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These are momentous times. An alarming picture of a future under IMF control was painted on RTE's Primetime, the country's equivalent of Newsnight, last night.
The programme reported from Latvia, Greece and Hungary, which have accepted bailouts in the past.
But despite Ireland's deep banking problems and the lack of confidence in our country's managers, Ireland is not a complete basket case.
People are not rioting on the streets, they are not looting supermarkets and last time I checked, people had shoes on their feet.
Exports are up and multinationals like Google still flock to the country for its low corporate tax of 12.5% and educated workforce.
And if you have enough gloom and doom for the week, take a look at this: the Irish Times today has an ingenuous antidote, with its arts editor Shane Hegarty launching a campaign on Twitter to find the #greatthingsaboutireland.
But with finance minister Brian Lenihan confirming that IMF officials are arriving in the department of finance tomorrow (if they are not already there as has long been rumoured), it's worth examining the experience of living with the IMF.
The former economics minister of Argentina, Martin Lousteau, has been in Ireland to witness events unfold first hand. He was in power between 2007 and 2008, but in 2002 was an adviser in the country's central bank where the IMF had a permanent presence.
He tells me: "Argentina was a big big mess. We defaulted, we had four presidents in one week, people were hungry, it was a big big mess, we had riots in Buenos Aires, 26 people were killed.
"We had 20% unemployment – it jumped from 14% to 18% in three years. Poverty jumped to 50%.
"There was a 13% cut in all public sector wages.
"In some places bartering kept the economy going and we had NGOs feeding mothers and children.
"We forget now. That was in 2001/2002 and we say 'that was the hand that God dealt us'."
The IMF's buzzword at the time, says Lousteau, was "expansionary fiscal contraction" – in other words, you cut so hard, the only way back is up.
"You show that you have the guts to cut down expenditure so far and you will have growth."
The IMF was so reviled, he says, that "children who were naughty were told I'll get the IMF after you" by parents. And when the present government was elected in 2007, it played on those fears with an advertising campaign promising no child would ever know again what the three letters IMF signify.
"It's very difficult to predict what's going to happen in a mess. In Argentina, a lot impacted from the panic," Lousteau says.
"Between January and November 2001,we lost almost a fourth of our deposit base. Everybody would take the money and take it out of the bank. In October, the government said don't worry. A month later, on December 3, all the deposits were frozen. We called it 'the little fence' for current accounts and 'the big fence' for long-term deposits. In the case of the current accounts you couldn't take your deposit out for a whole year – you could only take out 1,000 a month."
Argentina defaulted on its debts in December 2001 and the depression that set in didn't lift for four years, when the country restructured its debt and paid bond-holders 30 cents in the dollar.
Listening to fellow economists at the Kilkenomics festival, Lousteau saw parallels with Ireland but not in the order of play. "We defaulted first, then had a depression. It's the reverse for Ireland. You are in a depression and then …" he said.
Although a social nightmare, he says (with the benefit of hindsight) that the catastrophe was good for the country. It paid a high price, but it was so bad it "washed away" the underlying causes of the problem – corrupt politics.
"This huge nuclear bomb, this Armageddon, it adjusted the public sector wages, realigned the exchange rate and reduced the debt bill by 70%.
"It washed away the causes of the problem."
But he says Ireland is in a dangerous place – if it is not careful it will not address the causes of the problem and will store up a new banking crisis for the future.
"The truth is when these events get too big, nobody can predict what's going to happen and you know it's going to be chaotic. So the bad thing is political leaders don't want chaos, so what he does is postpone the inevitable and then it's more chaotic."
Head of economics at the University of Galway, Professor John McHale, told RTE's Primetime last night that the IMF is not as brutal as it was in the 1990s when it "decimated" social systems.
But the programme's report on Latvia was far from comforting. It joined the EU in 2004 but its economy went into reverse in 2008. Unemployment rocketed to 25% (Ireland's is at 13.5%) and the economy shrank more than 25%. The IMF came to the rescue with a €7.5bn loan.
Public sector wages were cut by 20% immediately. The number of staff in government departments were cut by between 10% and 15%, hospitals were closed, university fees went up and a property tax was introduced.
Today Brian Lenihan was at pains to say, as was the governor of the central bank last week, that the IMF/EU mission won't do anything different to what the Irish government already plans in terms of cuts of €15bn over the next four years.
