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Nate's Numbers: the numbers convergePosted: November 5, 2012 Filed under: 2012 elections, open thread, polling | Tags: Nate Silver, Number Krunching, open thread, Polling, polls, Punditry, Sam Wang, Statistics 60 Comments
I've spent all my professional life drenched in numbers and statistics so Nate Silver's numbers fascinate me. It's probably the same reason they drive Republicans and pundits to distraction. Unraveling trend is easier with numbers than hateful, wishful thinking motivated by political piety. So, Karl Rove got on TV–probably trying to save what's left of his credibility–saying that Romentum was stopped by Sandy. Romentum was a bit of canard and it turns out so is Sandy. Silver tries to discern the possible factors behind the recent numbers and looks at the Sandy Factor. That's a relatively simple task for any one with a database and a background in trend analysis.
When the hurricane made landfall in New Jersey on Oct. 29, Mr. Obama's chances of winning re-election were 73 percent in the FiveThirtyEight forecast. Since then, his chances have risen to 86 percent, close to his highs on the year.
But, while the storm and the response to it may account for some of Mr. Obama's gains, it assuredly does not reflect the whole of the story.
Mr. Obama had already been rebounding in the polls, slowly but steadily, from his lows in early October — in contrast to a common narrative in the news media that contended, without much evidence, that Mr. Romney still had the momentum in the race.
Moreover, there are any number of alternatives to explain Mr. Obama's gains before and after the storm hit.
- Mr. Obama was adjudicated the winner of the second and third presidential debates in surveys of voters who watched them.
- The past month has brought a series of encouraging economic news, including strong jobs reports in October and last Friday.
- The bounce in the polls that Mr. Romney received after the Denver debate may have been destined to fade in part, as polling bounces often do following political events like national conventions.
- Democrats have an edge in early voting based on states that provide hard data about which party's voters have turned out to cast ballots. Some voters who were originally rejected by the likely voter models that surveys apply may now be included if they say that they have already voted.
- Both Mr. Obama and Mr. Romney have been running lots of advertisements, which could have some effect, especially in the swing states.
- Mr. Obama's voter-targeting operation may in fact be stronger than Mr. Romney's and may have begun to show up in the polls.
- Mr. Obama's approval rating is at 49 or 50 percent in many surveys, a threshold that would ordinarily predict a narrow re-election for an incumbent.
- Some elections "break" toward one or another candidate at the end as undecided voters tune in and begin to evaluate their decision.
Each of these hypotheses could merit its own article. But the point is that the causes for Mr. Obama's gain in the polls are overdetermined, meaning that there are lot of variables that might have contributed to the one result.
If I had told you in January that Mr. Obama's approval rating would have risen close to 50 percent by November, and that the unemployment rate would have dropped below 8 percent, you likely would have inferred that Mr. Obama was a favorite for re-election, with or without a hurricane and what was judged to be a strong response to it.
Whatever the causal factors, Nate's numbers look good for the President. Sam Wang–a Princeton number kruncher–says it not only looks like the President will hold his office but that the US Senate might see a Democratic Pick up of two. This is an important firewall for those of us that care about things like Supreme Court appointments and getting rid of the filibuster silliness that has allowed the Republicans to basically thwart governing. Dems may pick up Nebraska and Pennsylvania.
Rather than responding to the analysis, the Republicans continue to attack Nate Silver the man. You may recall this Krugman piece last week about The War on Objectivity.
Brad DeLong points me to this National Review attack on Nate Silver, which I think of as illustrating an important aspect of what's really happening in America.
For those new to this, Nate is a sports statistician turned political statistician, who has been maintaining a model that takes lots and lots of polling data — most of it at the state level, which is where the presidency gets decided — and converts it into election odds. Like others doing similar exercises — Drew Linzer, Sam Wang, and Pollster — Nate's model continued to show an Obama edge even after Denver, and has shown that edge widening over the past couple of weeks.
This could be wrong, obviously. And we'll find out on Election Day. But the methodology has been very clear, and all the election modelers have been faithful to their models, letting the numbers fall where they may.
Yet the right — and we're not talking about the fringe here, we're talking about mainstream commentators and publications — has been screaming "bias"! They know, just know, that Nate must be cooking the books. How do they know this? Well, his results look good for Obama, so it must be a cheat. Never mind the fact that Nate tells us all exactly how he does it, and that he hasn't changed the formula at all.
This is, of course, reminiscent of the attack on the Bureau of Labor Statistics — not to mention the attacks on climate science and much more. On the right, apparently, there is no such thing as an objective calculation. Everything must have a political motive.
This is really scary. It means that if these people triumph, science — or any kind of scholarship — will become impossible. Everything must pass a political test; if it isn't what the right wants to hear, the messenger is subjected to a smear campaign.
Any kind of scholarship has become challenging under Republican fanaticism as witnessed by the attacks on evolution, climate change, and the economic analysis that shows there is no such thing as an economic benefit created by low marginal tax rates for the rich. Just ask scientists trying to get grants to study things like stem cell research. Fox gets people to believe anything. Science causes them to retreat to their medieval churches and scream about intervention by celestial beings. (So, if gawd caused Sandy to take out NJ and NY because of Gay Rights, does this mean gawd caused Sandy to give Obama momentum? Ask Grand Inquisitor Pat Robertson about that one.)
Yes, Nate's numbers took another upward tick this morning. He also believes that Obama will take the popular vote which should be dismaying to all those journalists that keep wanting to turn this into a white knuckle election.
Silver also added to Obama's likely number of electoral votes on Monday. He now sees the president winning 307.2 to 230.8 for Mitt Romney, a tiny tick higher than he saw the race on Sunday.
He also sees Obama capturing the popular vote, taking 50.6 percent to Romney's 48.5.
Rachel Maddow insists the Republicans see the numbers and actually believe that their man Mittster is a goner. The blame game has already begun. Haley Barbor blames Sandy. Lindsey Graham is actually looking at numbers and notices that that demographics are not in their favor.
QUOTE OF THE DAY: "If I hear anybody say it was because Romney wasn't conservative enough I'm going to go nuts. We're not losing 95% of African-Americans and two-thirds of Hispanics and voters under 30 because we're not being hard-ass enough."
– Politico quotes Sen. Lindsey Graham (R-SC), saying that demographics would be the only reason for a hypothetical Mitt Romney loss Tuesday.
Yeah, the blame game has begun and the election isn't even over. Funny he should bring up Hispanic and black voters. Latino Decisions found that Hispanics support President Obama in historic numbers — 73 percent. They believe that's enough to carry President Obama to victory in four swing states and ultimately to win re-election. Those four swing states are Nevada, Colorado, Virginia, and Florida.
This is closer to the truth. Republican policy statements as expressed by "severely conservative" Mitt have driven off the young, hispanics, blacks, and women. I had MSNBC on mute most of the weekend when they were following the candidates around. All you have to do is look at the people used as back drops for candidates' speeches to realize who really is on the losing side of US demographics. This is part of the numbers game. You can't build momentum or fantasize a trend based on capturing an ever decreasing slice of the American Pie.
Which brings me to the post election deconstruction that should occur in the Republican party. When will the actually give up on the Southern Strategy? We've seen more race baiting in this election that I've frequently wondered if the ghost of George Wallace is running the Romney campaign. We've seen attacks on women's rights that make me wonder if Republicans know that women got the right to vote. We've seen support of policies that are based on show us your papers that remind me of old NAZI movies. You can't attack and demonize the majority of the electorate and expect the numbers to come in for you. You also can't build policy on attacking scientific models and theories. The Republican party has definitely shown that its plan for America is to try to recreate the past no matter what the cost.
All I can say is go Nate go! Win one for Ada Lovelace and the country. There are easy ways to figure out which events contribute to trend and what's just random. People should pay more attention to Nate Silver and a lot less attention to the likes of Karl Rove and Haley Barbour. Nate Silver bet Joe Scarborough $2000 that his analysis was right saying "Occam's Razor: Pundits are useless". That's something I completely grok.
"I think I get a lot of grief because I frustrate narratives that are told by pundits and journalists that don't have a lot of grounding in objective reality,"
60 Comments on "Nate's Numbers: the numbers converge"
I'll be glad when Nate can hang his hat on an Obama win. I'll also be glad when we can exhale.
Dak, you should be on Rachel with this post, I wish I could put on paper what you have written here today. I think these things, but you always amaze me. I am just wanting more people to here this!
Southern Strategy women's rights old NAZI movies The Republican party has definitely shown that its plan for America is to try to recreate the past no matter what the cost".
You put all of that in one paragraph ……. I love ya! I am proud of you.
Me too!
I know this is not this thread but just wanted everyone here to see it.
Also Nate Silver is going to be on Colbert tonight.
Final ABC/WaPo Tracking Poll:
Obama 50
Romney 47
Happy Birthday Kat!! Great posts today as always.
happy birthday…………..don't know where we would be without you Dak
OK some one explain why a campaign would do this except they really don't give a damn about NJ


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Persons with disabilities who require alternative means of communication for program information (e.g., Braille, large print, audiotape, American Sign Language, etc.) should contact the Agency (State or local) where they applied for benefits. Individuals who are deaf, hard of hearing or have speech disabilities may contact USDA through the Federal Relay Service at (800) 877-8339. Additionally, program information may be made available in languages other than English. For more information please visit the following web page at FNS/USDA. These institutions are equal opportunity providers.
CalFresh provides assistance to low-income households and can help buy nutritious foods for better health. For CalFresh Information, call 1-877-847-3663 or go to the CalFresh Website. You may also apply on the e benefits website. This website is maintained by the State of California Department of Social Services. Frequently Asked Questions regarding Benefits.

