ramp like a champ
I'm looking like a stamp
Sex is war, no bugle tootin
Until my weapon of choice (boing) is saluting
The vitamin rainbow, pumping my main flow
Night to day glow, and so I say so
I love a dessert, I lather my body
A little fat yogurt, uh-huh, oh yeah, hey!
[Verse 3]
Erectile dysfunction?
I say without compunction
At this particular junction
Ain't my malfunction
No prostate's gonna state the state my prostates gonna be in
Restate the ? not the state I'm trying to be in
And years before my time
Put me out the pasture
No longer the master
And that is a disaster
We're talking sun city, well shove the pity
Cause I ain't living in limp dick city
I eat tons of keil, I eat it like a whale
My six pack abs ? Chippendale
I'm the go go
Cilantro my mo-jo, collard greens my green ho
Beets, organic meats, gluten free wheat's


text:
Friedman, Harvey and Dorismae, Lectureship on Aging | Vertical File Collection, Bernard Becker Medical Library Archives
Collection Overview
Biographical/Historical Note
Scope and Contents of the Materials
Administrative Information
Access Restrictions: The Vertical File Collection is open and accessible for research.
Use Restrictions:
Users of the collection should read and abide by the Rights and Permissions guidelines at the Bernard Becker Medical Library Archives.
Users of the collection who wish to cite items from this collection, in whole or in part, in any form of publication must request, sign, and return a Statement of Use form to the Archives.
For detailed information regarding use of this collection, contact the Archives and Rare Book Department of the Becker Library (firstname.lastname@example.com).
Preferred Citation: Item description, VF02432 [Friedman, Harvey and Dorismae, Lectureship on Aging], Vertical File Collection, Bernard Becker Medical Library Archives, Washington University School of Medicine.
Box and Folder Listing
Browse by Box:
[Box 76],
660 S. Euclid Ave., Campus Box 8132, St. Louis MO 63110
Phone: 314.362.7080 Fax: 314.454.6606
©2019 Washington University School of Medicine, St. Louis, Missouri
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Copyright ©2011 The University of Illinois at Urbana-Champaign


text:
|Assistant Coach
|Alma Mater:
|Graduating Year:
|1st Year
Cory Burleson is in his third season as an assistant coach for the UC San Diego baseball program in 2015. He was named to the position in September of 2012.
At his first collegiate coaching stop, Burleson is tasked with directing the outfielders while assisting with the catchers and the offense. He handles groundskeeping duties at Triton Ballpark and baseball operations. During the 2015 season, Burleson serves as the third base coach, having coached first base in 2014.
Burleson spent the 2012 summer guiding a U-16 team based out of Denver, Colo. He coached for president Tad Reida in the CageRat Baseball Academy program. For the 2013 summer, Burleson was the pitching coach and coordinator for the Green Bay (Wis.) Bullfrogs of the Northwoods League.
In addition to his duties within the baseball program, Burleson is the department's internal relations intern during the 2014-15 academic year, taking an active role with the Triton Ballpark renovation project, among other tasks.
Recruited to Lincoln, Neb., by then Nebraska assistant coach and current Triton head coach Eric Newman, Burleson was a four-year starting catcher for the Cornhuskers from 2009-12.
As a senior under former Major League Baseball player and current Nebraska head coach Darin Erstad, Burleson capped his Husker career with his best season at the plate. He batted .273 with career highs in hits (36), runs (25), RBI (22) and steals (3). He also tied his career high in doubles with seven, matching the output from his junior year. Burleson was one of the top defensive catchers in the Big Ten Conference, throwing out 20 would-be base-stealers in 38 attempts and being charged with just two errors in 246 chances for a .992 fielding percentage.
Originally from Midland, Texas, Burleson attended Midland Christian High School, where he was a four-year starting catcher and three-year starting quarterback. He comes from a baseball family, with his father, Jerry, a coach at Artesia (N.M.) High School and his younger brother, Bryant, a senior for the Texas Tech Red Raiders.
Burleson was a two-time academic all-conference honoree and 2011-12 Big Ten Sportsmanship Award recipient. He earned his bachelor's degree from Nebraska in finance in May of 2012, and completed a master's in sport management online from Texas A&M in the summer of 2014.
Burleson resides in Mission Valley. He is engaged to be married during the 2015 summer to Julie Brechtel, a former softball player at Nebraska and a current graduate student at USC.


text:
mb_enterprises necesitaba un nuevo diseño de logotipo y lanzó un concurso de diseño en 99designs.
Un ganador ha sido elegido entre 53 diseños de 19 diseñadores freelance.
CREATIVE IDEAS NEEDED. We are launching a new organic beverage company called Bad Ass Organic. We are building a product line of organic beverages starting with a soda product. We are passionate about organic food and want to make products for everyone. Typically, marketing for organic food has a soft and feminine feel. We want to keep the organic message, but remove any sissy effects.
BAO Soda Tagline: "Soda with Life"
I have run two contests ... I'vechosen two winners ... and I've paid them both. 99designs is a great serviceand we look forward to getting a top notch logo. We will provide plenty of feedback, so youknow what we are looking for all along the way. Ideally, this will be the first of several designs we need to completethe entire product line and we would like to work with the designer on anongoing basis.
We are looking for a logo torepresent our company called Bad Ass Organic and our first product. Our first product is a soda line called BAOSoda. The soda will be in multipleflavors (e.g., cola, root beer, ginger ale) so we need to be able to tweak thedesign to represent the different flavors.
The logo needs to reflect the companyattitude. We are not looking for any herbs, flowers, vines or leaves. BAO Soda is not a sissy soda, but one packedwith nutritious and natural ingredients that make you feel good. Coke and Pepsi drinkers will gladly droptheir addictions to these sugary drinks and reach for BAO Soda, because ittastes just as good and is also good for you. We do not want to turn off the "organic crowd" but rather grow themarket to include people that may have previously ignored organicproducts.
Creative ideas are welcome andencouraged. As possible inspiration, thetwo owners ride motorcycles (choppers, not street bikes) and are looking tointegrate motorcycles in the design. Ifyou want to try something without bikes, that is cool too.
The name BAO Soda needs to be in thelogo. You can use lower case or upper case letters for b, a, and o. The company name, Bad Ass Organic, should notbe in the logo.
You can use cola, root beer or gingerale as an example for how the name (i.e., BAO Soda) will be aligned with the flavor.
Some logos to look at as examples includeStarbucks or Fizzy Lizzy.
It would be nice if this logo wouldwork on both a white and black background, since the product (e.g., the soda)will be light and dark
- Clean, Professional
- Must Scale.Readable Large or Small
- Not necessarily a "must have," but the tagline "Soda with Life" would be nice.
- Key words to focus on forinspiration would be: organic, cool, trendy, flavorful.
- A refreshing beverage feel. A soda logo feel
- No photoshop
- No pictures
- No stockgraphics, images, or icons
- No pastel colors
- Nothing vulgar
Descubre cómo funciona un concurso de diseño u obtén más información sobre nuestros servicios de diseño de logo.


text:
The Orlando Magic plan to interview Michael Malone, Brian Shaw, Quin Snyder and Jacque Vaughn for their head-coaching vacancy, the Orlando Sentinel has learned from league sources.
Although it's unclear whether additional candidates are on the list or will be added to the list, Magic general manager Rob Hennigan likely will conduct first-round interviews with Malone, Shaw, Snyder and Vaughn over the next seven days.
Malone and Shaw have been rumored as potential candidates for the Magic ever since the team fired Stan Van Gundy on May 21.
Malone, 41, spent this past season the Golden State Warriors' lead assistant coach. He was the lead assistant coach for the New Orleans Hornets in 2010-11 and was an assistant coach for the Cleveland Cavaliers for the five seasons before that. He's a son of former Magic assistant coach Brendan Malone.
Shaw, 46, just completed his first season as the Indiana Pacers' associate head coach, and before that, he was an assistant coach with the Los Angeles Lakers for seven seasons. Shaw played 14 seasons in the NBA, including three for the Magic in the mid-1990s.
In an NBA.com survey of league general managers at the beginning of the 2011-12 season, the GMs were asked to name the league's best assistant coach. Malone finished first, with 29 percent of the vote. Shaw placed second, with 21 percent of the vote.
Snyder, 45, is an assistant coach with the Lakers and previously worked as an assistant coach with the Philadelphia 76ers. He also has worked as the head coach of the NBA Development League's Austin Toros and as the head coach at the University of Missouri.
Vaughn, 37, has been a San Antonio Spurs assistant coach for the past two seasons. He played 12 seasons in the NBA, including the 2002-03 season for the Magic.
Although WKMG has reported that San Antonio Spurs lead assistant coach Mike Budenholzer and Oklahoma City Thunder assistant coach Maurice Cheeks are the frontrunners to fill the Magic's vacancy, they are not expected to receive interviews, the Sentinel has learned.
Hennigan would like to have a coach in place by the end of July.
He will whittle the list after he completes his initial interviews, and then Magic CEO Alex Martins will meet with those candidates. Ultimately, finalists for the job will travel to Grand Rapids, Mich., to meet members of the DeVos family before a hire is made.
Hennigan mostly has kept mum about the qualities he's seeking in a coach, but he was asked recently if previous head-coaching experience will be a plus.
"It's important," Hennigan answered. "I think anytime you want to hire a coach certainly a guy who's been in that seat before is important. I don't think it's the most important thing. But I do think it's an important piece to the puzzle.
"I think also in conjunction with that is someone who's played the game in the NBA, who played the game at a high level."
Malone, Shaw, Snyder and Vaughn have not worked as NBA head coaches.
Martins has said team would emphasize NBA championship experience in their GM and coaching searches.
Hennigan worked in San Antonio's basketball operations department when the Spurs won the NBA title in 2007, and Vaughn played on that Spurs team.
Shaw won three NBA titles as a player and two as an assistant coach, all with the Lakers.