A poll tax – a property tax of €80 a month – has already been mooted and seems inevitable as do more cuts in public sector pay and social welfare. So the devil will be in the detail.


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subset: commoncrawl


text: Wilfred Marube has an experience of over a decade in training, public relations and corporate communications. He is a member of the Public Relations Society of Kenya and Marketing society of Kenya. Wilfred is a graduate of B Ed- English and Literature (Moi University), M.A.- Language and communication(Egerton University), Diploma in Public Relations Management ( KIM), Executive Diploma in Marketing ( MSK) and finalizing his PhD in communication studies( Moi University) with focus on development and participatory communication. Mr. Marube is currently the head of public relations and communication at the City Council of Nairobi.


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Dispute Resolution Framework
The Telecommunications Law, By-Law and licenses set out processes for resolving different types of disputes that arise in the telecommunications sector. The Communications Regulatory Authority (CRA) manages dispute resolution on issues related to interconnection and access , for disputes between the service providers, and disputes between service providers and their stakeholders.
In February 2015, the Communications Regulatory Authority (CRA) published the Dispute Resolution Procedures replacing the Dispute Resolution Rules issued in 2010. The Procedures are intended to produce a more effective mechanism in handling disputes between Access Seekers and/ or Providers. It is also aimed at providing clarity and transparency around the entire process, commencing from the initial submission of a dispute until the determination of a final decision.
- Broadly, the Procedures set out:
- The form and manner in which a dispute may be referred to CRA
- The information that CRA expects to receive during the course of the proceedings
- The process for investigation and conclusion of a dispute
- The remedies available to CRA
The full text of the Dispute Resolution Procedures is available at this link.


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EXTREME DUTY TROLLEY WHEELS, for LightRail 3.0, 3.5 and 4.0.
A suggested upgrade item for moving multiple lights +/or heavier loads such as heavier LED, Plasma or any light with an internal ballast weighing 35 pounds or more..
They are engineered with steel on steel ball bearing with a nylon over molded tire for smooth quiet operation. Rated up to 60 lbs per set and come with a lifetime warranty!
— Ships from HF: OR, CA, CO, MI, PA USA.


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Our Delivery Charges England, Wales & Southern Scotland Orders under £50 £3.50 Usually within 2-5 days Orders over £50 FREE Usually within 2-5 days Nominated day or Saturday delivery* £6.00 Select your day, on orders placed before 14:00 Click & Collect Furniture FREE FREE Same day at our Norwich stores on orders placed before 14:00 Expected within 5 weeks Scottish Highlands, Channel Islands and Northern Ireland View postcodes in this bracket All orders £5.00 Usually within 2-5 days Nominated day or Saturday delivery Furniture N/A N/A Unavailable to these postcodes Overseas (any non-UK address) At present we do not deliver overseas. Gift Cards, Book Tokens and Event Tickets £2.25 Furniture Small and flat packed items will be sent via Parcelforce, Direct Distribution or via our delivery vans. Large items will be sent by our specialist 2 man delivery partners. The delivery company will contact you within 7 days to organise delivery of your items. 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You have the right to cancel this contract within 14 days without giving any reason. To cancel your order prior to dispatch please telephone the Internet Order Help Desk on 01603 697150 or e-mail firstname.lastname@example.com. Click here for our cancellation policy Refunds Refunds will be against the card used to make the original payment following inspection of the returned goods, normally with in 7 working days. Return documentation is included with the goods when dispatched to you. We recommend that a verifiable method of return delivery is used, as proof of posting cannot be taken as proof of receipt. Please see our Returns Policy for items which are exempt from the right to cancel If goods are not returned and received by us in perfect condition we reserve the right to withhold or refuse any refund, pending enquiry, and/or make only a part refund Returning Furniture Please be aware that furniture purchased in store at Jarrold is exempt from the above terms. 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text:
Board of Directors meeting agendas are typically over-crowded, and cybersecurity often falls to the cutting room floor if not kept top of mind with the right influencers [year-round].
The question, is how do you get this topic on the agenda and have it properly interpreted into messaging that resonates with their thinking?
In this video Hadi Hosn, SecureWorks Head of Security Strategy and GRC Consulting in EMEA, gives the #1 tip to getting security on the board agenda and understanding the requirements of their thinking.