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This Washington Post editorial discusses a serious and under-reported problem in Iraq — the disqualfication of more than 500 would-be candidates in the upcoming parliamentary election. Most of the disqualified candidates are Sunnis who once supported the Baath party to one degree or another. Some of them are prominent leaders, including the current defense minister and the head of a major political bloc.
As the Post argues, if the ban stands, it will mean that an election that once promsed to be one of the most free in the history of the Arab world has been badly degraded. In addition, the ban might reignite the sectarian warfare that looked as if it might tear Iraq apart before the 2007 surge. There is already some evidence that this is occurring.
The Post urges the Obama administration to use its leverage to have the ban moderated so that legitimate Sunni leaders can get back on the ballot. And it notes that Vice President Biden has been "working the phones" for this purpose.
But it's not going to be easy. Prime Minister Maliki appears to support the ban, though president Talibani does not, and has asked for a ruling on its legality.
There have been times in the past when the political situation in Iraq was in crisis and the U.S. was able to knock heads and hammer out a settlement. But that was before we began our rush to exit the country. As things stand now, it's not clear that we have any real levearge in Iraq.
Thus, Biden can talk from now until election day (for him, a labor of love). Unless the Shiite leaders reach the counterintuitive (for them) conclusion that they have more to lose than to gain by excluding so many opposition candidates, it will be to no avail.
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ATLANTA (AP) — More than 14,000 rock and folk music fans braved steady rains over the weekend at the inaugural Shaky Knees Music Festival near downtown Atlanta.
The festival featured indie folk music heavyweights such as Band of Horses and The Lumineers, as well as soul-rock standouts Vintage Trouble. Here is a look at the performers who entertained thousands of music fans who donned plastic ponchos and galoshes to deal with the muddy field and torrential downpours.


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2011 ACM Winners: Miranda Lambert, Taylor Swift Honored
Taylor Swift can add yet another trophy to her already-full mantle. The 21-year-old singer won Entertainer of the Year at the 2011 Academy of Country Music Awards, held Sunday evening in Las Vegas.
The big winner of the night, though, was Miranda Lambert, whose Grammy-winning song 'The House That Built Me' took home awards for Single Record of the Year, Song of the Year and Video of the Year. Lambert also won Top Female Vocalist of the Year for the second year in a row.
Fresh off their success at the Grammys in February, Lady Antebellum racked up the award for Album of the Year, as well as Top Vocal Group of the Year.
Taste of Country has more on the night's top winners. Head over there now for the scoop on the ceremony, plus see photos from the red carpet.
Entertainer of the Year: Taylor Swift
Album of the Year: 'Need Your Now' – Lady Antebellum
Single Record of the Year: 'The House That Built Me' – Miranda Lambert
Song of the Year: 'The House That Built Me' – Miranda Lambert
Top Male Vocalist of the Year: Brad Paisley
Top Female Vocalist of the Year: Miranda Lambert
Top Vocal Duo of the Year: Sugarland
Top New Solo Vocalist of the Year: Eric Church
Top New Vocal Duo or Group of the Year: The Band Perry
Top New Artist: The Band Perry
Video of the Year: 'The House That Built Me' – Miranda Lambert
Vocal Event of the Year: 'As She's Walking Away' – Zac Brown Band feat. Alan Jackson


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The recipient of the 2008 Templeton Prize for Progress Toward Research or Discoveries about Spiritual Realities was Michael Heller, a Polish cosmologist and Catholic priest, currently Professor in the Faculty of Philosophy at the Pontifical Academy of Theology in Krakow. John M.
The antievolution bills recently introduced in the Florida legislature continue to elicit opposition. The bills closely resemble a string of similar bills in Alabama – HB 391 and SB 336 in 2004; HB 352, SB 240, and HB 716 in 2005; HB 106 and SB 45 in 2006 – as well as a model bill that the Discovery Institute's Center for Science and Culture, the institutional home of "intelligent design" creationism, recently began to promote.
NCSE's executive director Eugenie C. Scott is to receive the honorary degree of Doctor of Science from the University of New Mexico on May 17, 2008. In a letter to Scott, the university's president David J. Schmidly wrote, "This degree is a reflection of the high regard in which you are held and acknowledgement of the vast accomplishments you have made in your career."
Malcolm McKenna, a retired curator of vertebrate paleontology at the American Museum of Natural History and a Supporter of NCSE, died on March 3, 2008, in Boulder, Colorado, according to the obituary in The New York Times (March 10, 2008). Born on July 21, 1930, in Pomona, California, he attended the California Institute of Technology and Pomona College, before graduating in paleontology at the University of California, Berkeley, where he also earned his Ph.D.
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As we all learned in driver's ed, operating a motor vehicle is not a right, like voting or publicly speaking your mind. Driving is a privilege. And that privilege is earned by mature, sober, careful behavior and an active concern for the safety of not only yourself, but your passengers, other drivers and their passengers, and pedestrians.
The State Assembly passed a bill last month — A.6657 — that would require back-seat passengers to buckle their seat belts. The bill is now in the Senate Rules Committee. We support it, and hope the Senate takes it up quickly when the Legislature begins its session. There is already a state law that requires drivers and front-seat passengers who are 16 and older to wear seat belts. Making rear-seat passengers also buckle up makes good sense to us. We urge you to contact your state senator and ask him to vote the bill out of Rules and onto the floor, and to support it.
Roadways are dangerous places. Speed limits are mostly ignored. Too many drivers endanger themselves, their passengers and the occupants of other vehicles by texting, dialing, using apps and doing other distracting activities. Many young drivers act like young people do when they're not driving, incautiously testing their skills and daringly pushing limits. Mature drivers, with their years of experience, often think they can operate their vehicles on virtual autopilot while chatting on cell phones, checking the weather on an app, listening to Garmin give directions — and focusing least on their most important task: driving carefully.
With all that going on, a minimum responsibility all drivers have is to protect their passengers by making them buckle their seat belts. And 28 states and the District of Columbia now require rear-seat passengers to buckle up.
Research by the National Highway Traffic Safety Administration has found that lap/shoulder seat belts, when used properly, reduce the risk of fatal injury to front-seat occupants by 45 percent, and reduce the risk of moderate to critical injury by 60 percent. What's effective for front-seat passengers will surely be protective of those in the back seat as well.
The American Automobile Association estimates that 33,000 people are killed in motor vehicle crashes each year. And according to the NHTSA, car crashes are the leading cause of death for children age 4 and for those of every age from 11 through 27 in the U.S. Seat belts are the single most effective means of reducing the risk of death in a crash, having saved more than 250,000 lives since 1975 on our roads — more than 12,700 in 2009 alone, AAA says.
We encourage you to contact your state senator and tell him to vote for the bill.


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Ticket kiosk at Powell Street Station to be temporarily closed
As part of our station modernization program, the ticket kiosk at Powell Street Station will be closed from Wednesday, April 4, 2012, through Wednesday, April 25, 2012, reopening on Thursday, April 26, 2012. We apologize for this inconvenience.
For service, please visit the MyTransitPlus ticket kiosks in the Embarcadero, Montgomery and Civic Center BART Stations. Thank you for your patience.
If you need language assistance services, please call BART's Transit Information Center at (510) 465-2278.