text:
Harold Camping, rapture prophet: 'We were wrong'
The 90-year-old Alameda preacher who convinced hundreds of followers that the world was going to end last May now acknowledges he was wrong.
"Events in the last year have proved that no man can be fully trusted," Harold Camping wrote in a letter Thursday to listeners of his evangelical Family Radio program, "Open Forum." "Even the most zealous of us can be mistaken."
Camping had predicted that the rapture would occur at 6 p.m. May 21, 2011. Believers would rise to heaven while the rest would be left to wander a godforsaken planet until Oct. 21, when Camping promised a fiery end to the world.
But nothing happened.
Standing on his front porch the next morning, Camping said he was "flabbergasted" that the rapture he'd predicted had not come.
He promised an explanation after prayer and talks with friends. But Camping had a stroke less than a month later and was hospitalized until September. Once he was released, Camping said both the rapture and the end of the world would take place Oct. 21.
"I really am beginning to think as I've restudied these matters that there's going to be no big display of any kind," Camping said at the time. "The end is going to come very, very quietly."
Once again, the date came and went without incident.
"God has humbled us through the events of May 21," Camping wrote Thursday. "We have learned the very painful lesson that all of creation is in God's hands and he will end time in his time, not ours!
"We humbly acknowledge we were wrong about the timing."
Will Kane is a San Francisco Chronicle staff writer. Twitter: @WillKane. firstname.lastname@example.com


text:
Meghan McDevitt and Tom Keegan – July 20, 2012 – Lawnhaven
Lawnhaven Pocono Wedding Venue
Tom and I, first met at my favorite pub. I was there with my sister Marissa. Tom was there with his friend Jayme, who is a very close friend of my entire family. Tom and Jayme came over to talk and before long Tom asked me to go on a date with him... to church! After forcing myself to stop laughing, Tom explained that many years prior, Tom and his family would sit behind me and my family at church. He explained that church "wasn't so bad when he was able to sit behind me". I was very flattered by his adorable story but was not interested. Whenever I saw him over the next four years and would refer to him as "church boy."
One night, I was out celebrating my friend's the new job. I was just concluding my rant about how I didn't even want to look at a guy when Tom walked in. It was a moment I will never forget. He's eyes widened and he just smiled at me. We spent the entire night talking and drinking dirty martinis (my favorite). Tom and I have never looked back.
The first wedding Tom and I went to as a couple was at Stroudsmoor. It was amazing and we compared every other wedding to it.
Tom proposed to me a few weeks before our 3 year anniversary. It was a complete surprise because I thought he was going to "pop the question" while we were in Disney celebrating.
We didn't plan anything for two months. Tom and I knew what type of wedding we didn't want but we weren't exactly sure on what we did want. Finally one night we started making progress on our "vision" for our wedding. As we discussed what we wanted and pictured, Tom realized it was Stroudsmoor! We made an appointment and knew it was perfect for us within minutes. I remember, as Linda gave us a tour, Tom tugged at my sleeve and whispered, "Let's get married here." Stroudsmoor was definitely the rustic elegance we were looking for.
We planned our amazingly perfect wedding in four months. We used Stroudsmoor's services for everything! We would not change one second of our wedding...not even the rain.


text:
A survivalist and his 5-year-old captive remained in a bunker in southern Alabama on Saturday as top hostage negotiators continued to communicate with the man. NBC's Gabe Gutierrez reports.
A young Alabama boy entered his fifth day Saturday in a bunker with the man accused of snatching him off a school bus.
Police said Saturday that the boy, aged 5, has toys, coloring books, and medication in the underground bunker where he is being held by Jimmy Lee @!$%#s, the Associated Press reported.
The boy has Asperger's syndrome and attention deficit hyperactivity disorder, a state representative said earlier this week.
Dale County Sheriff Wally Olson said that @!$%#s, 65, told police he has an electric heater and blankets in the underground bunker on his property in Midland City, Ala. Olson would not say whether @!$%#s has made any demands, according to the AP.
"I want to thank him for taking care of our child," Olson said. "That's very important."
While little has changed in the standoff since Tuesday afternoon and authorities are keeping tight-lipped, former FBI hostage negotiator Clint Van Zandt said on the TODAY show that patience may pay off.
First picture emerges of man believed to have taken 5-year-old hostage in Alabama
"85 percent or more of standoff situations like this end nonviolently," Van Zandt said on Saturday. Police have said that they are communicating with @!$%#s in the bunker through a PVC ventilation pipe.
"The reality is he hasn't been hurt," Van Zandt said of the young hostage. "Law enforcement doesn't want to do anything precipitously that could cause anybody to be hurt at this time when the talking cure will likely work in this situation."
A police source confirms to NBC News that this is the suspect, Jimmy Lee @!$%#s, 65.
@!$%#s' neighbor Michael Creel told the AP that the man, who has been described as a paranoid Navy veteran, showed off the ventilation pipe after installing it about a year ago.
"He was bragging about it," Creel told the AP. "He said, 'Come check it out.'"
The first picture of @!$%#s emerged Friday. The man served just over four years in the Navy, a U.S. military official confirmed on Friday, and received several awards including a good conduct medal.
Meanwhile, the Dale County community mourned school bus driver Charles Albert Poland, Jr., 66, this weekend. Police say Poland was shot and killed while trying to stop the child from being taken off his bus on Tuesday.
Son says bus driver in Alabama hostage crisis gave life for 'his children'
Aaron Poland, the son of the slain bus driver, told TODAY that his dad died a hero.
"Every time a child got on my dad's bus, they were no longer their parents', they were his. He considered them his children," Poland said, his voice quavering with emotion. "I know that's the reason why my dad took those shots, for his children, just like he would do for me and my sister."
The Associated Press contributed to this report.
Mourners lined up to pay their respects to bus driver Charles Poland, who died on the job last week, protecting the kids on his bus after it was boarded by Jimmy Lee @!$%#s. @!$%#s continues to hold Ethan, 5, hostage in an underground bunker after snatching him from the bus. NBC's Gabe Gutierrez reports.


text:
It's not often I fall in love with a perfume, like, really fall in love in love with one. I don't know what it is that makes a scent really work with someone…but when I smell it, I know!
Molton Brown's Navigation Through Scent is the brands first foray into fine fragrances. I went to the launch a few weeks ago and each perfume was presented with in its own little alcove, and each represents a real geographic location.
I am not a perfume connoisseur – I am not going to be the best person to explain these scents to you in the most expert way but I'll tell you how they smell to me!
Iunu – Egypt. A favourite with a lot of ladies on the night – this one is a good unisex scent and had a masculine edge to it. It was quite gingery to me, like how I'd imagine George Clooney to smell. It was also very peppery! I think this is one that I'd learn to love.
Singosari – Indonesia. Very gingery and strong – I did find this one the hardest to settle on my skin, in that is was very pungent. It's extremely rich and I do like that it's bold – you know when you light an incense and you take the first whiff up your nose? it's that kind of strength.
Apuldre – Kent, England. The thing I remember the most from this perfume was berries, and grass. Smelt better on my once it had settled down as it was quite strong when first sprayed. Then it was quite 'soily'!
Rogart – Canada. Probably the most interesting scent of the lot – I could smell maple syrup and something that was like burning wood. Maybe it's the imagery that helped, but I could picture a little wooden hut in a field, and the guy from Due South inside. This was a very very sweet scent I found.
Then…there is Lijiang.
A note about the packaging – it is unisex, like the fragrance. The box and bottles are both unisex.
I love the crystal style bottle – reminds me of an expensive bottle for whiskey and the lid which you turn to spray is handy (although I keep going to pull it!).
When I smelt all the perfumes and wondered around with various cards, I realised that there was one perfume overwhelmingly that belonged to me – Lijiang. This perfume is for me, the most feminine by far, floral and definitely smells quite a bit lighter after a bit of wear.
A top note of pink berries & vegetal notes. Middle notes of Osmanthus absolute & white tea. A base of white musks & vetiver.
I could smell flowers and tea. Tea. Lot of fresh tea.
I chose this scent to take home in the end because although the others are nice and so interesting, there was only one that it could be! Mr C says it smells like CK one. I guess there is a bit of that in there – but a richer, more tea like, elegant finish.
I love this fragrance it's a pleasure to put on in the morning! All of the scents in this range are so unusual it's worth going to smell them for yourself because every one is so different.
If you visit the Molton Brown Facebook Page they have a quiz which you can take to get a sample of a perfume which suits you the most!