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Veteran Links (Texas)
Request Texas National Guard Military Records
Texas National Guard State Ribbons Order Form
Request Texas National Guard License Plates
Texas Military Department Jobs
Texas Military Department Transition & Employement Assistance
Texas State Veterans Cemeteries
Veteran Designation on Driver's License
Veteran Links
Veteran Benefits Administration
Request Military Awards From Active Component Service
Veterans On-line Application (VONAPP)
Department of Veterans Affairs
Federal Benefits for Veterans and Dependants
US Court of Appeals for Veteran Claims
Department of Veterans Affairs Cemeteries
Vocational Rehabilitation and Employment Services
Drivers License Fee Waived For Certain Disabled Veterans
Resources for Veterans in the USA
Retiree Links
Request Texas National Guard Military Records
Texas National Guard State Ribbons Order Form
Request Texas National Guard License Plates
Find a Military ID Cards location near you
Soldier for Life (Retirement, Education, Employment, He and Wellness, Benefits)
Tricare Retiree Dental (Changing at the end of December 2018)
Request Military Awards From Active Component Service
Afterburner Newsletter - Air Force Retirees
Army Echoes Newsletter - Army Retirees
Request Active Duty Military Records
Guide to Survivor Benefits
This guide provides answers to many questions about the Survivor Benefit Plan/ Reserve Component Survivor Benefit Plan (SBP/RCSBP), and the Retired Serviceman's Family Protection Plan (RSFPP), other annuity programs, and Voluntary Separation Incentive (VSI) payments to beneficiaries. If an annuitant has specific questions concerning the annuity, please write to the address listed below, or call our toll-free number: 1-800-321-1080. Overseas customers may call commercial (216) 522-5955. Our toll-free fax number is 1-800-982-8459. To expedite a prompt response, please include the annuitant's name and Social Security Number (SSN) as well as the member's name and SSN on all correspondence
Survivor Benefit Plan (SBP) Automatic Coverage and Options
There appears to be some confusion regarding the application of the automatic coverage provision under the SBP and the SBP options available to the military retiree after retirement. The information below serves as guidance on these issues.
Automatic Coverage & Options Available After Retirement
Member with Beneficiaries at Time of Retirement: Automatic SBP coverage applies only at retirement. The automatic coverage provision does not apply to any situation which may occur afterthe member's retirement date. Automatic coverage does apply to any member who, on the date of retirement, 1) did not make a valid SBP election, that is, an election for full coverage, less than full coverage, or declined coverage; and, 2) has a qualified spouse and/or child beneficiary.
Member with No Beneficiaries at Time of Retirement: A member who has no eligible beneficiaries at retirement is still required to complete the SBP election which is part of the form DD2656. There is a block on the form to indicate the member has no eligible dependents. Automatic coverage does not apply in this case since the member has no eligible beneficiaries. However, if such member later acquires a spouse and/or child he may elect SBP coverage. The member would have one year from the date of acquisition to make an election to cover the newly acquired beneficiary. An election may be submitted by letter provided the intent to elect is clear and all information required for an election is provided. A letter which only notifies us that the member has married does not constitute an election of SBP.
Again, there is no automatic coverage for any spouse and/or children newly acquired after retirement. In order to provide SBP coverage in this situation, the member must submit a valid positive written election which is received within the election period prescribed by law.
Member Who Had SBP Coverage for Spouse and/or Child at Retirement: A member fitting this situation who later loses a beneficiary will have SBP coverage suspended during the period in which there is no eligible beneficiary. Should the member subsequently acquire a new beneficiary in the same class as was originally provided at retirement, the original coverage is reinstated for the newly acquired beneficiary. Reinstatement of coverage is not the same as automatic coverage. The reinstatement will happen when that person qualifies as an eligible beneficiary. For a new spouse, it would be on the first anniversary date, or sooner, if a child is born of the marriage. For a new child, it would be the date of birth (if a natural child) or the date of acquisition (if other than a natural child).
It should be noted that upon acquisition of a new spouse the member does have the option to either increase the level of coverage (base amount), or to terminate coverage. Such a request must be made by the member before the first anniversary date of the marriage.
Keep Retired Pay Records Current
It is very important to review your retired pay account information and ensure that it is current; things such as correct mailing address, marital status and designated beneficiaries, to name a few.