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Team Associated's Renezeder Wins the Pro-4 Championship!September 24, 2013
Event Name: Lucas Oil Off Road Racing Series
Event Location: Las Vegas Motor Speedway
Event Date(s): September 20, 2013 to September 21, 2013
The Lucas Oil Off Road Racing Series headed back out to "Sin City" to do battle at the famous Las Vegas Motor Speedway for the second visit of the year, but this time it was to run under the lights. The 2013 LOORRS season is winding down to the final rounds and the action was sure to be wild.
The racing action kicked into gear in Friday's evening hours and the Pro 4s were the first class up and it was Team Associated drivers, Kyle LeDuc and Carl Renezeder, starting off on the front row. Kyle looked like a man possessed as the green flag waved, showing the speed that won him the 2012 Pro 4 championship. The battles behind Kyle are where all the action was taking place. Renezeder and fellow off road veteran Rob MacCachren were battling for the second-place spot. Rob was able to put a good pass on Carl and slid into control of the second position. A couple laps later, MacCachren would suffer a mechanical issue that would send him to the hot pits and end his race for the evening. As the race was winding down, a caution would stack the field back together. Kyle's huge lead was taken away and a new race had begun with less than a handful of laps to go. When they got back to green, Renezeder had found some speed and Kyle had to work hard to keep Carl behind him. As they went through the chicane, Carl saw an opening on Kyle and in a door-to-door move with Kyle he was able to take the lead as they came to the white flag. Kyle was right on the back bumper of Carl and was fighting to get it back, but Carl was able to hold on to take the win.
Pro 2 came to the line late in the night and looked to re-energize the fans that hung out for the finale. The Pro 2 class is usually one of the cleaner classes and showcases the driver's true talents and ability to drive close, but something got into the water before Friday's main event and that all got tossed out the window. As the green flag flew, the fiberglass followed. Team Associated's Robby Woods was looking to put on a show for his hometown fans, and was doing so by running out in the lead pack following Marty Hart who was leading the majority of the race, until Robby made a door-to-door pass for the lead, but a small bobble would give it back to Hart. Woods kept pushing and was clearly the fastest truck in the field, but a mechanical issue ended his night early. With the carnage continuing, the cautions continued to fly and driver positions changed rapidly. When all was said and done, Carl Renezeder was able to stay alive and fight from the back to the front several times and secured a third-place finish behind Rob MacCachren and Marty Hart, who was able to hold off for the win.
Saturday's schedule was shaken up due to severe wind, causing the Lucas race officials to delay the start time in hopes that the wind would subside. After about an hour delay, the wind settled enough to allow racing to resume. With the delay, Pro 2 started the evening (this class usually ends the program) and the fans got a heck of a race to watch. Carl Renezeder jumped out to the early lead, closely followed by Robby Woods. The front runners kept close to each other, without change, until Robby Woods got a good run going down the back stretch and made a clean move and passed Carl in turn four as they entered the chicane. Robby got into clean air and began to control the race. Robby's lines were tight and fast, not allowing Carl, the crafty veteran, to take the lead back as the laps started to count down. Woods and Renezeder took the white flag together, nose to tail. Robby had one lap to go to collect his first career win and while landing the table top on the backstretch, his transmission gave way, handing the lead to a hard-charging Carl.
Modified Kart followed the Pro 2s, and this class features some young, talented kids that are sure to be the future of off road racing. When the race started, a pile up in turn one collected the #508 Team Associated sponsored kart of Brooke Kawell, leaving her with a flat tire and forcing a restart. On the restart it was Gavin Harlien and his #555 Team Associated kart out front. Behind Gavin was last year's points champion Myles Cheek with Cole Mamer in third. When the green dropped, Gavin pulled a small gap on Myles and kept it on all four, not giving Myles the opening he was looking for. Gavin went on to pick up his second win of the year over Myles and Cole Mamer.
Pro 4 would round out the night's race program, and with a win the night before, Carl Renezeder was looking to clench his 9th short course championship. Kyle LeDuc was looking to spoil Carl's fun and did all he could to get out front and keep his championship hope alive. At the start, Kyle was looking as he might be able to push the point's battle to the final round, until he over-rotated in turn four and give up the lead to Rob MacCachren. After the spin, Kyle fell back several places, but a caution came out allowing him to regain his thoughts and make another charge to the front. When the action got back under way, Kyle refocused and started to make quick work of those in front of him. Kyle got back into second, following race leader Rob MacCachren, when coming through the chicane he over-rotated again, causing him to roll onto the K-rail. Renezeder moved back into second with the mistake, and it was looking like his championship was within arm's reach. Carl gave Rob a good race, but didn't look to make a move as a second-place finish would secure his championship. Rob kept it rubber side down and grabbed his first win in the Pro 4 class of the season. Carl came across the line in second followed by Todd LeDuc in third. With the second-place finish, Carl Renezeder clinched his 2013 Pro 4 championship. The entire Renezeder family and team was there to celebrate the win and crowing of the 2013 Pro 4 champion Carl Renezeder.
The final round of the Lucas Oil Off Road Racing Series will be in Southern California at Lake Elsinore Motorsports Complex where drivers will have one more chance to collect a win in the 2013 season. There will be nothing for those to lose and everything to gain. Round 15 will be taking place on October 26th. After all the racing is finished and the champions are crowned, Lucas will be running the Challenge Cup, which will pair the Pro-Lites against the Pro Buggies, and the main event will be Pro 4s vs. Pro 2s for a large cash prize. It will be the biggest race of the year and a must-see!


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It's my dad's birthday today – he would have been eighty one. So I was thinking of something to write and looking through a box of stuff I have saved after he passed away three years ago
I came across these petrol ration coupons.
In the wake of the 1973 Arab-Israeli war, the Arab oil-producing countries (OPEC) imposed an embargo on supplies to the US on 20 October 1973. For the wider world, oil prices went through the roof, from around $3 a barrel before the war to over $11 by early the following January.
My readers of a certain age may well have a memory of the three-day week – power cuts, flickering candles and the early shutdown of television at 10.30pm every night. I have been employed in an office all my life and I can remember the lights and the heating going off and having to work by candlelight.
Early in December 1973, the chancellor, Anthony Barber, told the cabinet that the country was facing its worst crisis since the second world war, triggered by the decision of Arab oil producing states to quadruple the price of oil, coupled with an overtime ban by the miners and the power industry workers.
On December 4, Peter Walker, the trade and industry secretary, told the cabinet that falling coal stocks at the power stations would make indiscriminate electricity blackouts inevitable by the end of February unless emergency action was taken.
Mr Walker said that the government's policy of denying the Americans the use of UK airbases during the Yom Kippur war may have put Britain at the top of the list of countries regarded as friendly by the Arab states, but it had not stopped the cabinet having to consider petrol rationing.
More than 18 million petrol ration books were printed, 12 million "supplementary coupons", 20 million forms and 7 million envelopes, and distributed to post offices and motorists to beat the Christmas rush.
In the event the petrol rationing books / coupons were not used.
Here's a link to an article in the The Guardian from 2004 about the oil crisis and a veto on the Queen