text:
Tiger Print Lightweight Hooded Jacket
€1,550.00 €1,550.00
All orders will be fulfilled by Burberry Limited and shipped from the UK. When you place your order, you accept Burberry's Terms and Conditions and confirm that you have read our Privacy Policy.
Size Guide
Use the Size Guide to choose the best fit for your measurements.
In this window, you can find out the shipping and returns options for the selected item.
In this window, you can contact customer service for further assistance.
- A lightweight hooded jacket in tiger-print nylon, highlighted with runway motifs from Riccardo Tisci's debut collection for Burberry.
- Jacket length: 76cm/29.9in. This is based on a size UK 8 as proportions change slightly according to size.
- Model's height: 176cm/5ft 9in
- Outer: 100% polyamide
- Trims: 100% leather; 100% polyamide
- Linings: 100% cupro; 100% polyamide
- Press-stud closure
- Chest flap pocket, chest patch pocket, front patch pockets
- Drawcord hood
- Press-stud cuffs
- Machine wash
- Made in Italy
Item 45605101
Complete the Look
Palladium-plated Hoof Earrings
Recommended Products


text:
I'm so excited about my new top....it's totally different than anything I've ever worn, but I love it.
I've seen this pattern reviewed on various sewing blogs, most recently Lori's blog. I have liked all Lori's versions but wasn't sure if the style would suit me. I bought the pattern anyway, but procrastinated making it. The deciding factor to sew it up was when Jessica bought me a ready-to-wear top of a similar style, and I found that I really liked it...a big surprise to me, because I didn't think I would. This style works well to hide my "I've had seven babies" midsection (ie, no waist, and a fluffy tummy).
Pattern changes on my version:
- I made the armhole/sleeve to be a higher cut.
- Next, because I did not want the neckline to be too loose, I moved in my center fold (to make it smaller) by 1/2" on both front and back pieces, taking out a full inch from both front and back. I added an extra 1/2 inch to each side seam at both the bodice and the waistband to accommodate my midsection.
- I reduced the width of the neckband. The pattern is 2 inches wide; I cut my neckband to 1 and 1/2 inches wide and applied it as instructed. I like the narrow band better.
- The instructions call for gathering the waistband side seams before sewing them together. Instead, I sewed the side seams and then used clear elastic in the seam allowances to gather the sides. Using clear elastic worked quite well.
On another note, I have waaaayyyy too much hair and it's driving me crazy. Any ideas of how I should cut it? If I go too short it will stick out in a frizzy, fluffy bush, lol. So I definitely need a haircut--but don't know what I want. Seriously, if you have hair style suggestions for thick wavy hair that's not curly but not straight, let me know.


text:
My Multiplex Fun Cub with recommended powerpack arrived today from Great Hobbies - undamaged
I must say, as a former nitro guy, there's something unnatural with a 55 inch wingspan aircraft that weighs less than a nitro engine by itself.
Not sure aircraft will be ready this weekend (looks like strong winds again) but hopefully I'll have plane up and ready by following weekend.
While I've built several kits before, this is my first electric and first foamie. Intent is to get an instructor take it up for its first flight. I'll ensure it meets advertised CG


text:
These Paper Mache Pencil Boxes come one box to a package, and are 9.0" x 2.75" in size. Paper Mache Recipe Boxes can be painted, and are great for group activites such as birthday parties or classroom events.
We also carry a complete line of Beads, Birdhouse Kits, Christmas Crafts, Craft Supplies, Kids Crafts, Misc Crafts, Wood Craft Kits, and much more.


text:
Apok's barbed wire fence is hilarious. The last I camped with them, '08, was the year they had problems with the MV. After we put up the fence, our Espy neighbors complained to the Rangers that Apok was being unburny by not letting them cut through and put up a very dangerous fence.Snow wrote:We've almost always backed up to TC and once I dressed in camo/guns to infiltrate your barbed wire (rubber) and snuck into the Kantina for a drink with the Kurnel. All this, sadly, went un-noticed as I was hoping to get "caught." I suppose I should have staged a coup. that's a random story but it was fun.
The barbed wire is made from heavy cotton thread and latex paint, kinda like Apok, all bark no bite(unless you ask nicely). But it fit very well with their camp's theme and worked until the Rangers let the cat out of the bag.
If you want to invade Apok it needs to be done in a big manner, better if it is accompanied with a soundtrack. Back in '06 the Kiwi's did a great job, even lobbed PBR granades into tents to keep potential combatents from entering the fray.
(sorry for any misspelled words, my phone's spellcheck is not cooperating right now and I still have fat fingers from the Playa)