Failure to update retired pay records when the retiree married, divorced, remarried, became widowed, or acquired a child could result in Survivor Benefit Plan (SBP) benefits being denied or life insurance paid to an unintended recipient.
In the case of divorce with SBP coverage involved, there is a one year notification period to DFAS, to include a copy of the court decree, for benefits to stay in force for a former spouse.
Make sure your spouse or designated beneficiaries know what benefits to expect or not and are prepared by maintaining a file of information that will be needed upon your death.
Some account information can be updated yourself through myPay. Other changes and notifications should be mailed or faxed to:
Defense Finance and Accounting Service
U.S. Military Retirement Pay
P.O. Box 7130
London, KY 40742-7130
Fax: (800) 469-6559
Defense Finance and Accounting Service
U.S. Military Annuitant Pay
P.O. Box 7131
London, KY 40742-7131
Fax: (800) 982- 8459
Please include your Social Security number or that of deceased member and sign the request.


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As a little girl, the world to me was only as far south as Boston, far east as the Toronto Beaches, as far north as Pacific Mall and as far west as the Junction. Why? Well Boston is where my mum's siblings reside – often visiting once a year, the beaches is where my sisters often took me every summer, what Asian did not know go to Pacific Mall and every Saturday was our weekly visits to Gong Gong and Po Po's (Grandpa & Grandma) in the Junction.
As a little girl, my parents were ageless and smart. They knew almost everything about life and I swear, they were psychic. In the mornings, I use to wake up and sneak into the kitchen to eat chocolate chip cookies. My stairs are notorious for creaking, however, I found a clever way to walk on it which would stabilize the wooden boards beneath. I would step on the edges ever so lightly where the support is firmest. This I mastered as my mum couldn't even hear it as she sewed away directly below. But the part that was always the hardest, was opening the pantry door. As soon as she heard that door open ever so slightly, "Ah Fongggg" will come traveling up the stairs. And all I can think to myself is, 'Damn it, not again.'
Fong is my name in Chinese.
Although my parents were ageless and smart, my grandparents on the other hand were wise and immortal. They were old, but immortal. They would live forever in that giant house down the street from Annette. It would always be a pleasure coming home to see their shoes at the front door only to be welcomed by the best two people in the whole entire world.
Now Doctor, I know you save people and solve things. And I know you're not real. But if you were, I'd make it my one wish to go back to when I was a little girl, when death was non-existence, when the word healthy was the definition of every person in the whole entire world (even if it was only from the Beaches to the Junction) and when I could always look forward to their shoes aligned neatly at the front of my home.
Yours truly,


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Driver-less Cars of the Future Will Be So Safe That We Can Make Them Out of Glass
At the recent London Design Festival 2014, a local designer named Dominic Wilcoxrevealed his beautiful conceptual design of the future. As driverless technologies are well underway in the R&D process, Wilcox took the thought a step further implying that cars of the future will be so safe that we can make them out of glass! He presented a beautiful life-size concept car design made of colorful stained glass. He called his creation, which was commissioned by Dezeen and MINI Frontiers for their exhibition on the future of mobility, Stained-glass Driverless Sleeper Car of the Future. You can take a nap while your beautiful, computer-operated car takes you where you need to go!
Wilcox explains:
In the future it will be safer to drive in a driverless car than it will in a manual car, therefore we don't need the protection systems that are built into contemporary cars. We can just have a shell of any design.
These lovely pictures were taken by photographer Sylvain Deleu. See this innovative design live in London at The Sorting Office until September 21st, 2014. Check out more work by Dominic Wilcox on his website.


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Remarks: Sen. Joe Lieberman
Remarks As Prepared for Delivery: U.S. Sen. Joe Lieberman:
Thank you for that warm welcome. I am honored to be here.
We meet tonight in the wake of a terrible storm that has hit the Gulf Coast but that hurts all of us, because we are all members of our larger American family.
At times like this, we set aside all that divides us, and we come together to help our fellow citizens in need.
What matters is certainly not whether we are Democrats or Republicans, but that we are all Americans.
The truth is, it shouldn't take a hurricane to bring us together like this.
Every day, across our country, millions of our fellow citizens are facing huge problems.
They are worried about their homes, their jobs, and their businesses; they are worried about the outrageous cost of gas and of health insurance; and they are worried about the threats from our enemies abroad.