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Here's a good paper on how to measure nanoparticles by AFM: [http://ncl.cancer.gov/NCL_Method_PCC-6.pdf]
Here's a good paper on how to measure nanoparticles by AFM: [http://ncl.cancer.gov/NCL_Method_PCC-6.pdf]
Revision as of 10:59, 19 June 2013
- 1 Paul Gruenbacher
- 2 Notebook
- 2.1 Friday May 10
- 2.2 Sunday May 12
- 2.3 Monday May 13th
- 2.4 Friday May 24th
- 2.5 Monday May 27th
- 2.6 Tuesday May 28th
- 2.7 Wednesday May 29th
- 2.8 Friday May 31st
- 2.9 Monday 6/3
- 2.10 Tuesday 6/4
- 2.11 Wednesday 6/5
- 2.12 Thursday 6/6
- 2.13 Friday 6/7
- 2.14 Monday 6/10
- 2.15 Tuesday 6/11
- 2.16 Wednesday 6/12
- 2.17 Thursday 6/13
- 2.18 Friday 6/14
- 2.19 Monday 6/17
- 2.20 Tuesday 6/18
- 2.21 Wednesday June 19th
- 3 References
Paul Gruenbacher
Will be using links to google drive for files.
Friday May 10
Submitted cadnano design of 66 helices, non-hollow: 66helicev1. Will note that midline there are scaffold crossovers that do not have staples nearby, had incorrectly assumed that staple crossovers must avoid scaffold crossovers. Design approximately 40 nm long and less than 22 nm in diameter.
Sunday May 12
New version edited by Dr. Castro.66 helices, 2-layered, hollow: 66helicev3. Will note that diameter remains the same, is approximately 44 nm long now. Should not be an issue
'Literature review': It seems well accepted that crossing the nucleolus membrane is the greatest challenge facing gene therapy. The calcium phosphate protocol as it stands only takes us to the cytosol, so the origami must be designed to be able to enter the nucleus. A paper in 1988 used a range of gold particles to measure the size able to enter through the nuclear pore complex [1] and found that the limiting size was 26 nm. This suprised some people as some viral proteins are larger than this, including Hepatitis nucleocapsids. A later paper in 2002 used gold particles with protein complexes to measure a larger size of 32-36nm [2]. We would need to add targeting sequences or nuclear localization signals to the origami. For NLS peptide conjugation to the DNA staples, one would need DNA staples with a free alkylamino group of a thymine. see A_single_nuclear_localization_signal_peptide_is_sufficient_to_carry_DNA_to_the_cell_nucleus
However, peptide conjugation and staple modification is making things more complicated than what is desired or allowable. Perhaps nuclear targeting sequences would be better, though a recent paper in 2011 downplays their importance [3].
Monday May 13th
Ordered staples for version 3 origami design edited by Castro. The templated had now been decreased to the smaller 7202 template strand. Will be leaving for Puerto Rico while waiting for staples to come in, will be back on the 23rd.
Corresponded with Dr. James Adair at PennU and Dr. Robert 'Bob' Lee of OSU pharma.
Literature Review: In general, some better indications that the origami can be functionalized with cationic peptides to facilitate transport across the nuclear pore complex.
Rosenecker's lab synthesized a tetramer of the PKKKRKV NLSV404 peptide sequnece spaced by glycine residues. (may want to consider finding a lab that has a Applied Biosystems Automatic Synthesizer) [4]. A strong paper, with luciferase transfection assay, in situ flourescent hybridization to detect plasmid dna, and a nuclear import assay with labelled BSA-BODIPY to be transported by the peptide. The SV40 NLS sequence they used is the minimal sequence observed on the T-antigen of SV40 necessary. HeLa cells showed 58.9% transfection within 2 hours, much faster than with the dendrimer alone or with a mutant peptide control. Analysis shows quicker localization of the peptide-plasmid in the nucleus, indicating that the peptides probably helped transport the plasmid across the pore if not through the cytosol as well. Early cell transfection also indicates a lessened need for mitosis to occur. Also did a nuclear transport inhibition protocol by Sebestyen [5], which had also showed linking of peptides to plasmids by the cyclo-propayrroloindole cross-linker. Remember: Karyopherins are the proteins found to accumulate in the nucleus.
Dr. Davis at Case Western also showed that DNA was binded the nucleolin receptor on the cell membrane, and may indicate a new cellular uptake mechanism with increased transfection efficiency. The DNA nanoparticles were formed by PEGlyated polylysine. We probably don't want to pursue this mechanism, but the effectiveness of the internalization and transport of the nanoparticles is impressive [6]. Davis had also demonstrated using PEGylated=polylisine/lipid/DNA nanoparticles that are 10-20nm in diameter and 100 nm in length that are rapidly uptaked and concentrated into the nucleolus. This confirms the aspect ratio hypothesis, and they also seem to enter by nucleolin as well. Davis also mentions compacted DNA being known to increase efficiency so long as the ellipsoidal diameter of the compacted DNA does not approach 25 nm, and this was shown in non-dividing cells[7].
Dr. Dean is one of the first to have described the nuclear targeting sequence of the SV40 DNA itself. In his review [8], Dean describes several methods to nucleari import of nonviral vectors. The classic SV40 NLS PKKKRKV. He also describes the 72 bp sequence that is shown to have binding sites for a number of mammalian transcription factors that are usually brought into the nucleus for RNA transcription. Reich has also idenifitied NFkappaB binding sites that show enhanced expression as well. Using the DNA sequences have an obvious advantage to us in that it eliminates the need for the peptides, but I'm worried that the origami will not allow for the normal binding of the factors even if the binding sequence is spaced and free. Also, we also need to take pore size into account, peptides keep the origami size efficiently small. If we use the peptides, it is important we use a mutant peptide as a negative control. It is also important we check to make sure there is no immunogenic response to the peptide. Dean also mentions the use of the amphiphathic trans-cyclohexane-1,2-diol to disrupt hydrophobic region of central NPC channel. If the DNA origami were to have methyl groups around it, it may help as well.
Side Note: DNA origami would probably be an effective way of DNA labelling, so that only certain regions are exposed for labelling such as biotinylation. Especially if we have a single loop exposed from the origami after it is coated with a protective polymer or such. Then only that loop would be treated with the label.
Friday May 24th
DNA staples arrived two days ago. Pooled the staples into four groups of pre-stock: 96 core staples from plate p086, 41 core staples from plate p085, 13 neighbor staples, and 13 polymer staples. The polymer staples are thus named in the event we want to replace them with staples that will polymerize the origami into filaments.
Created 500nM working stock solution of all staples, and used template strand M13mp18. Final concentrations were 20 nM staples, 10 nM template. The DNA origami was folded under a magnesium screen, ranging from 12, 14, 16, 18, 20, 22, 24, 26 mM. Two groups of 2.5 and 4.5 day ramps were then created. There were 16 groups total.
Monday May 27th
Memorial day, harumph harumph.
- Need to schedule TEM this week, get Nanotech West Access, Order calcium phosphate materials
- Literature notes:
- <300nm is the accepted size for tumor extravasation. keep this in mind for any sort of drug delivery mechanism.
- Zhao Xiaobin makes some good points about FR-targed gene therapy as a target for cancer cells.
- Ward et al. shows that the PEG spacer for the folate is important for efficiency.
- There are groups showing FR-targeting with polyplexes, cationic lipids, PEI. FR-targeted liposomes seem to be more efficient than lipofectim commercial products.
- Concerns: endogenous levels of folic acid and competition, intratumoural diffusion, size, insufficient levels of FR on the tumor cells. Coadministration with anti-estrogen or retinoid receptor ligands which can increase FR expression.
Tuesday May 28th
Found a good protocol for using PEG-bis amine with NHS-activated folate [9]. Scheduled TEM training next week. Called Sigma for their materials, here is a list of what I'm thinking at the moment.
|Igepal CO520
|~500 ml
|1 litre
|Calcium chloride
|100 g
|Disodium Phosphate
|100 g
|Sodium Citrate
|25 g
|Sodium Metasilicate
|25 g
I don't think the metasilicate is absolutely necessary, it seems that it is mostly served as a nucleation agent to help the precipitation of the CPNP, though I also thought it helped limit the size of the particles. I do know it is in Adair's protocol, and there is literature of CP-silicate nanocomposites that exist elsewhere, so I think it's safe to go ahead and make sure we have it.
Also note: Motskin shows cytotoxicity of hydroxyapatite nanoparticles with concentrations above 250 μg/mL, which I'm not too worried about especially systemically [10], especially since they were microparticles and they drowned the cells in them, and KEster had shown doped CPNPs had no cytoxicity.
possible flourescence schemes
- Propidium iodide - intercalating flouroescent, excitation 535 nm, emission 617 nm. Good DNA origami marker.
- Dope CPNP with flourescein, lanthanides, or Cy3 amidite (Adair)
- Indocyanine green (ICG) as an infrared flourophore for animal trials (Adair bioconjugation)
- Dr. Castro mentioned YOYO-1 and SYBR Gold. These seem to be ultrasensitive Nucleic acid detection and quantitation, mostly due to their precise proportion of dye to the bases.
Finally, I am still thinking there is a huge potential of origami in DNA engineering, by organizing the DNA origami in such a way that the encoding domains are protected while other non-important regions are exposed to some sort of steric-hindered chemical treatment.
Literature Review:
Nongradative intracellular trafficiking of highly compacted polymeric DNA nanoparticles 2011 by Kim and Nanoparticles of Compacted DNA Transfect Postmitotic Cells 2003 by Liu
Both of these papers are highly suggestive that compacted DNA is a good thing for the gene transfer. Both labs use 30-mer lysine polymers linked to PEG. The cationic lysine condense the DNA into compacted shapes. Liu had shown that compacted DNA up to 25 nm showed good nuclear transfection efficiency, which is limited by the size of the DNA (after 9.9 kb). Liu also suggests that the polylysine incorporated into the condensed DNA binds to alpha importin with equal affinitiy as an extended SV40 large T antigen NLS, which he points to two immunosorbent binding assays as proof[11]. That's quite a statement.
In general, it seems that poly Lysine or pLy can form toroids of 80-100 nm with DNA. This structure depends on a lysine/nucleotide ratio of 0.4. Chan's lab[11] had shown that the polylysine works when coupled with T-ag NLS (P101) works for importin binding and transfection efficiency.
Wednesday May 29th
Don't forget Dr. Jim Lee has his general lab meetings on 4pm Sunday afternoons.
Talked to Adair. Sent me a patent that summarizes much of the work. Funny enough, they are using 200 micron glass beads that one would use in road paint to help make them reflect light at night! So that actually makes the stationary phase of the HPLC a lot cheaper, since these are easier to obtain.
Found HPLC at Dr. Poirier and Dr. Ottesen's labs. I've suggested taking 200 micron glass beads from Polysciences Inc.[7], and packing them into a 5x55 mm column. The glass beads are cheap, and they would seem to be effective by Adair's suggested Van Der Waals chromatography in which the calcium phosphate will bind to the glass beads in a nonionic eluent of ethanol. If this is sound theory, then we're looking at a rather simple HPLC protocol. Need to hear back from Ottesen on how she sets up the HPLC and what sort of spectrophotomoter and such I could be using. 280 nm is the absorbance for Igepal CO-520 apparently.
Lab work Storage: The 16 groups using 7249 template, 2.5/4.5 days are stored in the fridge in a box with my name labelled on them. Ran the origami in gel. See OhioMOd2013:Methods/gel electrophoresis for more details. I stored pieces of gel that contain each band of fully folded origami away in the fridge. Uploaded picture of the gel below. Will note that lane 12 has a band that is slightly raised, that is the one I took out of the gel. All other gel pieces were taken from the second band observed. You'll notice that lanes 3-5 and lanes 12-15 have the best looking bands. There is significant agglomeration to the later bands where there was increased Magnesium concentrations.
Lane 1 DNA 1kb ladder. Lane 2 DNA phage 7249 control. Lanes 3-10 2.5 day folding, 12-26 magnesium conc. from left to right. Lanes 11-18 4.5 day folding, 12-26 magnesium conc. from left to right.
Friday May 31st
Not much to do for Thursday or Friday. Simply waiting on others.
- Maleimide-PEG-NH2 from PEG creative works.[8]
- Microemulsion notes: Igepal CO-520 is a nonionic surfactant. Read The colloidal stability of flouresecent calcium phosphosilicate nanoparticles. This paper is the best justification for using Igepal, and using Citrate as the dispersion. Citrate serves as a highly charged small molecule effective for dispersion. Paper discusses whether the particles will irreversibly agglomerate with drying.
- Read the DLVO theory. DIscusses the distribution of charges in ionic solutions. Also discuses double layers forces to explain phenomena found in the colloidal sciences.
- Use this for DNA mass/molar calculator
- [9]
- So if I use 20 nM template concentration for my folding, with 100 ul final volume, then assuming 14 kb structure size at the end, and assuming 100% folding efficiency, then there is 9.09 ug of origami total, yeech, we can get reporter plasmids for 10 mg. I'd like a 100 μg, but it looks like I'm going to be using a smaller range for loading. Maybe something like 0.1, 0.5, 1, 2 μg for every 1 μg of nanoparticle. I can up the concentration by not bothering with magnesium screen and only use 16 magnesium concentration.
- Since 3x10-3 Molar phosphate concentration and 500x100-3 Molar calcium for the microemulsion method, then phosphate will be a limiting factor by no more than 1:1 molar ratio, unless we get monomer crystallization.
- Should probably first measure mass of calcium phosphate nanoparticles without DNA loading.
Monday 6/3
Still waiting on others. Here's a picture of the nuclear pore complex taken by Dr. Martin Goldberg from Durham University.
Left image: Cytosol side of the membrane. Composed of the cytoplasmic ring, of eight 50 nm long cytoplasmic filaments, with a gap of 70 nm. Right image: Nucleus side of the membrane. Composed of nuclear filaments joining to create a basket-like ring with a gap of 30-50 nm. The central pore itself has a gap of 45-50 nm. The nucleoporin is composed of around 50 different nucleoporins, which occur in multiples of eight.
It's such a good picture I went ahead and made a page simply for the nuclear pore complex.
Tuesday 6/4
Well at least the order was approved, still waiting for delivery for materials.
Composed paper asking donation/collaboration from CLL group. Will probably send to Dr. Byrd with Chris Lucas as cc.
Will continue working on Adobe illustrator and on the website.
Wednesday 6/5
Performed crunch-n-squeeze purification on the gels I had cut last week. Most of the lab does their purification soon after the cutting of the gel, but I see no problem with storing the origami in a gel for over a week. Added to protocol in methods.
Will do TEM grid Uranyl stain with Carl today, added protocol in methods. I stained purified lanes 4 and 5. Its stored in the top left cabinet with my name and information taped. 4 carbon grid was significantly damaged, so check hte periphery for that. lane 5 carbon grid was well done, but since it was the second grid performed there may still be more crystallization. Hopefully all goes well Friday. I just need one picture of the folded structure.
Literature Reivew: Went over Maitra's paper [12] in which she states calcium phosphate can be considered a third generation nanoparticle. Instead of Igepal they used Aerosol OT from sigma, and n-Hexane rather than cyclohexane. A good sign is that she only adds 2.94 μg of pDNA to a 25 ml AOT microemulsion of 70 μL 0.35 M phosphate, which was then added to another 25 ml AOT microemulsion containing 70 μL of 1.36 M calcium solution. They were mixed at 20 drops per minute with continuous stirring at 4 °C for 12 hrs. Then reaction is dissolved in absolute ethanol by vortexing. Solution was pelleted for half an hour at 8000 rpm at 4 °C. The pelleted nanoparticles were washed in ethanol three times, then disperesed in distilled water, and dialyzed overnight using 12 kD dialysis membrane bag.