text:
The procedure begins with a high yield transcription reaction based on the Ambion MEGAscript technology, to synthesize two complementary RNA transcripts from template(s) supplied by the user. The RNA strands are hybridized either during or after the transcription reaction to form dsRNA. Next, DNA and any single-stranded RNA (ssRNA) are removed with a nuclease digestion. Finally, the dsRNA is purified with a solid-phase adsorption system to remove protein as well as monoand oligonucleotides. The dsRNA produced can be introduced into the organism of interest by a variety of means, including injection, electroporation, or chemically-mediated transfection, depending on the organism being studied.
RNAi, the phenomenon by which long dsRNAs specifically suppress expression of a target gene, was originally discovered in worms (Fire 1998), but this phenomenon has now been found in a large number of organisms, including flies (Misquitta and Paterson 1999), trypanosomes (Ngo 1998), planaria (Sánchez-Alvarado and Newmark 1999), hydra (Lohmann 1999), and zebrafish (Wargelius 1999). The RNAi mechanism is currently being investigated, but it appears to work through smaller dsRNA intermediates. The parent dsRNA is broken down into these smaller fragments in vivo, and this siRNA directs a post-transcriptional breakdown of the targeted mRNA (Zamore 2000). An unusual feature of this process is that it works non-stoichiometrically and can spread between cells (Fire 1998). RNAi is a powerful method to investigate gene function through suppression of gene expression.
20 dsRNAs.
|10 mL
|Nuclease-free Water
|any temp*
|Filter Cartridges
|room temp
|Collection Tubes
|room temp
|40 μL
|T7 Enzyme Mix T7 RNA polymerase, RNase Inhibitor Protein, and other components in buffered 50% glycerol
|40 μL
|10X T7 Reaction Buffer
|40 μL
|ATP Solution (75 mM)
|40 μL
|CTP Solution (75 mM)
|40 μL
|GTP Solution (75 mM)
|40 μL
|UTP Solution (75 mM)
|1 mL
|10X Binding Buffer
|4 mL
|Elution Solution
|40 μL
|45 μL
|DNase I
|100 μL
|10X Digestion Buffer
|12 mL
|2X Wash Solution Add 12 mL 100% ethanol before use
|20 μL
|Control Template (500 ng/μL)
* Store Nuclease-free Water at –20°C, 4°C or room temp
Gene-specific template(s)
A transcription template(s) is needed with T7 RNA polymerase promoters positioned to transcribe sense and antisense RNA corresponding to the target RNA.
For dsRNA purification
- 100% ethanol: ACS grade or better
- Equipment to draw solutions through the Filter Cartridges: Use either a microcentrifuge capable of at least 8,000 X g, or a vacuum manifold with sterile 5 mL syringe barrels mounted to support the Filter Cartridges.
To assess the reaction products
Reagents and equipment for agarose gel electrophoresis
A. Choosing the dsRNA Sequence
RNAi experiments are typically done with dsRNA 400 bp and larger; 200 bp is the minimum size of dsRNA recommended for RNAi. Typically templates for transcription of dsRNA for use in RNAi correspond to most or all of the target message sequence.
B. Strategies for Transcription of dsRNA
RNAi experiments requiredouble-stranded RNA (dsRNA). Since the T7 RNA polymerase used in this kit synthesizes single-stranded RNA (ssRNA), use one of the following strategies to produce dsRNA:
- Prepare one DNA template with opposing T7 promoters at the 5' ends of each strand, and use it in a single transcription reaction to synthesize dsRNA without a separate annealing step.
- Use two DNA templates that are identical except that a single T7 promoter sits at opposite ends of the region to be transcribed. With this strategy, the templates can both be added to a single reaction. Although both templates should be transcribed at the same rate, if they are not, the final dsRNA yield will be dictated by the template with the lower transcription efficiency. Alternatively, the two templates can be transcribed in separate reactions to make complementary RNA molecules, which are then mixed and annealed. For the annealing step, the two transcripts can be mixed in precisely equimolar amounts. Note, however, that if the templates are transcribed in separate transcription reactions, this kit contains enough reagents to produce only 10 different dsRNAs.
Figure 1. T7 Polymerase Promoter: Minimal Sequence Requirement
The +1 base (in bold) is the first base incorporated into RNA. The underline shows the minimum promoter sequence needed for efficient transcription.
C. PCR Templates
Amplification strategies to add T7 promoter sequences to DNA
T7 promoter sequences can be added to DNA using PCR to generate templates that can be directly added to the MEGAscript RNAi Kit transcription reaction. Begin by synthesizing PCR primers with the T7 promoter sequence appended to the 5' end of the primer. The T7 promoter-containing PCR primers (sense and antisense) can either be used in separate PCRs, or in a single PCR to generate transcription template for both strands of the dsRNA. The two strategies for adding T7 promoter to DNA are shown below:
Figure 1
- Calculate the annealing temperatures of the entire PCR primer (with the T7 promoter site) and the gene specific portion of the PCR primer separately.
- Since the first cycles of PCR use only the 3' half of the PCR primer(s), the gene-specific part, the annealing temperature for the first 5 PCR cycles should be ~5°C higher than the calculated Tm for the gene-specific region of the primer. We have found that using the calculated annealing temperature for the initial cycles often results in synthesis of spurious PCR products.
- Once some PCR product is made, subsequent primer annealing events (cycle 6 and thereafter) use the entire primer site; therefore use the calculated Tm for the entire PCR primer plus ~5°C for subsequent cycles.
- We recommend using primers at 100 nM in the PCR mix. Higher concentrations may result in synthesis of primer dimers.
Check PCR products on a gel before using them in this procedure
PCR products should be examined on an agarose gel prior to in vitro transcription to estimate concentration and to verify that the products are unique and of the expected size.
D. Plasmid Templates
Cloning strategy
When using plasmid templates in the MEGAscript RNAi Kit, it is best to make two separate clones with the same target region in both orientations.
Figure 2. Cloning in plasmids
Note: Although some dsRNA templates have been cloned in plasmids containing opposing T7 promoters (on either side of the linker region Kamath 2001, Timmons and Fire 1998, Morris 2001), these constructs will result in synthesis of dsRNA with significant stretches of RNA corresponding to the polylinker region of the plasmid.
PCR products can be cloned into plasmid vectors using any of the following strategies:
- Amplify the target by PCR and ligate the product into a PCR vector with a T7 promoter. Identify plasmids with the insert in both orientations with regard to the T7 promoter.
- Or, include the T7 promoter sequence at the 5' end of one or both of the PCR primers, then perform PCR to incorporate them into the fragment. Finally, ligate the PCR product into a PCR cloning vector (one that does not include a T7 promoter).
- Or, include both a T7 promoter and a restriction site at the 5' end of one or both PCR primers to incorporate them into the fragment during PCR. Ligate the PCR product into a cloning vector using the added restriction sites.
Plasmid linearization
Plasmid templates must be linearized downstream of the insert to create a transcription termination site—the RNA polymerase will literally fall off the end of the DNA molecule. Linearize each template, then examine the DNA on a gel to confirm that cleavage is complete. Since initiation of transcription is the rate limiting step of in vitro transcription reactions, even a small amount of circular plasmid in a template prep will generate a large proportion of transcript, wasting much of the synthetic capacity of the reaction.
Note: We routinely use all types of restriction enzymes. However, there has been one report of low level transcription from the inappropriate template strand in plasmids cut with restriction enzymes leaving 3' overhanging ends (produced by Kpn I, Pst I, etc.; Schenborn and Mierindorf, 1985).
Figure 3 Linearized plasmids
After linearization
Terminate the restriction digest by adding each of the following:
- 1/20 volume 0.5 M EDTA
- 1/10 volume of either 3 M NaOAc or 5 M NH4OAc
- 2 volumes of ethanol
Mix well and chill at –20°C for at least 15 min. Then pellet the DNA for 15 min in a microcentrifuge at top speed. Remove the supernatant, respin the tube for a few seconds, and remove the residual fluid with a very fine-tipped pipet. Resuspend in TE (10 mM Tris-HCl pH 8, 1 mM EDTA) at a concentration of 0.5–1 μg/μL.
Plasmid DNA purity
DNA should be relatively free of contaminating proteins and RNA. The greatest yields of dsRNA will be obtained with very clean template preparations. Most commercially available plasmid preparation systems yield DNA that works well in the MEGAscript RNAi Kit. Alternatively, a DNA miniprep procedure that generally yields high quality template is presented in section V.D.
Note that DNA from some miniprep procedures may be contaminated with residual RNase A. Also, restriction enzymes occasionally introduce RNase or other inhibitors of transcription. When transcription from a template is suboptimal, it is often helpful to treat the template DNA with proteinase K before performing the transcription reaction.
A. Before Using the Kit for the First Time
Prepare the Wash Solution
1. Add 12 mL ACS grade 100% ethanol to the bottle labeled 2X Wash Solution.
2. Mix well, and store at room temperature.
We suggest crossing out the 2X from the bottle label after adding the ethanol. In these instructions this reagent will be called Wash Solution once the ethanol is added.
B. Transcription Reaction Assembly
1. Thaw the frozen reagents at room temp then place them in ice
Remove the T7 Enzyme Mix from the freezer and place it directly in ice; it is stored in glycerol and will not freeze at –20°C. Vortex the 10X T7 Reaction Buffer and the 4 ribonucleotide solutions (ATP, CTP, GTP, and UTP) until they are completely in solution. Once they are thawed, store the ribonucleotides (ATP, CTP, GTP, and UTP) on ice, but keep the 10X Reaction Buffer at room temperature. Microcentrifuge all reagents briefly before opening to prevent loss and/or contamination of any material on the rim of the tube.
2. Assemble transcription reaction at room temperature
Assemble the transcription reaction at room temperature in the order shown below. The following amounts are for a single 20 μL transcription reaction. Reactions may be scaled up or down if desired.
Important: The following reaction setup is recommended when the RNA produced will be ≥400 nt in length. For transcripts shorter than this, see section V.C. Optimizing Yield of Short Transcripts for modified reaction setup suggestions.
|to 20 μL
|Nuclease-free Water
|1–2 μg
|Linear template DNA Use either 1 μg of a template with opposing T7 promoters flanking the transcription region, or use a mixture of 1 μg of each template when the T7 promoter template is on separate molecules
|2 μL
|2 μL 10X T7 Reaction Buffer
|2 μL
|ATP Solution
|2 μL
|CTP Solution
|2 μL
|GTP Solution
|2 μL
|UTP Solution
|2 μL
|T7 Enzyme Mix
3. Mix thoroughly
Gently flick the tube or pipette the mixture up and down, then briefly microcentrifuge to collect the reaction mixture at the bottom of the tube.
4. Incubate at 37°C for 2–4 hr
The first time a new template is transcribed, the recommended incubation time is 2–4 hr. The optimal incubation time for a given template varies depending on its size and transcriptional efficiency. For transcripts <400 nt, a longer incubation time (up to ~16 hr) may be advantageous, since more transcription initiation events are required to synthesize a given mass amount of short RNA, compared to transcription of longer templates. Instead of doing overnight incubations, we suggest freezing the reaction overnight and then thawing and resuming the reaction the next day. Reactions are stable for several days when frozen at –20°C; however, storage will result in gradual loss of enzyme activity. To determine the optimum incubation time for maximum yield with a given template, a time-course experiment can be done. To do this, set up a transcription reaction, and remove aliquots of the reaction at various intervals (for example after 2 hr, 4 hr, 6 hr, and overnight incubation). Assess results by looking for a decrease in absorbance at 260 nm as the free nucleotides are incorporated into RNA, or by running 0.5 μL samples of the RNA on a gel stained with ethidium bromide
C. Annealing RNA to Maximize Duplex Yield
Include this annealing step for the following types of reactions:
- All >800 nt dsRNA synthesis reactions
- ≤800 nt dsRNA synthesis reactions when the two strands were synthesized from separate transcription templates (in the same or in separate transcription reactions).
Annealing the complementary RNA is often unnecessary for transcripts ≤800 nt made from a single template with opposing T7 promoters because RNA products in this size range will typically hybridize during the transcription reaction. With transcripts >800 nt, however, at least a portion of the transcripts form aggregates (presumably branched structures) rather than the dsRNA.
1. Mix the transcription reactions containing complementary RNA
- If sense and antisense RNA were synthesized in separate transcription reactions, add the entire contents of one of the reactions to the other. If desired, reserve a 0.5 μL aliquot of each template before mixing for gel analysis.
- If sense and antisense RNA was synthesized in a single transcription reaction, both strands of RNA will already be in a single tube; proceed to step 2
2. Incubate at 75°C for 5 min, then cool to room temperature
Incubate at 75°C for 5 min then leave the mixture on the bench to cool to room temperature. The RNA will anneal as it cools, forming dsRNA. Do not put the reaction on ice to cool.
3. Check 1/400th of the dsRNA on an agarose gel
Run 1/400th of the dsRNA on a 1% agarose gel (nondenaturing) to examine the integrity and efficiency of duplex formation.
- 1/400th of a 20 μL dsRNA solution is 5 μL of a 1:100 dilution.
- Dilute the gel samples in TE (10 mM Tris, 1 mM EDTA) or in gel loading buffer.
(Instructions for running a gel are in section V.B.) The dsRNA will migrate slightly slower than DNA markers of the same length.
D. Nuclease Digestion to Remove DNA and ssRNA
This DNase/RNase treatment digests template DNA and any ssRNA that did not anneal. RNase will not degrade dsRNA when using the reaction conditions specified below.
1. Assemble RNase digestion reaction on ice
The amounts shown are for a 20 μL transcription reaction; scale up if your transcription reaction was larger.
|20 μL
|21 μL
|Nuclease-free Water
|5 μL
|10X Digestion Buffer
|2 μL
|DNase I
|2 μL
The ssRNA will be digested after 15 min but allow the incubation to proceed for 1 hr to completely digest the DNA template. Do not continue this incubation longer than 2 hr.
E. Purification of dsRNA
This purification removes proteins, free nucleotides, and nucleic acid degradation products from the dsRNA.
Note: For the quickest dsRNA purification, preheat the Elution Solution to ~95°C before starting the purification procedure.
1. Assemble the dsRNA binding mix
Assemble the dsRNA binding mix by adding 10X Binding Buffer, water, and 100% ethanol to the dsRNA according to the table below.
|50 μL
|dsRNA (from step D.2 above)
|50 μL
|10X Binding Buffer
|150 μL
|Nuclease-free Water
|250 μL
|100% Ethanol
Gently mix the reaction by pipetting up and down.
2. Apply binding mix to the Filter Cartridge, and draw it through
Pipet the entire 500 μL dsRNA binding mix onto the filter in the Filter Cartridge, and draw it through by centrifugation or with a vacuum manifold.
Centrifuge users:
- a. For each dsRNA sample, place a Filter Cartridge in a Collection Tube. Use the Collection Tubes supplied with the kit.
- b. Pipet the entire 500 μL dsRNA mixture onto the filter in the Filter Cartridge. Centrifuge at maximum speed for 2 min.
- c. Discard the flow-through and replace the Filter Cartridge in the Collection Tube.
Vacuum manifold users:
- a. For each dsRNA sample, place a 5 mL syringe barrel on the vacuum manifold, load it with a Filter Cartridge, and turn on the vacuum.
- b. Pipet the entire 500 μL dsRNA mixture onto the filter in the Filter Cartridge. The vacuum will draw the lysate through the filter.
3. Wash the Filter Cartridge with 2 X 500 μL Wash Solution
Verify that 12 mL of 100% ethanol was added to the 2X Wash Solution.
- a. Pipet 500 μL of Wash Solution onto the filter in the Filter Cartridge. Draw the wash solution through the filter as in the previous step.
- b. Repeat with a second 500 μL of Wash Solution.
- c. After discarding the Wash Solution, continue centrifugation, or leave on the vacuum manifold for ~10–30 sec to remove the last traces of liquid.
4. Recover the dsRNA 2 X 50–100 μL Elution Solution
- a. The Elution Solution provided with the kit is 10 mM Tris-HCl pH 7, 1 mM EDTA. It is compatible with dsRNA injection, or 2X Injection Buffer can be added to the purified dsRNA for a final concentration of 1X Injection Buffer. Alternatively, the dsRNA can be eluted into any sterile low salt solution (≤30 mM), e.g. 5 mM KCl, 0.1 mM sodium phosphate buffer as used by Rubin and Spradling (1982).Transfer the Filter Cartridge to a fresh Collection Tube.
- b. Apply 50–100 μL (hot) Elution Solution to the filter in the Filter Cartridge.
Apply preheated (≥95°C) Elution Solution to the filter, or
Apply room temperature Elution Solution, close the tube lid over the Filter Cartridge, and incubate in a heat block set to 65°C or warmer for 2 min.
- c. Centrifuge for 2 min at maximum speed.
- d. Repeat steps b–c with a second 50–100 μL aliquot of Elution Solution collecting the RNA into the same Collection Tube. Most of the RNA will be eluted in the first elution. The second elution is included to recover any remaining RNA.
5. Quantitate and store the dsRNA
Quantitate the reaction product by measuring its absorbance at 260 nm and calculating the concentration The dsRNA is stable when stored at –20°C in Elution Solution.
6. Check 1/400th of the purified dsRNA on an agarose gel
Run 1/400th of the dsRNA on a 1% agarose gel (nondenaturing) to examine the integrity and efficiency of duplex formation.
- 1/400th of 100 μL elution volume is 2.5 μL of a 1:10 dilution
- 1/400th of 200 μL elution volume is 5 μL of a 1:10 dilution
- Dilute the gel samples in TE (10 mM Tris, 1 mM EDTA) or in gel loading buffer
The dsRNA will migrate slightly slower than DNA markers of the same length.
A. Use of the Control Template
The Control Template is a linear dsDNA fragment with opposing T7 promoters. It yields a 500 bp dsRNA product.
Positive control reaction instructions
- Use 2 μL (1 μg) of Control Template in standard MEGAscript RNAi Kit reactions
- Incubate the transcription reaction for 2 hr.
- Skip section III.C. Annealing RNA to Maximize Duplex Yield as the sense and antisense RNA strands will hybridize during the transcription reaction.
- Follow the procedure as described in sections III.D and III.E starting on to purify the dsRNA.
- (Optional) Run 1–2 μg of the positive control reaction product on at 1% agarose gel to verify that the dsRNA is 500 bp.
Expected yield from the control reaction
The yield of RNA from the positive control reaction should be 50–80 μg of a 500 bp dsRNA.
What to do if the positive control reaction doesn't work as expected
If the yield of RNA from the control reaction is low, something is probably wrong with the procedure, the kit, or the quantitation.
- Double check the RNA quantitation
- When assessing yield by UV spectrophotometry, be sure to use TE (10 mM Tris-HCl, 1 mM EDTA) to blank the spectrophotometer and dilute the RNA.
- To confirm that the quantitation is correct, verify the yield by an independent method. For example if UV spectrophotometry was used to assess yield, try also running an aliquot of the reaction on an agarose gel and comparing its intensity to a sample of known concentration.<
- Consider repeating the positive control reaction. If the yield is indeed low by two different measurements, there may be a technical problem with the way the kit is being used. You may want to consider repeating the positive control reaction. If you are certain that the procedure was carried out correctly, and the control reaction does not give the expected results, contact technical support
B. Low Yield
The amount of RNA synthesized in a standard 20 μL transcription reaction should be 50 μg or more; however, there is a great deal of variation in yield among different templates. If the yield is low, the first step in troubleshooting the reaction is to use the Control Template in a standard MEGAscript RNAi Kit transcription reaction to determine if the problem is with the template, the reagents,or procedure.
Neither my template nor the control reaction works
If the positive control does not work, it could be an indication that something is wrong with the kit; call Ambion's Technical Support group for more troubleshooting help.
The control reaction works,but my template gives low yield
Figure 5
If the transcription reaction with your template generates full-length, intact RNA, but the reaction yield is significantly lower than the amount of RNA obtained with the Control Template, it is possible that
contaminants in the DNA are inhibiting the RNA polymerase. A mixing experiment can help to differentiate between problems caused by inhibitors of transcription and problems caused by the sequence of a template. Include three reactions in the mixing experiment using the following DNA templates:
1. 2 μL Control Template
2. 1–2 μg experimental DNA template
3. a mixture of 1 and 2
Assess the results of the mixing experiment by running 0.5 μL of the transcription reaction on an agarose gel.
Transcription of the Control Template is inhibited by your template. (See Figure 6.a)
This implies that inhibitors are present in your DNA template. Typical inhibitors include residual SDS, salts, EDTA, and RNases. Proteinase K treatment followed by phenol:chloroform extraction frequently improves template quality. Proteinase K treatment Treat template DNA with Proteinase K (100–200 μg/mL) and SDS
(0.5%) for 30 min at 50°C, followed by phenol/chloroform extraction and ethanol precipitation. Carry-over of SDS can be minimized by diluting the nucleic acid several fold before ethanol precipitation, and excess salts and EDTA can be removed by vigorously rinsing nucleic acid pellets with 70% ethanol before resuspension.
Adding your template to the reaction with the Control Template does not inhibit synthesis of the control RNA.
(See Figure 6.b.) This indicates that the problem may be inherent to your template.
Check the amount and quality of template Template quantitation may be inaccurate. If quantitation was based on UV absorbance and the DNA prep had substantial amounts of RNA or chromosomal DNA, the amount of template DNA may be substantially less than the calculated value.
Also, check an aliquot of the template DNA on an agarose gel to make sure it is intact and that it is the expected size. If there is even a small amount of circular template in the transcription reaction it will reduce the yield of dsRNA (see section Plasmid linearization).
Extend the reaction time. Another parameter that can be adjusted is reaction time. Extending the standard 2–4 hr incubation to 6–10 hr,
Change your priming region.
Some sequences are simply inefficient transcription templates. If you get low RNA yields, even after checking the template and trying overnight incubation of the transcription reaction, it may be necessary to prepare a transcription template from a different region of the gene. Often simply moving the transcription start point can overcome problems with inefficient transcription; typically there are several regions of the gene that will transcribe with equal efficiency. Also, transcription efficiency may be higher when the transcription template contains 2–3 bases of purines immediately following the GGG sequence at positions +1 to +3 in the T7 promoter sequence • Use 2 separate templates Sometimes the template will simply not transcribe well with opposing promoters. In this case, the two strands of RNA need to be made from separate templates and annealed after synthesis
C. Multiple Reaction Products, Transcripts of the Wrong Size
Reaction products produce a smear when run on a gel
This problem is usually seen with single-strand transcriptions. If the RNA appears degraded (e.g. smeared), remove residual RNase from the DNA template preparation before in vitro transcription. Do this by digesting the DNA prep with proteinase K. The RNase Inhibitor in the transcription reaction can only inactivate moderate RNase contamination. Large amounts of RNase in the DNA template will compromise the size and amount of
transcription products.
Reaction products run as more than one band, or as a single band smaller than expected
Premature termination of transcription
If gel analysis shows multiple discrete bands or a single band smaller than the expected size, there may be problems with premature termination by the polymerase. Even if transcription of only one of the strands was prematurely terminated, the single-stranded portion of the duplex will be digested during the nuclease treatment, resulting in a shorter than expected dsRNA.
Possible causes of premature termination are sequences which resemble the phage polymerase termination signals, stretches of a single nucleotides, and GC-rich templates.
Termination at single polynucleotide stretches can sometimes be alleviated by decreasing the transcription reaction temperature (Krieg 1990). We suggest testing incubations at 30°C, 20°C and 10°C, but be sure to increase the reaction time to offset the decrease in yield caused by incubation at suboptimal temperatures.
There is a report that single-stranded binding (SSB) protein increased the transcription efficiency of a GC rich template (Aziz and Soreq, 1990).
Reaction products are larger than expected
Products occasionally run as two bands after the nuclease digestion and dsRNA purification; one at the expected size, and one that is double the expected size. If this occurs, check the size of the transcription template on a gel to verify that it is pure and sized correctly. dsRNA that contains a double-sized band can be used for RNAi with no problems, in fact double-sized bands are sometimes seen from the Control Template
Multi-strand aggregates are present in the mixture
Larger than expected bands or ethidium bromide staining in the wells could be seen as a result of aggregates of multiple RNA strands. These can be denatured by heating the solution to 75–100°C for ~3 min, then allowing it to cool to room temperature. Be sure that RNA is in a solution containing at least 1 mM EDTA (such as the Elution Solution supplied with the kit) for the heat treatment.
- Aziz RB and Soreq H (1990) Improving poor in vitro transcription from GC-rich genes. Nucl. Acids Res.
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, and Mello CC. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–11.
- Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, and Ahringer J (2001) Effectiveness of specific
RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome
Biol.2(1):RESEARCH 0002.
- Krieg PA (1990) Improved Synthesis of Full-Length RNA Probes at Reduced Incubation Temperatures. Nucl. Acids Res. 18: 6463.
- Lohmann JU, Endl I and Bosch TC (1999) Silencing of developmental genes in Hydra. Dev Biol.
- Milligan JF, Groebe DR, Witherell GW, and Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7
RNA polymerase and synthetic DNA template. Nucl. Acids Res. 15: 8783–8798.
- Misquitta L and Paterson BM (1999) Targeted disruption of gene function in Drosophila by RNA interference
(RNA-i): a role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci USA. 96(4):1451–6.
- Morris JC, Wang Z, Drew ME, Paul KS, and Englund PT (2001) Inhibition of bloodstream form Trypanosoma brucei gene expression by RNA interference using the pZJM dual T7 vector. Mol Biochem Parasitol. 117(1):111–3.
- Ngo H, Tschudi C, Gull K, and Ullu E (1998) Double-stranded RNA induces mRNA degradation in Trypanosoma
brucei. Proc Natl Acad Sci USA 95(25):14687–92.
- Rubin GM and Spradling AC (1982) Genetic transformation of Drosophila with transposable element vectors. Science 218(4570):348–53.
- Sanchez Alvarado A and Newmark PA (1999) Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc Natl Acad Sci USA 96(9):5049–54.
- Schenborn ET and Mierendorf RC (1985) A novel transcription property of SP6 and T7 RNA polymerases:
dependence on template structure. Nucl. Acids Res. 13: 6223–6236.
- Timmons L and Fire A (1998) Specific interference by ingested dsRNA. Nature 395(6705):854.
- Wargelius A, Ellingsen S, and Fjose A (1999) Double-stranded RNA induces specific developmental efects in zebrafish embryos. Biochem Biophys Res Commun. 263(1):156–61.
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cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101(1):25–33.