But when they look to Washington, all too often they do not see their leaders coming together to tackle these problems.
Instead they see Democrats and Republicans fighting each other, rather than fighting for the American people.
Our founding fathers foresaw the danger of this kind of senseless partisanship. George Washington himself -- in his Farewell Address to our country -- warned that the "spirit of party" is "the worst enemy" of our democracy and "enfeebles" our government's ability to do its job.
George Washington was absolutely right. The sad truth is -- today we are living through his worst nightmare, in the capital city that bears his name.
And that brings me directly to why I am here tonight. What, after all, is a Democrat like me doing at a Republican convention like this?
The answer is simple.
I'm here to support John McCain because country matters more than party.
I'm here tonight because John McCain is the best choice to bring our country together and lead our country forward.
I'm here because John McCain's whole life testifies to a great truth: being a Democrat or a Republican is important.
But it is not more important than being an American.
Both presidential candidates this year talk about changing the culture of Washington, about breaking through the partisan gridlock and special interests that are poisoning our politics.
But only one of them has actually done it.
Only one leader has shown the courage and the capability to rise above the smallness of our politics to get big things done for our country and our people.
And that leader is John McCain!
John understands that it shouldn't take a natural disaster like Hurricane Gustav to get us to take off our partisan blinders and work together to get things done.
It shouldn't take a natural disaster to teach us that the American people don't care much if you have an "R" or a "D" after your name.
What they care about is, are we solving the problems they are up against every day?
What you can expect from John McCain as President is precisely what he has done this week: which is to put country first. That is the code by which he has lived his entire life, and that is the code he will carry with him into the White House.
I have personally seen John, over and over again, bring people together from both parties to tackle our toughest problems we face --to reform our campaign finance, lobbying and ethics laws, to create the 9/11 Commission and pass its critical national security reforms, and to end the partisan paralysis over judicial confirmations.
My Democratic friends know all about John's record of independence and accomplishment.
Maybe that's why some of them are spending so much time and so much money trying to convince voters that John McCain is someone else.
I'm here, as a Democrat myself, to tell you: Don't be fooled.
God only made one John McCain, and he is his own man.
If John McCain was just another go-along partisan politician, he never would have taken on corrupt Republican lobbyists, or big corporations that were cheating the American people, or powerful colleagues in Congress who were wasting taxpayer money.
But he did!
If John McCain was just another go-along partisan politician, he never would have led the fight to fix our broken immigration system or to do something about global warming.
But he did!
As a matter of fact, if John McCain is just another partisan Republican, then I'm Michael Moore's favorite Democrat.
And I'm not.
Senator Obama is a gifted and eloquent young man who can do great things for our country in the years ahead. But eloquence is no substitute for a record -- not in these tough times.
In the Senate he has not reached across party lines to get anything significant done, nor has he been willing to take on powerful interest groups in the Democratic Party.
Contrast that to John McCain's record, or the record of the last Democratic President, Bill Clinton, who stood up to some of those same Democratic interest groups and worked with Republicans to get important things done like welfare reform, free trade agreements, and a balanced budget.
Governor Sarah Palin, like John McCain, is a reformer who has taken on the special interests and reached across party lines. She is a leader we can count on to help John shake up Washington.
That's why the McCain-Palin ticket is the real ticket for change this year.
The Washington bureaucrats and power brokers can't build a pen strong enough to hold these two mavericks.
And together, you can count on John McCain and Sarah Palin to fight for America and to fight for you! And that's what our country needs most right now.
What we need most is not more party unity in America but more national unity!
Especially at a time of war, we need a President we can count on to fight for what's right for our country -- not only when it is easy, but when it is hard.
When others were silent, John McCain had the judgment to sound the alarm about the mistakes we were making in Iraq. When others wanted to retreat in defeat from the field of battle, when Barack Obama was voting to cut off funding for our troops on the ground,
John McCain had the courage to stand against the tide of public opinion and support the surge, and because of that, today, our troops are at last beginning to come home, not in failure, but in honor!
Before I conclude, I ask the indulgence of those in this hall tonight, as I want to speak directly to my fellow Democrats and Independents who are watching.
I know many of you are angry and frustrated by our government and our politics and for good reason.