To determine pDNA encaspulation efficiency, she ran 3 to 9 μg of DNA until distinct DNA bands were observed outside of the nanoparticles. Also ran zeta potential at every pH point. This is a pretty good paper to follow, and I may try to adapt this to Adair's protocols.
Also, I found a paper I hadn't seen before [10] recently published in Jan 2013. They also use the double reverse microemulsion approach using a different non-ionic surfactant ethylene glycol monobutyl ether. There microemulsion was combined for several minutes as well, and citrate was used as the dispersing agent for 10 minutes. THey then centrifuged at 14000 rpm for 15 minutes, and then washed with ethanol and PBS twice to remove residual ethanol. Then dispersed in PBS. Huh, maybe we can do away with HPLC for the time being.
They also measure loading efficiency by agarose gel elecrophoresis and ethidium bromide staining. They loaded with antisense oligonucleotides, so smaller loading. To make sure citrate is on surface, use characterization by the FTIR spectra. USe X-ray Diffraction to determine the phase.
Here's a dissertation paper on nonviral delivery[11]. Random notes from it: HA1, GALA, and melittin are peptides with well-known ph-sensitive endosome lysis. Luciferase is the most reliable indicator of gene transfer. Can use bioluminescent imaging to quantify the luciferase expression in vivo. BLI uses a camera to detect photon emission at 610 nm. There are little false-positive results. I could simply test to see luciferase in the liver.
John Wolff, Professor of Pediatrics and Medical Genetics, U of W-Madison: "Nuclear targeting is the grand problem facing the non-viral gene therapy community". Jesse Gelsinger first to die from adenovirus gene therapy. Can try using H1 linker histone to help wrap the DNA origami? Useful for delivery possibly. See: Peptide nucleic acid staples. Also, photo-chemical coupling of peptide to DNA.
Oh, and the items were finally shipped yesterday!
Thursday 6/6
Waiting on materials. Will try to further prepare materials prior to their arrival.
Friday 6/7
Did TEM images with Carl. Will upload quality pictures shortly. Will probably take some dimensioning as well. Structures seem well-folded with a few structures attaching to one another at the ends. This is possibly due to how the structure was edited by Dr. Castro for the possibility of of being filamentous.
I also started another DNA folding for bulk production. Used the same folding excel sheets as last time on Friday two weeks ago. Will run at 16 mM MgCl2 and 2.5 day thermal ramp. Will use 20 nM scaffold and 50 μL total volume for each PCR tube. Will run two of these sets for 16 total, and 800 μL of around 20 nM origami. This suggests a 78.99 μg yield assuming ~100% folding. For my initial reaction runs next week, I can use a small range of 0.1, 0.5, 1, 2 μg of DNA for 4 different reactions, thus using 14.4 μg.
Here are the images taken. 1st picture forgot to add captions to image. There are some structures that are sitting vertically with the hollow end visible. This is somewhat suprising and is rarely seen in origami structures, so a lucky find!
Monday 6/10
Finished bulk production of structures, they're sitting in 4°C currently.
Will be meeting with Dr. Poirier with Dr. Castro today, will be discussing use of the HPLC. Currently I feel that I should explore other purification whether by pelleting and evaporation or by other means before I try to adapt to an expensive and complicated HPLC method.
Stock Solutions
|Molar Mass
|Measure Out
|110.98 g/mol
|0.5549 g in 50 ml
|1x10-1 M
|141.96 g/mol
|0.425 g in 50 ml
|122.06 g/mol
|0.05 g in 50 ml
|8.2x10-3 M
|294.10 g/mol
|0.0147 g in 50 ml
|1x10-3 M
I had the materials arrived today. Did an initial reaction today as a trial. I created the stock solutions using ddH2O and filling up the falcon tubes up to 50 ml. Then sonicated the calcium, phosphate, and metasilicate in the sonicator for 10 minutes. Mixed by vortexing.
- Added 4.06 ml of Igepal in 9.94 ml of cyclohexane to two 50 ml beakers.
- Began stirring, then added the calcium, phosphate, and metasilicate per protocol. No DNA origami was added. ALlowed to equilibrate for 30 minutes. The cyclohexane had evaporated significantly in this time.
- Mixed the microemulsions
- Quenched the reaction after two minutes with the citrate solution.
- Let the mixture continue for ten minutes with the citrate
- Ended microemulsion by adding 50 ml ethanol.
- Saw no particulates, so I transfered to a 50 ml centrifalcon tube(orange) and centrifuged for 10 minutes at 2,000 rpm. No pellet was formed. I do not believe I will be able to perform pellet purificaiton for the nanoparticles, since I believe that the origami and the nanoparticles will not pellet since they are so small.
Tuesday 6/11
Need to rethink the CPNP microemulsion protocol. Will start with just aqueous solutions.
- Trial 2 (50 ml falcon tube)
- Same as trial 1, used bulb pipette to mix the two microemulsions. Stored in a centrifalcon tube.
- Trial 3
- Made aq solution particles with no citrate present. Added 65 μL of the stock solutions to a 1.5 tube, and 1 ml of ddH20. Vortex. Will wait to see any visible particulate formation.
- Trial 4
- Same as trial 3 but added citrate after mixing reaction for 8 minutes. Should image.
- Trial 5
- Added 65 μL of the stock solutions, 50 μL of the origami non-purified after folding, 420 μL of ddH2O, and NO citrate was added.
- Trial 6
- To a 15 ml falcon tube with ~9ml of ddH2O, I added 100 μL of each stock solution (0.1M Calcium, 0.06M phosphate, 0.08 M silicate, plus 50 μL origami. After 2 minutes, 250 μL citrate was added.
Trial 5 showed significant aggolomeration of the DNA and calcium phosphate, so much so that it was cloudy and visible. It's somewhat nice to see, but I wonder if it's just the DNA agglomerating due to the high salt concentration or the calcium phosphate forming as well.
Trial 6 shows no visible particulates. Maybe it's working with nanoparticles, maybe it's too dilute.
Therefore, there is a shift in strategy: Focus on Aqueous reactions For tomorrow: Three groups of different stoichometry, three groups of DNA loading.
Wednesday 6/12
Will be meeting with Yun tomorrow at Nanotech West. Let's try to make a lot of samples. May want to include 1 ml of the microemulsion in cyclohexane. Let's try 994 ul of cyclohexane, 406 ul of igepal, and add 6.5 ul each of the calcium and 125 ul of the diluted phosphate/silicate.
Sample Group 1, aqueous: Created 9 groups of origami/CPNPs. Added DNA first to tube, then calcium followed by mix of phosphate and silicate for 1 ml total. Mixed for 1 minute at RT by vortex. Quenched with citrate for 1 minute. Sonicated for 15 minutes.
|quench 1 min
|0.5nM origami
|6.25 mM Ca, 3.75 mM PO4, 492 μM SiO3
|6.25 mM Ca, 375 μM PO4, 49.2 μM SiO3
|6.25 mM Ca, 37.5 μM PO4, 4.92 μM SiO3
|225μM citrate
|0.5 nM origami
|625 μM Ca, 375 μM PO4, 49.2 μM SiO3
|625 μM Ca, 37.5 μM PO4, 4.92 μM SiO3
|625 μM Ca, 3.75 μM PO4, 0.492 μM SiO3
|22.5μM citrate
|0.05 nM origami
|625 μM Ca, 375 μM PO4, 49.2 μM SiO3
|625 μM Ca, 37.5 μM PO4, 4.92 μM SiO3
|625 μM Ca, 3.75 μM PO4, 0.492 μM SiO3
|22.5μM citrate
Ran samples of these down the ethidium bromide gel. See lanes below.
|7249 pDNA
|0.5nM DNA/6.25nM Ca control
|0.5nM DNA/3.74 nM PO4 control
|0.5nM DNA/6.25nM Ca/10:6
|0.5nM DNA/6.25nM Ca/100:6
|0.5nM DNA/6.25nM Ca/1000:6
|0.5nM DNA/625μM Ca/10:6
|0.5nM DNA/625μM Ca/100:6
|0.5nM DNA/625μM Ca/1000:6
|0.05nM DNA/625μM Ca/10:6
|0.05nM DNA/625μM Ca/100:6
|0.05nM DNA/625μM Ca/1000:6
As one can see, lanes 14-15 of the 0.05 nM DNA are not visible due to the too dilute DNA. Lanes 7-9 seems to have the least free DNA, and lanes 11-13 have more visible bands and thus more free DNA. I suppose I'll take Lanes 7 and 9 on carbon film and see if I can see them on TEM.
It doesn't seem like this was a good assay. Hopefully DLS/Zetasizer will be more enlightening tomorrow on how the particles are.
Thursday 6/13
Will be going to zetasizer/dynamic light scattering training with Yun Wu today at 4:30pm. For the zetasizer I will be bringing specifically be a sample of a microemulsion that was in a 15 ml falcon tube: 3 ml of Igepal, 7 ml of cyclohexane, with 395 ul of water, 23 ul each of stock calcium, phosphate, and silicon, amounting to 464 ul of aqueous solution total. 80 ul of citrate was then added and the falcon tube was revolved for around 1.5 hours. This will be to measure the stability of the microemulsion.
For the dynamic light scatterer I will be measuring the tubes that were done yesterday at varying calcium/phosphate ratios and DNA loading. I will have sonicated these tubes again for 10 minutes prior to measurement, this will be the third time these particles were sonicated.
Finally, I will bring tubes of calcium phosphate nanoparticles that were synthesized as 6.25 mM calcium and 3.74 mM phosphate with and without citrate. This will be to see if we can measure a change in the zeta potential of the nanoparticles to confirm citrate binding.
Edit: Never mind, the training was rescheduled for Tuesday next week.
Friday 6/14
Started a 2.5 day folding for more bulk DNA. For one of the PCR strips I accidentally added 20ul of scaffolding to the first two tubes, and the 6th and 8th tubes have a little less scaffold as a result. These tubes were marked. Otherwise, the folding should be finished on Monday.
Here's a recent video from Adair's Keystone speech this year: [12]
Monday 6/17
- Purified bulk DNA from last week by PEG centrifugation. Pooled into 4 groups of 200 ul volume for the origami, added 200 ul of 15% PEG, and centrifuged for 30 minutes at 14.7k RPM. I then resuspended the pelleted origami/PEG in 200 ul of 1x FOB and 10 mM MgCl2.
- Will be trying to schedule AFM as well at nanotech west. Will need to think of ways to prepare the nanoparticles. Here's a good pdf on sample prep and AFM[13]
- Also prepared the stock solutions of calcium and phosphate again, they had a pH of around 10, which is somewhat surprising on how basic the solutions are by themselves. I hope this isn't affecting the DNA, but at least we definitely know now that the solutions are basic enough for CPNP synthesis. The phosphate and metasilicate solutions were combined together into one stock solution.
Tuesday 6/18
- Degassed water for one hour in the sonicator.
- Trial #1 - did microemulsion 10 ml in volume. used new solutions of calcium/phosphate/silicate. Reacted for 2 minutes. Citrate for 10 minutes. 4 ml of ethanol added. centrifuged 8,000 rpm for 30 minutes. Washed with ethanol twice. Redispersed in 1 ml of water. The pellet looked suspiciously gel-like, which is not the sort of pellet I've gotten from aqueous reactions only. I suspect it's mostly Igepal precipitate, but the pellet was rather small as well. I'll run a second trial, and if it does the same thing then I'll try using 50 ml falcon tubes, so that I can add signicantly more ethanol.
- Trial #2 - did microemulsion 10 ml in volume. new solutions were used plus 25 ul of the PEG purified bulk DNA was added. I equilibrated the phosphate/silicate/DNA for 30 minutes by rotary machine then added the calcium. reacted for 2 minutes, and then added the citrate. Quenched with 4 ml of ethanol. Continued for 10 minutes. Then centrifuged 8,000 as before. Problem: When I washed with ethanol, the pellet washed away with it. There was significantly more precipitate this time, and they definitely looked like Igepal precipitate to me. Which is perturbing...
- Trial#3 - did another microemulsion, but did not quench it with alcohol. I got good precipitate from it! I washed it three times with hexane, and then dried. It resuspended well in water too, with cloudiness at 500 ul. No DNA origami involved.
- Trial #4 - I replicated trial #3. Ran reaction for around 2 minutes, citrate 10 minutes, 8,000 rpm for 30 minutes. Hexane washed pellet. Dried for 10 minutes. Resuspended in 1 ml. This was a good concentration, not as cloudy as trial #3. GREAT!
- Finally, I performed four 1 ml aqueous reactions of CPNP with and without DNA origami, and with or without citrate dispersion. Mixtures were 712 ul of ddH2O, 62.5 ul calcium, 125 ul of PO4/SiO3, and 225 ul of citrate. They were reacted for around 2 minutes, and citrate was then added.
Here are the zetasizer results. I'll upload the DLS data tomorrow.
Trial #3 No origami, CPNP by microemulsion. Zetasizer after 5 rounds.
|Zeta Potential(mV)
|Rel Residual
|Std Error
Trial #4 +origami, CPNP by microemulsion. Zetasizer after 5 rounds.
|Zeta Potential(mV)
|Rel Residual
|Std Error
Aqueous CPNP, no microemulsion, +DNA but no citrate. Zetasizer after 5 rounds.
|Zeta Potential(mV)
|Rel Residual
|Std Error
^ This is good, what we should be seeing. Without citrate, the CPNPs should have a more positive zeta potential. The microemulsion CPNPs also demonsrate a negative zeta potential that is less than -30 mV, which is a good sign for dispersion! I now need to tighten up the protocol to gain more monodispersed particles, and I also need to effectively demonstrate the zeta potential changing with and without citrate!
Here's a good paper on how to measure nanoparticles by AFM: [14]
Wednesday June 19th
Apparently, the blue polystyrene falcon tubes are not compatible with my microemulsions! I added cyclohexane, Igepal, and water...but the microemulsion was cloudy throughout the reaction with very bad precipitation after centrifugation. I've been using the more expensive polyethylene centrifuge falcon tubes in the past, which have always had great clear micreomulsions.
Ran four different CPNP microemulsion groups today. With and without citrate, with and without DNA origami. The emulsions are a little bigger now, I'm using a falcon tube each for microemulsion A and B, and then mixing them together in a 50 ml falcon tube. So the reactions are about twice as large now, but now I'm adding equilibrated microemulsions A and B together, rather than adding the calcium or phopshate directly into the microemulsion. Hopefully this will lead to better equilibrated reaction conditions during the actual mixing, and I'll see better monodispersed particles.
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- van Gaal EV, Oosting RS, van Eijk R, Bakowska M, Feyen D, Kok RJ, Hennink WE, Crommelin DJ, and Mastrobattista E. DNA nuclear targeting sequences for non-viral gene delivery. Pharm Res. 2011 Jul;28(7):1707-22. DOI:10.1007/s11095-011-0407-8 |
- Ritter W, Plank C, Lausier J, Rudolph C, Zink D, Reinhardt D, and Rosenecker J. A novel transfecting peptide comprising a tetrameric nuclear localization sequence. J Mol Med (Berl). 2003 Nov;81(11):708-17. DOI:10.1007/s00109-003-0483-2 |
- Sebestyén MG, Ludtke JJ, Bassik MC, Zhang G, Budker V, Lukhtanov EA, Hagstrom JE, and Wolff JA. DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA. Nat Biotechnol. 1998 Jan;16(1):80-5. DOI:10.1038/nbt0198-80 |
- Chen X, Kube DM, Cooper MJ, and Davis PB. Cell surface nucleolin serves as receptor for DNA nanoparticles composed of pegylated polylysine and DNA. Mol Ther. 2008 Feb;16(2):333-42. DOI:10.1038/sj.mt.6300365 |
- Liu G, Li D, Pasumarthy MK, Kowalczyk TH, Gedeon CR, Hyatt SL, Payne JM, Miller TJ, Brunovskis P, Fink TL, Muhammad O, Moen RC, Hanson RW, and Cooper MJ. Nanoparticles of compacted DNA transfect postmitotic cells. J Biol Chem. 2003 Aug 29;278(35):32578-86. DOI:10.1074/jbc.M305776200 |
- van Steenis JH, van Maarseveen EM, Verbaan FJ, Verrijk R, Crommelin DJ, Storm G, and Hennink WE. Preparation and characterization of folate-targeted pEG-coated pDMAEMA-based polyplexes. J Control Release. 2003 Feb 21;87(1-3):167-76.
- Motskin M, Wright DM, Muller K, Kyle N, Gard TG, Porter AE, and Skepper JN. Hydroxyapatite nano and microparticles: correlation of particle properties with cytotoxicity and biostability. Biomaterials. 2009 Jul;30(19):3307-17. DOI:10.1016/j.biomaterials.2009.02.044 |
- Chan CK, Senden T, and Jans DA. Supramolecular structure and nuclear targeting efficiency determine the enhancement of transfection by modified polylysines. Gene Ther. 2000 Oct;7(19):1690-7. DOI:10.1038/sj.gt.3301275 |
- Bisht S, Bhakta G, Mitra S, and Maitra A. pDNA loaded calcium phosphate nanoparticles: highly efficient non-viral vector for gene delivery. Int J Pharm. 2005 Jan 6;288(1):157-68. DOI:10.1016/j.ijpharm.2004.07.035 |