text:
Bring On the Next American Revolution
There are few human beings who have the cultural depth, breadth, and perspective of 96-year-old Grace Lee Boggs. Born during World War I to Chinese immigrant parents, she has lived through the Great Depression, World War II, and the Civil Rights, anti-war, women's, gay rights, and disability dignity movements, then 9/11 and the Afghanistan and Iraq wars. She effectively transformed herself into an African American through her marriage to black autoworker Jimmy Boggs and her immersion in the Detroit community over the past 60 years. Much of her new book, The Next American Revolution, reads as her philosophical autobiography, charting how experience shaped her political ideas, using the first person and the collective "we" grounded in her community activism.
Co-author Scott Kurashige, the award-winning author of The Shifting Grounds of Race: Black and Japanese Americans in the Making of Multiethnic Los Angeles, collaborated as an editor, shaping the book by combing through an archive of Boggs-written articles, pamphlets, speeches, and correspondence. His introduction gives perspective for the ensuing narrative.
Nearly a century ago, Detroit was the high-technology capital of the world. In the 1930s and 1940s it was the vanguard of organized labor. By the 1960s the Motor City exported Motown music, and 20 years later pulled off the same trick with techno. Today, Detroit is arguably the most devastated post-industrial community in America. If you pay attention to the mainstream media, pretty much all that is left of Detroit is crime-ridden, Rust Belt woe—with a big helping of divisive racial posturing.
Boggs and Kurashige agree that Detroit's days as the world's industrial giant are over. That's pretty much the end of their concord with conventional perspectives. They describe a transformation of vision and perspective going on in Detroit that is much more than a matter of rhetorical jiu jitsu. And they put it in historical perspective:
"We are in the midst of a cultural transition as far-reaching as that from hunting and gathering to agriculture 11,000 years ago and from agriculture to industry 3,000 years ago."
Their view is that the struggle now is about what direction the world will take from here. To make that direction positive for the majority of people, nothing less than revolution is necessary. Not a violent takeover of the political-economic state, but rather a revolution in our ways of thinking and acting on a personal and community level, a transformation from victims to empowered citizens.
TNAR is partly a celebration of the sort of democratic community movements the authors see emerging as a critical force for creating the new Detroit, a new United States, and indeed a new world. They cite a number of organizations as evidence, like the Detroit Coalition Against Police Brutality. Led by a former Black Panther, it pushes for police accountability and works to reduce policing by resolving neighborhood conflicts. The Beloved Communities Center in Greensboro, N.C., is another. It uses truth and reconciliation to heal the community, where five racial-justice marchers were killed in 1979. An international example is Mexico's Zapatistas, who defied NAFTA in favor of development grounded in their own culture and needs.
There are points where TNAR strays toward cataloguing what might be considered modest community achievements. But part of the "Why?" of the book is to show how these components weave together into a nontraditional, nonhierarchical force that defies efforts to control the stumbling world economy through political and military domination.
There are lessons here for activists that make this slim volume a handbook for personal, and therefore social, transformation. That, more than anything, is the revolution that Boggs seeks.