You may be thinking of voting for John McCain but you're not sure. Some of you have never voted for a Republican before and in an ordinary election, you probably wouldn't.
But this is no ordinary election, because these are not ordinary times, and John McCain is no ordinary candidate. You may not agree with John McCain on every issue.
But you can always count on him to be straight with you about where he stands, and to stand for what he thinks is right regardless of politics.
As President, you can count on John McCain to be a restless reformer, who will clean up Washington and get our government working again for you!
So tonight, I ask you whether you are an Independent, a Reagan Democrat or a Clinton Democrat, or just a Democrat: This year, when you vote for President, vote for the person you believe is best for the country, not for the party you happen to belong to.
Vote for the leader who, since the age of 17, when he raised his hand and took an oath to defend and protect our Constitution, has always put our country first.
So, let's come together to make a great American patriot our next great President!
Web Politics Editor
September 2, 2008; 10:42 PM ET
Categories: Conventions , Primary Source , Republican Party
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what i know about Palin:
bans books and threatens to fire the librarian (just made the NY times - no trash tabloid there!)
requires loyalty oaths
says NO to global warming - it simply is not a problem we have to address
mayor of SMALL town (folks i am a hick and my town was 5 times bigger!) now $27 MILLION in debt
KEPT and SPENT the bridge to nowhere money - pure hypocracy - NOT maverick
better than Bush in ignoring the environment
NO Foreign POlicy experience - oh wait she lives close to russia (thanks cindy mcsame)
member of AIP that wants to leave the US
vetos alternative energy plans - yes that is right in line w/ mcbush/mssame
been out of the country ONCE
few weeks ago did not know what the VP did for crying out loud
laughs out loud w/ radio host6 while he calls another woman a "bi--h" and cancer when the woman is a cancer survivor! very very compassionate there
mcsame melanoma 4 times wants this a heart beat away - not for this woman!
Posted by: lets talk palin | September 3, 2008 4:49 PM | Report abuse
Palin's church says "Jews deserve to be victims of terrorism."
Palin's church leader (Brickner) also described terrorist attacks on Israelis as "God's judgment of unbelief of Jews who haven't embraced Christianity." Palin was in church that day.
Posted by: Anonymous | September 3, 2008 2:38 PM | Report abuse
Im not a lib, I'm a GOP member. Lieberman is not someone I would ever trust.
Posted by: Anonymous | September 3, 2008 2:35 PM | Report abuse
This is great. Liberals being liberals.
Can't you see the love? The compassion? Oh yes, and the tolerance.
Any Jewish folks in the audience? See the love the left has for you?
Any gay folks? See how your fellow leftists REALLY feel about you?
Keep talking libs. It's very revealing...
Posted by: Observer | September 3, 2008 1:37 PM | Report abuse
Liarbearmanpig should return to the pack of wolves that raised him and McCain!
Posted by: Anonymous | September 3, 2008 12:51 PM | Report abuse
King Judas is still in politics!?
Posted by: Anonymous | September 3, 2008 12:49 PM | Report abuse
I am glad the Americans can remember the last eight years because after watching last night you would think the Republicans think we are stupid. John McCain has no impact on the Nation outside of his military expereince. That is sad because it has been 40 years since that time.
Posted by: concerned | September 3, 2008 9:04 AM | Report abuse
Here is my question to you: Should the 18 yr old kid that got Gov. Palin underage daughter pregnant be charged, with a crime like other teen in this Country? There are teen in this Country currently serving jail time, for commenting the same offense of having sex with an underage minor that this kid is being accused of. Why isn't the law being followed in this case? Is there a double standard for a VP candidate?
Posted by: Dan | September 3, 2008 8:25 AM | Report abuse
To begin, I love most of the comments above. Last night was a whole lot of sucking up!!!!! I give my respect to McCain heads down for servicing our country... I look up to him for it, and would like to do the same one day. But I do not believe that everyone who serves a country has the right to be a president. Everyone who spoke last evening spoke of his service, which is not a problem, but that not what being a president is all about... I sat with my family to watch the last night, and watched in disbelieve... I can't believe what the speakers were saying. Most of them believe that the eco is good. But I have 3 brothers, and 3 sisters, and my parents brought all of us here to have the American promise. We were doing fine until about 7 years ago, when my mother got pegent with my little brother... Lil Johnny was born 3 months early due to complications with my mother's blood pressure, and doctors telling her the baby was growing when it was dead flesh growing, and not the baby. Anyhow, our lives have changed, my mother was told by the government that she could no longer work, and if she did the government would not take care of my little brother. The only way that she can work is if she divorces my dad.