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NetWellness is a global, community service providing quality, unbiased health information from our partners. NetWellness is commercial-free and does not accept advertising.
Wednesday, September 9, 2015
How Can I Relieve the Asthma Cough in My 5 Yr
help with asthma cough when inhaler is not working
If a cough is not relieved by using a rescue inhaler (albuterol or xopenex), there are 3 possible reasons:1) The medicine isn't actually being inhaled correctly. (The inhaler is empty of medication and is just spraying propellant, or the way the child is inhaling the medicine isn't actually getting it to his/her lungs).2) The asthma flare triggering the cough no longer is just due to airway spasm (which rescue medicines treat) but has worsened to the point of airway swelling and mucus plugging. (Steroid medicine is needed - soon - to fix this.)3) The cough isn't from asthma, after all.Make sure the inhaler has not expired and has not been used for more puffs than the label says it contains. Make sure your child is inhaling the medicine correctly. (At 5 years of age, he/she probably needs a mask style spacer to do this, and should be taking 6-7 breaths per puff.) If these issues are not the problem, then he/she needs to see the doctor to help figure out if the problem is #2 or #3.
Elizabeth D Allen, MD
Clinical Associate Professor of Pediatrics
College of Medicine
The Ohio State University


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Season 0, Episode 37
Amazing Appetizers
It's a first-course feast, as Guy Fieri digs into amazing appetizers. From Miami to Kansas to the Pacific Northwest, he's grabbing the bomb tots, jalapeno poppers, unique fritters, and even Rocky Mountain oysters. Plus, grape leaves and wicked wings in New England, while things get spicy in New York with atomic fried mac and cheese. Get ready to keep starting things off with this sharable one-hour special.
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Leander ISD is coordinating with the five schools impacted by the city of Austin's water boil notice to ensure students and staff stay safe. More info: www.leanderisd.org/lisdalerts
Leander ISD's Board of Trustees (school board) sets the policies by which LISD operates. Much public school policy is set by state and federal law and listed within the Texas Education Code. These "Legal" policies and a set of corresponding "Local" policies make up the Leander ISD Policy Manual, available online for all parents and community members.
Board Policy Manual On-line
Student Handbooks, Code of Conduct and Dress Code
Electronic Communication and Data Management - Acceptable Use Policy
Check Policy
Checks written to the school or any school organization must include on the front of the check the driver's license number and a valid telephone number. Post-dated and temporary checks are not accepted.
In the event that a check written to any Leander ISD campus, club, or organization is returned unpaid by a bank, Leander ISD or it's agent will redeposit your check electronically. Additionally, Leander ISD may electronically collect a returned check fee of $30.00 plus applicable sales tax. This fee is in addition to any fees your bank may charge. The use of a check for payment is your ACKNOWLEDGEMENT AND ACCEPTANCE of this policy and its terms.
Engage, inspire, achieve for lifelong success.