text:
Events at Green Jeans
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3600 Cutler Avenue Northeast
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An indoor/outdoor "courtyard style" commons, surrounded by local businesses in a relaxed and social environment.
A year round Farmers Market, Tap Room/Beer Garden, Coffee shop, Chocolate & Wine bar, Flower/Spice depots and local food concepts will combine with a Hydroponic Farm to create a unique and fun environment.
Green Jeans will offer "grow your own in a Bins" for year round hydroponic vertical farming, built and shipped to your home, school, restaurant or business.
ONGOING: Pints & Planks (Yoga) every Monday & Thursday at 6 PM


text:
Spokane, Washington - Spokane, Washington - Providence Medical Group is seeking an excellent BC/BE Palliative Care Physician with inpatient palliative care experience. Full-time position (0.8 FTE). No call. Inpatient team consultation model with limited outpatient clinic. Competitive compensation; comprehensive benefits. Join a well-established, interdisciplinary team with Providence Medical Group's mission-driven Supportive Care Team, featuring expansion potential that is embraced by both the community and the local hospital system. Enjoy a healthy work/ life blend in Washington's second largest city, Spokane.
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Who You'll Work For
Providence Health & Services (PH&S) is a not-for-profit system of 34 hospitals, more than 600 clinics and hundreds of locally driven programs across five western states: Alaska, California, Montana, Oregon and Washington. Providence is committed to improving the health of the communities it serves, with a special emphasis on its mission to serve the poor and vulnerable. PH&S is a partner organization of the Providence St. Joseph Health family and is proud to be an Equal Employment Opportunity organization.
Job ID Number: 8641
Facility Name: Providence Medical Group - Eastern Washington
Location Name: Spokane, WA
Brand Name: Providence Health & Services
Provider Profession: Physician/Surgeon
Provider Profession: Nurse Practitioner
Medical Specialty: Palliative Care
Job Setting: Medical Clinic
Type of Role: Clinical
Email: email@example.com
Phone Number: (509) 474-6605
Schedule: Full Time
CP: Yes
CB: Yes
NP: No
PA: No
HC: Yes
IS: No
YM: No
Let's Talk!
You've got questions? I've got answers! Reach out today, and together, we'll find a job that checks all your boxes.
Express Interest
Mark Rearrick
Provider Recruiter
(509) 474-6605


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I have a workflow that creates a new task at the time of creation of a new item in a given list. This new task is assigned to a user when created. Then, this task can either be reassigned or completed. If the task is completed, the workflow ends. If the task is reassigned, the task should be completed, another task should be created assigned to another user, and the workflow continues.
I have the issue that when the second task is created(first task is reassigned) the workflow ends and the onTaskChanged of the second task is never hit.
Any Ideas?


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Professor, Chair of Engineering Science & Physics
Deb Dudick is a Professor at Corning Community College, and currently serves as Department Chair of Engineering Science and Physics. She has been a faculty member since 1988. Prior to that, she worked at North American Philips Corporation and Corning, Incorporated.
Deb has taught in various programs including Engineering Science, Mechanical CAD Design Technology, Manufacturing Technology, and Fine Art. Her current teaching includes coursework in Art (Drawing, Printmaking), Engineering (Engineering Mechanics - Statics, Mechanics of Materials, Dynamics, Orientation), and Technology (Technical Mechanics, Strength of Materials).
Deb has studied Art, Architecture, and Engineering at Virginia Polytechnic Institute and the State University of New York at Oneonta, in addition to holding degrees from Corning Community College, Clarkson University, and Binghamton University. Her art has been exhibited regionally and internationally.
Her hobbies include cross country skiing, ice boating, sailing, cycling, caving, hiking, and traveling.
- MS in Industrial and Systems Engineering from Binghamton University-Watson School of Engineering
- BS in Mechanical and Industrial Engineering from Clarkson University
- AS Engineering Science from Corning Community College


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One of the 8 photos in my first ever exhibit. It's only for my class final but I'm still ultra stoked about it.
In the Art in Abandonment photo essay.
1 response
Jasmine McGill gave props (15 Dec 2008):
this is amazing..
Also by Bethany Crawford
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The Shell Graduate Programme
As a technical graduate, you'll embark on a three-year training programme that offers talented individuals a comprehensive introduction to various areas of Shell's business and operations within the Technical area of the Shell Graduate Programme. It's a robust platform from which you'll be perfectly positioned to become pioneers in the field of Electrical Engineering can establish a long and fulfilling career.
Joining the Programme you'll work alongside the industry's leading technical experts and visionaries, and could gain experience on some of Shell's most groundbreaking engineering projects. Within Shell you'll find a supportive work environment, working with teams across the globe. You'll be encouraged to take an innovative approach, while developing your skills and technical capabilities.
Electrical Engineering
Shell's Electrical Engineers' responsibilities include troubleshooting and resolving electrical issues, designing hardware and software, and monitoring power distribution.
An Engineering job at Shell is not only a chance to progress your career with a global energy leader and expand your horizons, but an opportunity for you to make an impact on the future of the industry. As an Electrical Engineer, you'll play a key role in successful project delivery and work alongside fellow creative thinkers, problem solvers and innovators keen to push boundaries. We'll ensure that you are constantly developing your expertise and abilities.