Just like Obama said in his speech, it's not their fault they just don't get it; they just don't understand what's going on.
Joe, you are a trader of parties, but its ok. We will change, and I have hope that Obama will do so. I say thank God now that you did not win when you run, because all of us would have been confused in the middle of your term. God works in ways will never understand, and I know he will work now.
Posted by: Mary-Jane Williams | September 3, 2008 8:16 AM | Report abuse
natasha, you just posted an angry comment from someone on the left making fun of the Republicans for condemning the "angry left."
Posted by: David | September 3, 2008 12:55 AM | Report abuse
Tonight was like watching "Night of the Living Dead" with all the geezers out there gumming away their praise and full of senile hostility condemning the "angry left." WOW.
Posted by: natasha | September 3, 2008 12:15 AM | Report abuse
Lieberman proved that he is first and foremost an Israeli than he can be anything from a neoconservative to a gay lover of john McCain[or McSame]. I would rephrase what he said to independents reagan-democrats I want to say to u that i'm for abortion and same-sex marriage and loves caucusing with democrats I want your children's blood america's blood for israel the reason we went to war on Iraq and I support it even based on a lie that there were'nt any WMD gov.Palin what a woman she is althoug i disagree what everything she believes in but Israel first sorry country first
Posted by: asd. joe | September 3, 2008 12:14 AM | Report abuse
Typical traitorous heeb who cares what some freak lefty thinks. Lieberman just wants us to kill off more towelheds so the jews can take over but here in america he wants to let the beans and the wetbacks in the country which is ruining everything. THANK GOD for the ICE raids, I say beat them, imprison them , then send them back
Posted by: Frank | September 2, 2008 11:52 PM | Report abuse
He should have just spent his 20 minutes licking a picture of McCain. Man-crush.
Posted by: DemoDevil | September 2, 2008 11:52 PM | Report abuse
Didn't he just copy the main tenants of Obamas speech when it come to talking about America being one??
Personally I prefer to vote for Obama as he is actually leading in this area rather than one old man turning up to his fellow pensioners party
Posted by: JO L | September 2, 2008 11:49 PM | Report abuse
HEY LEWISSHAPIRO : Idiot Jew Boy it's not spelled racsium it's racism you moron. How about you & fag lover liebermann go take your jewy arses our of here
Posted by: Greg | September 2, 2008 11:49 PM | Report abuse
Here is the real problem
Its not Mcain nor the new VP on the block, its the old Cheney, Rove, and Rummy in the background
They really messed this country up and will still be there,
Sad thing is I don't trust Obamas inexpereince, possiby Bidens
Posted by: Larry | September 2, 2008 11:47 PM | Report abuse
You folks just need to bend over and take where you like it: Right in the pooper! First, you Liberals campaign against Joe Lieberman, because he supported The Surge. He lost his primary, back in '06. Heck, even Hilary and Schumer campaigned against him. He ran in a heavily Democratic State and won as an Independent. He still votes 88% of the time with the Democrats! If someone in this Senate votes 88% of the time, with George W., you Liberal Trash would label that Senator a war criminal, pig, facist, nazi, torturer, etc. (look at Turban Dick Durbin!). So, you've got an Independent Senator that votes with you 88% of the time. What has happened since Mormon Harry and Mafia Nancy took over in 2007? Absolutely Nothing! They promised, "Peace in our time." It's the least productive US CONGRESS SINCE 1861, folks. At least those legislators had an excuse! Now what? You've got a Jewish Senator that leans left and all of the sudden, it's screwing up your buzz, because he's not towing your Party Line! Do yourself a favor: Put down your pipe for a while and put away the KY Jelly! You Loved It When Lincoln Chaffee jumped ship, didn't you! Well, bend over and enjoy it! I'm sure you know how!