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posted by Tony Steidler-Dennison on Thu 10th Jul 2008 16:37 UTC
The Linux desktop has come a long, long way, but there are still times when I have to use the command line. (I am a hardcore user, after all.) But even though I'm used to typing, spending hours upon hours with my fingers at the keyboard, I still grow tired of typing the same commands over and over. To reduce that tedium, I always add aliases to my .bashrc file.


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18K gold and Original steel, or as customer request, smooth luster, don't fade for long time, no allergic
Jewelry Main Material
Stainless Steel
Products Feature
No allergy and no rust and nickel Free
In stock size, or Customized
Customer's logo is available
Over 10 years' experience
Order Type
Accept Mixed Order
Payment Terms
Paypal/bank/Western union/Secure Payment
Delivery Time
Within 24-72 Hours After Payment,mainly depend on the quantity.
Baicheng Jewelry has a wide varieties, attractive design and fashion style. The products include pedant, earring, ring, bracelet, necklace, anklet, bangle and garment accessories. The raw materials and electroplating raw materials are certified by SGS, the global authoritative certifying agency. The products comply with European and American environmental protection standard, and are sold well in the world.
Why choose BAI CHENG Jewelry
1. Our company was established in 2010 as a industry and trade integration company. we engaged in stainless steel jewelry business for 6 years and specialized in producing all kinds of stainless steel jewelry,Have professional designer team,Welcome to visit our factory at any time!
2. We alway execute the quality control over the whole production process and offer the factory direct price.
3. The newest styles will be recommended to you periodically,we can also provide customized service according to your specifications, sketches,ideas or samples.
4. The trial order or small order are welcomed.
5. The best after-sales service
Step 1: Pls contact us online by Trademanger or send us inquiry by email
Step 2: We will reply you in 24 hours
Step 3: After you confirmed order,we will make invoice for your confirm.
Step 4: After we received payment,we will arrange start production for you.
Step 5: Once order finished and shipped we will email you Tracking # to follow it.
Step 6: We also pay more attention to our after-sales service, if there is something wrong with the products during shipping, Take photos or videos of the problems and send to us,we will try our best to help you , but not including the man-made destroy. And we'll be patient to accept your advice about our products and then develop it.
Q1:Have any concern about our MOQ?
The MOQ is 50-100 sets/pieces depends on the jewelry type because of we are an industry and trade integration company and have our own designer team and professional technical operations staff,the factory could't start the production program if the order is not big enough.
Q2:How about the customized product?Any customized order is warmly welcome.Send us pictures and specification,we will check and make the sample as per your request,and we need charge the customize sample's fee,It will be returned when you place an order.In addition,if you want to change the specification,color or the material,contact us no hesitate.
Q3:Any discount possible if I place an order?We have different price ranges (discounts) based on different order quantities.Please contact our sales girl for detailed price information.
Q4:How about your shipping ? We usually ship by famous courier such as Fedex, UPS, DHL, TNT, EMS ect.
Q5:How long is the delivery time? Different products and quantities have different procedures and different timing. The production lead time is 20-35 days. We promise that we would try our best to finish all of your orders within the shortest time.For more information, please contact us directly.
Q6:How about the sample policy? Sometimes a free sample instock is possible and you just freight cost, or give us your courier No.
Q7:More information about payment terms?We uesd Paypal,T/T and Western Union ,Alibaba secure payment,and some others payment you asked is also accept.
Q8:How about the after-sales service?We also pay more attention to our after-sales service, if there is something wrong with the products during shipping,Take photos or videos of the problems and send to us, we will try our best to help you , but not including the man-made destroy.And we'll be patient to accept your advice about our products and then develop it.


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If you're a Sonos user making the move to BlackBerry 10, you won't have to worry about controlling your system from your new device. The folks at Tiggit have let us know that they are hard at work on their Tiggit Controller app for BlackBerry 10. With the app, Sonos users can control their system from a BlackBerry 10 device and have access to streaming services, media controls and much more.
Kevin is a new Sonos owner and has become a huge fan of the system (I know because he hasn't stopped talking about it for two weeks). He also motivated me to make my own low budget system using my BlackBerry PlayBook and a few BlackBerry Music Gateways in my house. Maybe when BlackBerry 10 comes out I'll convince him to buy me a Sonos setup so I can try this out for myself :-)


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LONDON (AP) — British medical technology company Smith & Nephew PLC said Wednesday it is buying Texas-based Healthpoint Biotherapeutics for $782 million.
Healthpoint Biotherapeutics, founded in 1992, is a privately owned company that focuses on acute, chronic and burn-related wound care.
Smith & Nephew CEO Olivier Bohuon said the acquisition of Healthpoint would strengthen his company's position in bioactive wound care.
"It brings material revenues from a fast growing product range, an attractive pipeline, and commercial and R&D capabilities upon which we will build," he said.
Smith & Nephew shares were down 0.8 percent at 653.5 pence in midday trading in London.
"Whilst the acquisition is broadly about strategy, expanding S&N's wound business, it takes S&N into riskier areas, with future performance linked to the outcome of clinical trials," said Sebastien Jantet, analyst at Investec Securities.
Travis Baugh, Healthpoint's president and chief operating officer, said the acquisition "will enable us to accelerate many of the initiatives we wanted to pursue and will provide development and promotional opportunities to our employees that would not have been available to them had we stayed an independent company."
Healthpoint, which has 460 employees, will continue to be based in Fort Worth.