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As the film "The Central Park Five" heads into the Film Independent Spirit Awards in Los Angeles on Saturday, where it is nominated for best documentary, its filmmakers can rest assured that at least one contest, the one that was taking place far from Hollywood in a New York City courtroom, is over. In a case closely watched by the documentary film and journalism community, a New York district court judge on Tuesday quashed a subpoena seeking access to outtakes from the film, saying that the plaintiff had established entitlement to a reporter's privilege.
The courtroom battle between the documentary's filmmakers--the prominent documentarian Ken Burns, his daughter Sarah Burns, and David McMahon--and New York City officials was the latest, unlikely chapter in one of the city's most notorious crimes. "The Central Park Five," which was released in November, tells the story of the conviction and eventual exoneration of five men in the infamous 1989 rape and assault of a jogger in Central Park. The film makes the case that conviction of the five teenagers on scant evidence and confessions (later disavowed) was a miscarriage of justice by the legal system and society, both of which sought quick resolution and to assign blame for an emblematic attack. The five men, who were cleared of the charges after another inmate confessed to having committed the crime, have been engaged in a civil dispute with the city for years, seeking damages for the seven to 13 years they spent in prison.
A subpoena filed in October, amending a broader version brought a month earlier, sought access to all of the film's unused footage featuring the five men, their family members, attorneys, and anyone with knowledge of the case, claiming the content was of use in the civil case. Florentine Films, the movie's distributor, filed an appeal to quash the subpoena on the grounds that the filmmakers were entitled to a reporter's privilege under the New York State Shield Law.
Shield laws, which are on the books in 40 states and frequently offer only qualified protection, have been at the center of many journalistic legal struggles in recent years. What differentiates the "Central Park Five" from many of the latest national security-related cases is firstly that the material sought did not come from a confidential source. Sarah Burns acknowledged that the threshold to overcome a reporter's privilege is much lower when it comes to non-confidential sources under the New York Shield Law, but that the protection exists nonetheless. While the subjects of the film did not ask to remain anonymous, they did not "give the interviews with the understanding that they would be investigative material in the case for the city," Burns told CPJ. "The city has the opportunity to ask them those questions when they depose them in the case. We felt that it was an unnecessary and unfair intrusion into our work to ask for this material," she added. "It is not our job to be investigators on their behalf." Judge Ronald L. Ellis agreed, writing in his decision that the city had "failed to overcome the reporter's privilege by making a showing that the information they seek pertains to a significant issue and is unavailable from other sources."
But the real distinguishing factor in the "Central Park Five" case stems from its medium: documentary film. Documentaries have long been closely related to traditional journalism, with films from "The Thin Blue Line" to this year's Oscar-nominated "The Invisible War," perfecting the art of the cinematic exposé. While the directors clearly felt they deserved protection as journalists under the reporter's privilege, there was enough uncertainty in recent legal precedents to create cause for concern.
The doubts stem from a 2011 decision in a case that pitted filmmaker Joe Berlinger against the Chevron Corporation. Chevron sought access to all 600 hours of outtakes in Berlinger's documentary "Crude," which centered on a lawsuit filed by Ecuadorans who accused Chevron of contaminating their water supply in the Amazon.
The contentious case again Berlinger included revelations that the idea for the film had been originally presented to the filmmaker by the Ecuadoran plaintiffs' attorney, and that he had removed a potentially incriminating scene after receiving a request to do so from the attorney. Berlinger said that he maintained editorial independence throughout the making of the film. But the language of the decision, which ruled that Berlinger could not claim a reporter's privilege because the documentary was not an act of "independent reporting," disturbed the documentary film community.
Documentary filmmaker Michael Moore told the New York Times that the decision could have much wider repercussions and worried that it might cause a "chilling effect." Professor Jon M. Garon at the Law and Informatics Institute wrote in a blog that in light of the court's designation of Berlinger as an advocate rather than a journalist, documentary filmmakers might need to rethink the practice of receiving support from nonprofit groups with similar advocacy goals or partnering in awareness campaigns. That technique, which has become a mainstay of an industry that often needs to fight for funding, distribution, and viewers, could imperil their ability to claim the independent journalist status necessary for a reporter's privilege, Garon wrote.
At the heart of the "Central Park Five" case is the filmmakers' clearly sympathetic portrayal of the five men. Classifying the directors as "advocates" for the Central Park Five would be no stretch of the imagination, and they have been vocal about their desire for a settlement favorable to their subjects in the civil case. An advocacy angle is often intrinsic to documentary film, where filmmakers choose stories that speak to them and spend much longer periods of time with their subjects. Does this relationship or lack of neutrality disqualify them for journalistic legal protection?
It does not, says Michael Donaldson, a lawyer specializing in documentaries who filed an amicus brief in the Berlinger case. "The case law is well established. One does not stop being a journalist just because he or she advocates for a position that is consistent with his or her writing," Donaldson told CPJ. "That unfortunate, rather imprecise, reference in the Berlinger decision is just that. It does not change the law."
Sarah Burns argued that "a lot of journalism is advocacy in one way or another. ... There is no such thing as total objectivity. Opinion columns are not objective." The more important distinction to make, she told CPJ, was that "there was no financial or editorial relationship between us and the subjects of our film." A side issue of whether Burns could be considered an independent journalist because she had interned at the law firm that was then defending the five men while in college was dismissed by the judge because the material sought had not been collected during that time. The key point to qualify for the reporter's privilege is whether the plaintiff's "intention at the time the information in question is gathered was for the purpose of disseminating the information to the public, " Judge Ellis wrote. Moreover, "having a point of view," he concluded, does not make the newsgathering process non-independent.
The outcome in the case has averted a "major setback," in the words of Donaldson, though he advised documentary filmmakers to be "wary" and that if "they want later to invoke a reporter's privilege they have to conduct themselves like a journalist."
What seems clear is that this won't be the last case of a documentary filmmaker invoking journalistic protection. Dwindling resources in media companies and the increased appeal of documentaries thanks to Internet distribution means journalists and filmmakers will likely increasingly look to film as a way to tell--or break--a news story. More subpoenaing of filmmakers in civil and criminal cases can't be far behind.
One sector of society that faces the harshest scrutiny in "The Central Park Five," is the New York City press, whose hysterical coverage the filmmakers portray as having amplified the drumroll for the convictions. Asked if she finds it ironic to be claiming journalistic protection for a movie that slams the media, Burns told CPJ that in the "Central Park Five" case, the press failed to fulfill its purpose of "asking questions and holding our politicians and society accountable." But the role of journalists is a "hugely important one in our society," said Burns, who considers herself part of that community, "so trying to do that role properly is noble."


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Autometer ES Sensor Temperature 1/8in NPT Male Replacement Short Sweep
SKU: am990344
Auto Meter offers the solution for any level of performance monitoring you need, whether you need only to monitor the critical systems like oil, water, and volt, or your needs have been expanded due to the addition of nitrous or boost. Auto Meter's broad line includes styling and quality levels for everything from sport compact, street, restoration and street rod, and all-out performance. Auto Meter is the most trusted name in performance gauges.


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This is the story of Nohar Rajasthan, a private eye descended from genetically manipulated tiger stock to become a moreau-a second-class humanoid citizen in a human world. Nohar retired from the private eye business ten years ago, and just wants to spend his remaining time in the peace and quiet of his wilderness homestead. Then a human lawyer asks him to take on a missing moreau case-and suddenly all hell breaks loose!
Product recommendations
by S. Andrew Swann
by Ulysses S. Grant
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Views: 135
You're so right!! It is scary because we can't use those cop-outs but it is so freeing!! And its true that every day we write a new page in our own book!!! It's good to be reminded of this.
@Ally-get that hot pink toothbrush girl!!
I can relate. The one thing about knowing there is no God, is all your choices are on you. You cannot blame use God as an excuse "it was God's will". When you don't believe in God you cannot cop out by saying "well, I'm gonna leave it in God's hands". You are totally accountable, and that can be a scary thing at times.
But the liberating part is, knowing that you have the power to make changes and no entity has already determined your path. Having the power of change and not predestination, is a wonderful thang, lol.
Hot Pink! Sweet! - makes me want a new toothbrush. I agree with everyone else = Well Said D Bookworm! Kimberlee, I was't brought up religious, but I did have a deeply entrenched "Fear of Mom" - Liberating yourself from any authority is scary and exciting, but wow the rewards!
Congrats, taking resposnibility means you get all the "credit" too! ;)
Hi Kimberlee! I understand where you're coming from. It's odd that my mother raised me Catholic but wasn't herself particularly religious, whereas I latched onto religion from a very early age. I think now it's because my family dynamics were all screwed up and I wanted someone to love me unconditionally, even if I had to delude myself.
Anywho, I understand that it's scary and liberating at the same time. I think liberty in and of itself is pretty scary. Being all "free" and shit makes things complicated and sometimes dangerous, but that's the price we all pay for living our own lives and generally being AWESOME.
Do what is meaningful to you. And always, always buy the hot pink toothbrush. :-P
© 2019 Atheist Nexus. All rights reserved. Admin: The Nexus Group. Powered by