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Fifty-one-year old Sarah Mupakati does not remember the last time she ate sadza, a cooked cornmeal. She opens her one-room tent which she shares with her five grown-up children - the corner that serves as her pantry is empty. It has been like that for the past month. The little she has been getting from barter trading is used up that same day. It is all too easy to understand her relief as she joins the queue to receive her first food rations from the World Food Program (WFP).
The food situation at Chingwizi Resettlement Camp had become dire, but thanks to a US$773,181 rapid response grant from the UN Central Emergency Response Fund (CERF), WFP was able to provide food for four months to some 15,625 people displaced by flooding in Masvingo province located in the south-east of Zimbabwe.
Risk of flooding
Due to above-average rainfall across Masvingo between mid-January and early February 2014, river levels in the catchment area of the Tokwe Mukorsi dam rose rapidly, causing widespread flooding which prompted the declaration of a State of Disaster and the issue of a Government Appeal. Some 20,000 people were reported to be at high risk of flooding in the basin area upstream from the dam.
While there were initial fears of the dam bursting (which would have placed an additional 40,000 people at risk), repairs to the dam were undertaken and water levels dropped as the rains let up. It is estimated that more than a thousand hectares of farmland were inundated, leading to a loss of more than 700 tonnes of crops. As part of the dam construction project, a phased relocation plan was drawn up to resettle people from areas of risk by October 2015.
Four months
At the start of the floods in early February, only 600 households had been relocated. The authorities subsequently relocated the remaining 3,125 households (15,625 people) to a designated resettlement camp at the Chingwizi section of Nuanetsi ranch some 120 kms away. Those moved were either affected or deemed at risk. Until June, food assistance was provided by well-wishers and charitable organisations but, as resources become more limited, food availability become a pressing issue. It was at this point that WFP stepped in.
"We're very grateful to CERF for this valuable support," says WFP Country Director Sory Ouane. "We just hope the government or other donors will be able to come on board after the four months."
For Sarah, it is not certain when she will have a place to call home but, for now, she is just happy to be safe and have food for her family.


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The Gulf of Mexico off the coast of Texas hosts commercial and recreational fishing and diving and plays an important role in local economies. It provides vital habitat for marine life of all types and links us with the ocean through an interconnected environment. Artificial reefs enhance this marine environment by providing additional habitat needed by marine life. The Texas Parks and Wildlife Artificial Reef Program, funded through industry partnerships and grants, creates and enhances this critical marine habitat to benefit Texans and the natural environment.
The Gulf accounts for 80% of all shrimp harvested,
62% of all oysters harvested and more than 1.4 billion pounds
of annual seafood production.
More than 140 petroleum platforms—with more on the way—have found new purpose as marine habitat in the Texas Artificial Reef Program.
Texas boasts 66 artificial reef sites ranging from 5 to 100 miles from shore in the Gulf of Mexico—that's 3,440 acres of prime fishing and diving adventure.
Seven reef sites within nine nautical miles of shore serve as accessible nearshore fishing and diving opportunities.
Red snapper, the most popular game fish in Texas Gulf waters, thrive around artificial reef sites. Scientific divers see red snapper at TPWD artificial reef sites during four of every ten visits to these locations.
With a few exceptions, the floor of the Gulf of Mexico is flat and bare except for artificial reef sites. Nearly 200 marine fish species have been seen on these complex, stable, and durable habitats among artificial reef structures.
Sixteen of 23 U.S. coastal states (or 70 percent) maintain artificial reef programs.
The Texas Clipper ship reef off South Padre Island generates more than $1 million for the local economy from anglers and $1.4–$2 million from divers. Anglers spend on average $460 per fishing trip, while divers spend upwards of $2,000 per dive.
Thirteen ships have been intentionally sunk as part of the Texas Artificial Reef Program, the largest being the USTS Texas Clipper. She's 473 feet long—that's 1.5 times the length of a football field.


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Election season for La Plata Electric Association will be competitive with one challenger already calling the cooperatives leadership too conservative and out of touch with its membership.
During a campaign event at Carver Brewing Co. on Thursday, Jack D. Turner called himself a centrist type who would be more reflective of this community.
Turner said Britt Bassett, who won election to the LPEA board last year on a green platform, elbowed him into the race.
One-third of the 12-member board is elected annually to three-year terms.
A native of Durango and nephew of former Durango Municipal Judge David Turner, the 50-year-old candidate teaches geology and sociology at Southwest Community College but has worked with rural utilities as a representative for the Skokomish Tribe in Washington state.
Turner would face Herb Brodsky, who lost his re-election last year to renewable-energy candidate Heather Erb in a squeaker. Brodsky later was reappointed to the board to serve out the term of Pam Patton, who was appointed to the Public Utilities Commission last June by Gov. John Hickenlooper.
Turner and Brodsky are competing for a District 4 (northern La Plata County) seat. Brodsky said it was too soon to comment.
I will be interested to hear what (Turner) has to say, Brodsky said in a telephone interview.
The member-owned cooperative primarily provides power for La Plata and Archuleta counties.
So far, incumbents Brodsky, Lindon Stewart for District 1 (Archuleta County) and Tom Compton for District 2 (southwest La Plata County) have returned petitions.
Harry Goff, the incumbent for District 3 (Durango), is not seeking re-election.
Michael Rendon, a former mayor of Durango, has said hes for running for the Durango seat.
Petitions with 15 signatures are due by Wednesday to qualify for the LPEA ballot, which would be mailed to members April 19 and must be returned by May 10.


text:
Press release 212/2017
6 December 2017
Finland conveyed its grave concern about the situation in the Korean peninsula and strong condemnation of the latest ballistic missile test conducted by North Korea on 29 November. Director General Kimmo Lähdevirta of the Finnish Ministry for Foreign Affairs delivered the message to Ambassador Kang Yong Dok in Helsinki on 6th December, 2017. The Finnish government urged the DPRK government to comply with the UN Security Council resolutions and immediately cease further provocations aggravating the situation. Finland also urged the DPRK to engage in a meaningful dialogue with its neighbours and the international community.
Inquiries: Arto Haapea, Deputy Director (East Asia), Unit for Eastern Asia and Oceania, tel. +358 50 566 0435.
The Foreign Ministry's email addresses are in the format email@example.com.


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Unfinished Business: Paid Family Leave in California and the Future of U.S. Work-Family Policy
(Cornell University Press, 2013)
This book documents the history and impact of California's paid family leave program, which began in 2004 and was the first of its kind in the United States. Drawing on original data from fieldwork and surveys of employers, workers, and the larger California adult population, the authors analyze in detail the effect of the state's landmark paid family leave on employers and workers. They also explore the implications of California's experience for the national debate about work-family policies. While business interests argue that paid family leave and similar programs impose significant costs and burdens on employers, the majority of California employers surveyed reported that its impact on productivity, profitability, and performance was negligible or positive. The authors lay out lessons for advocates in other states and localities. Ruth Milkman is a professor of sociology at the Graduate Center.
Purchase this book
Submitted on: OCT 31, 2013
Category: Faculty Books | Sociology


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WASHINGTON (MarketWatch) — Decisions made by the former head of bankrupt MF Global sealed the firm's fate and led to the investment firm's collapse and over $1 billion in missing customer funds, according to details released Wednesday from a report and investigation conducted by a House Financial Services Committee panel.
According to the panel's preliminary report, the firm's former CEO, Jon Corzine, a former co-chairman of Goldman Sachs GS, +0.69% who later became a senator and governor of New Jersey, created an authoritarian atmosphere. In addition, the report said that Corzine insulated the firm's trading activities from the company's normal risk management review process.
The report comes after New York-based MF Global filed for bankruptcy roughly a year ago on Oct., 31, 2011, leading to $1.6 billion in missing customer funds.
The firm failed after disclosing sizable exposure to derivatives in European sovereign debt and after questions emerged about whether the futures brokerage firm tapped into customer-segregated accounts for its own proprietary trading. Read about how after MF Global, lawmakers debate future reform
Details revealed Wednesday say that Corzine failed to maintain systems and controls necessary to protect customer funds.It added that Corzine "dramatically" changed MF Global's business model without "fully understanding the risks."
Specifically, the report notes that Corzine's decision to focus much of the firm's investments in European sovereign debt was a problem because they carried "enormous default and liquidity risks." It added that he failed to develop a corporate strategy for managing the risks.
The report details were released by the House Financial Services Committee's subcommittee on investigations, which is headed by Rep. Randy Neugebauer, Republican of Texas. Neugebauer said in a statement that the report is essentially an autopsy of how MF Global collapsed and what can be done to prevent future problems.
The panel said in a statement that its investigation of MF Global involved three hearings, more than 50 witness interviews and the review of more than 243,000 documents.
The full report is expected to include recommendations that Congress should consider legislation to impose civil liabilities on directors of certain types of investment firms who sign financial statements or authorize transfers from customer accounts. Read about how the Commodity Futures Trading Commission floated post MF Global protections
Such legislation could "restore investor confidence in the derivatives markets," the report said.
Corzine resigned from MF Global on Nov. 4, 2011, roughly 20 months after becoming the firm's CEO.
The full report is scheduled to be released by the committee on Thursday.