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Matt Bares needed a new print or packaging design and created a contest on 99designs.
A winner was selected from 63 designs submitted by 9 freelance designers.
Meissner Tierney Fisher & Nichols S.C.
MEISSNER TIERNEY FISHER & NICHOLS S.C. is a law firm with an exceptional history, which spans more than 150 years. Since its beginnings in 1848, the firm has been located in downtown Milwaukee, in the heart of the city's major financial, business and governmental district. Our offices are located on the 19th and 20th floors of the Milwaukee Center – an office, hotel and theater complex, across the street from historic City Hall.
MEISSNER TIERNEY FISHER & NICHOLS S.C. has a sophisticated practice, often found at much larger law firms. The firm's structure enables it to assure that client problems receive the attention of experienced attorneys. Long-term relationships with clients are fostered to provide continuity of representation, based on a well-grounded understanding of each client's personal and business history.
The firm's lawyers are able to closely interact with each other. By design, they are not isolated into specialty departments. This pooling of expertise and experiences leads to a comprehensive and creative approach to solving problems and meeting client needs. At the same time, the personal attention of the lead lawyer, well known to the client, is retained.
When a new attorney joins our firm we take that opportunity to send an announcement to our clients of the new hire. The current mailer is very dated and needs to be redone. Unfortunately we have very little unification between our very dated print media and our somewhat less dated website, http://www.mtfn.com.
Given we are a law firm the design needs to be constrained and very professional, however, I'd like to see some small contemporary flashes (if that makes any sense).
I'm attaching the old mailer, and a few pictures of the building (only because thats what they've always done but if you have better ideas by all means), if you're interested in any other art please let me know. Also included is what I would consider very poor redesign attempt that made by a local printer. It's the basic direction and he had some solid ideas one being that the mailer be a post card which I do like but I think we can do a lot better.


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Hickenlooper says decision on special session after floods 'a day or two' away
Gov. John Hickenlooper said Monday his administration is within "a day or two" from making a decision on if a special legislative session is needed to address issues in the wake of Colorado's massive and deadly floods.
"We're basically going out to every agency and asking them to push down into their bureaucracies … what are the issues you foresee and do we need legislation for any of these, so we're gathering that information," Hickenlooper said.
The governor added that to his knowledge there's nothing that "rises to the level" of needing legislation, "but that doesn't mean it's not there."
At a news conference last Thursday, Hickenlooper alluded to the possibility of a special legislative session and cited the need for potential legislation to address looting, contractor fraud and price-gouging.
Hickenlooper had said a decision would be made within a "matter of days."
Democratic leaders, including House Speaker Mark Ferrandino and Senate Majority Leader Morgan Carroll, said that while all options are open, nothing had been brought to their attention from the governor's office that would warrant the special session.
The legislature's budget includes money to cover 20 days of special session, estimated at $21,500 a day to handle legislative per diem, mileage and temporary staffers. Hickenlooper called a special session in 2012 after the Republican-controlled House let the clock run out on the second to last night of the session to kill a civil unions measure that had enough support to pass.
More than a dozen counties have been impacted by massive floods that have damaged thousands of homes and structures and claimed eight lives.
Follow Kurtis on Twitter: @kurtisalee


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This afternoon in a White House briefing, President Obama was asked what he thought of the contention of some Republicans that he's out of touch with average Americans.
He had a good answer (paraphrase):
I went to law school and college with the help of scholarships. So did my wife. We were still paying off student loans 9 years after we graduated.
I bought my first car for about $900. It had a big hole in the floor that allowed you to see the road, so I knew my wife wasn't marrying me for my money. We had credit-card debt we hadn't paid off. We had to start college fund.
Our personal finances--meaning not having to worry about paying bills at the end of the month or gas prices--weren't stable until fairly recently. I have a middle class upbringing and experience. In contrast to my Republican challenger...
How's that for a diplomatic answer?
It's a very diplomatic answer!
And persuasive.
If the election boils down to who's more in touch with average Americans, Obama will win in a landslide.
SEE ALSO: LIVE FROM WHITE HOUSE: Team Obama Socks It To GOP On The Economy


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Now with postgis2 which supports rasters by default is it possible to do a zonal statistics analysis?
I've google it but I haven't found anything solid? Is there a tutorial outthere to get me started?
Can anyone give me an sql example on how to do it?
An updated (simplified) query according the blog Aragon mention :
CREATE TABLE sum_pop3 AS
SELECT gid, SUM((ST_SummaryStats(ST_Clip(rast,1,geom))).sum)
FROM perez_grid, ls_den
WHERE ST_Intersects(geom,rast)
GROUP BY gid;


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Ok, enough of the sappy stuff - here's the good stuff.
We set off Saturday morning to head to the Brevard Zoo which has been a tradition for Natalie. We first started going on her 2nd birthday and have gone every year since.
This year, Christian, Anilyse and Grammy came with us!
Here's some photos of the day:
|My diva - looking fierce
|We love feeding this deer every time we go. She's so sweet!
|Of course we HAD to feed the giraffes!
|They had Christmas crafts set up around the park.
Natalie got another stuffed giraffe from the zoo (which is also a tradition). So, to date we have Daddy Giraffe (2nd birthday), Mommy Giraffe (3rd birthday), Baby Giraffe (gift from Aunt Jodi) and now Grammy Giraffe (4th birthday). My girl loves giraffes!
After the zoo, we drove home and put Nati down for a nap. We kicked it into high gear and decorated for her birthday party! This is the first year she was aware of parties and she was so excited!
|This is the cutest little hat! She was grumpy when I took the picture - made it even cuter!
|She loved this giraffe snuggle blankie and insisted in taking it immediately to her room to put on her bed!
|Adding to her collection.
|Yes, I'm a bad Mom, I got my Christmas baby a Christmas birthday cake. I'm told, that as she gets older, she will HATE any reference to Christmas associated with her birthday. Oh well. This year she LOVED it!
All in all, a great birthday! I'd say she had a great time! I'm slowly recovering from loosing my baby and am fulling embracing having a little girl! Can't wait for many more birthdays to come!


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The Group reached this conclusion following review of the latest data on pandemic influenza A(H1N1) 2009 vaccines, and the results are published in the WHO Weekly Epidemiological Record today.
Recommendations of the Strategic Advisory Group of Experts on Immunization on the influenza A (H1N1) 2009 pandemic, July 2009 [pdf 273kb]
SAGE also reviewed safety data on the H1N1 vaccines following administration of more than 350 million doses. "The data indicate a similar safety profile to that of seasonal vaccines," said Professor David Salisbury, SAGE Chair.
Other issues considered by the Committee during its first meeting of this year include recent progress towards polio eradication and a process for revising WHO's vaccine prequalification procedure, a service provided by WHO to ensure that vaccines purchased by United Nations agencies meet international standards of quality, safety and efficacy.
Robust and independent oversight of new strategic plan on polio eradication needed
SAGE recognized the substantial progress made in the last 12 months by national authorities in reducing transmission of wild poliovirus, particularly in northern Nigeria, northern India and many re-infected countries, but cautioned against excessive enthusiasm. Members emphasized the importance of ensuring robust and independent monitoring of major milestones of the Global Polio Eradication Initiative's new Strategic Plan for 2010-2012. They further recommended the rapid development, implementation and monitoring of corrective action in all countries in which a major milestone is seen to be at risk. SAGE recognized that insufficient financing is now the single greatest threat to polio eradication.
"Recent progress suggests that interruption of transmission of poliovirus is now within our grasp," said Salisbury, "It is critical that international immunization and development communities help close the US$ 1.3 billion funding gap for 2010-2012 activities so this momentum can be maintained."
Proposal to revise WHO's vaccine prequalification procedure
SAGE expressed support for a proposed process to revise WHO's procedure for prequalification of vaccines, but emphasized that the quality of the service must be maintained and resources be sufficient to meet increased demand. An improved process is needed in order to better meet the challenges of the increasing volume and complexity of prequalification requests from manufacturers. Working papers have been developed on issues such as programmatic suitability of vaccines, new approaches to testing, and regulatory oversight of vaccines manufactured in multiple sites. The broad consultative process on these papers is now under way, with the proposed revisions expected to be submitted to the WHO Executive Board in May 2011 for approval.
About SAGE
SAGE advises WHO on overall global policies and strategies, ranging from vaccine and technology research and development, to delivery of immunization and linkages between immunization and other health interventions.
The Group is comprised of 15 members, serving in their personal capacity. Members are acknowledged experts from around the world in fields relating to vaccines and immunization.
The Group meets at least twice a year, with working groups established for detailed review of specific topics prior to discussion by the full group.
For more information, please contact:
Alison Brunier
Communications Officer
Immunization, Vaccines and Biologicals
Telephone: +41 22 791 4468
E-mail: firstname.lastname@example.com


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I'm testing a new forum search which hopefully works better than the existing one. It currently only indexes public posts. Feedback welcome-- ngld
0 Members and 1 Guest are viewing this topic.
The installer shouldn't cause that kind of warning since it only sends HTTP(S)-Requests which is exactly your browser does. It doesn't behave exactly like a browser and you could detect it by scanning your internet traffic but I don't know why your friend's ISP would call it "abnormal behaviour". Most likely they detected traffic to known C&C servers or ports commonly used by trojans. If that's the case, the warning is warrented and you should make sure that your friend's computer(s) aren't infected.
FreeSpace\ Port.Silent\ Threat\:\ Reborn.Silent\ Threat\:\ Reborn\ Cutscene\ Pack\ (720p)=Version 1.4
FreeSpace\ Port.FreeSpace\ Port\ Media\ VPs.HD\ Cutscenes\ by\ MjnMixael\ (720p)=Version 1.0
Do I do anything with the file called ".JarClassLoader" that's right above the file you two have told me about?
PIe has the right answer. .... Delete the entire lines, but nothing else. Make sure the Installer isn't open while you're editing the file.
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