Posted by: mikewoolsey | September 2, 2008 11:42 PM | Report abuse
WOW you all are really fair and balanced ;)...I don't like any of the politicians but I do vote and I do not vote party lines. I vote for who is the best for our country. Now i still have not figured that out yet but I respect what Joe L had to say. He may have lost his primary but he still won the election and to me that says something about him. As far as Israel is concerned...there is a reason why no politician will touch that comment - figure it out. The Republican Jesus comment- even Christians will tell you that God makes that judgment not humans or Jesus. Turncoat??? 1st the dems didn't support him in his primary and 2nd they didn't support him because he sided with the right on some issues. Reality is that this is a blow to the democratic party and Obama's run for president but it may not be the best thing for McCain either. This election comes down to who is more into their party and Joe proves it. The republicans are more republican and would not vote for any Democrats but the Democrats are not as strong and will end up voting for a republican. Not to mention their is still racsium in this country and the reality is that the media is to PC to talk about it. My vote is for Powell he should be our president. The problem is he is to smart to run.
Posted by: LewisShapiro | September 2, 2008 11:35 PM | Report abuse
Wow. Just... wow. What engenders such hatred among you folks? Don't you all realize that this irrational us-v-them knee-jerk jingoism is the result of mass manipulation?
You have allowed yourselves to be distorted by partisan rhetoric, as surely as any member of the John Birch Society ever was. Get a handle on yourselves! Your rage is foolish and counterproductive.
The world and its complications are subtle -- as intelligent people (I hope), you should be embarrassed by the superficiality and foolishness of the comments preceding.
Joe Lieberman is a man of conviction, experience and political skill -- one of the few of his generation who has stood tall, alongside some of the great political lights of his time. I disagree with many of his policy ideas, but he was the final reason I voted Gore in 2000, which was tough for me.
Men like him and Ted Kennedy and some few of their generation transcend the short-term thinking of partisan power plays to represent true statesmanship, and I salute them.
Posted by: Ned | September 2, 2008 11:33 PM | Report abuse
Lieberman says to Democrats and independents:
"You may not agree with John McCain on every issue. But you can always count on him to be straight with you about where he stands, and to stand for what he thinks is right regardless of politics."
OK, let's say that's true. But if I disagree with him on most issues, while I agree with the other major-party candidate on most issues, should that count for less than the fact that McCain will always be straight with me? And what if I feel that the other major party candidate is also being straight with me also?
Posted by: yrral | September 2, 2008 11:31 PM | Report abuse
Who cares, Joe is still a FAG lover and a baby killer and WETBACK aider so he can screw off
Posted by: Tim in Atlanta | September 2, 2008 11:30 PM | Report abuse
Lieberman has officially become a republican, evidently, a 100% republican. (with his Palin endorsement.
Posted by: I hate them | September 2, 2008 11:28 PM | Report abuse
Wait, I thought Joe wasn't a Democrat any longer so how can he ask what a Dem like him is doing there? What a fraud.
No it's Joseph Lieberman (Opportunist - Connecticut)
Posted by: A democrat | September 2, 2008 11:20 PM | Report abuse
What did McCain promise Lieberman? He certainly wasn't for "post-partisanship." Secretary of State maybe?
I wonder why Lieberman even bothered running against Bush in 2000. They're so much alike.
Posted by: aBigSAM | September 2, 2008 11:18 PM | Report abuse
Joe is a Democrat that I have always respected. And He knows John McCain. Joe is willing to take the heat to back McCain. It says a lot.
Posted by: Kevin | September 2, 2008 11:15 PM | Report abuse
These men, McCain, Lieberman, Thompson, Old Bush, and deceased Reagan, of course, are irrelevant. They are old men of another day. This is 2008!!!
Posted by: pkb | September 2, 2008 11:15 PM | Report abuse
Snatch up the McCain/Palin buttons because they are going to be rare.
Someone must have a McCain/Who? button by now.
Posted by: K Ackermann | September 2, 2008 11:13 PM | Report abuse
Lieberman is going to hell in gasoline drawers. His comments were thinly veiled but I heard them loud and clear. Disgusting!! "this is no regular election" and "McCain is no regular candidate" calling on voters that are struggling to vote for Obama to not "break the mold" and vote the same demographic. Disgusting, I did not want to believe he was a bigot and turncoat but he proved it tonight. Lieberman is an independent and should turn in his democratic creds. Loser!! And will there be an original thought in the whole convention this week? If I hear "executive experience, tough, commander of her national guard, stronger resume than obama (that makes me throw up in my mouth), she cannot begin to touch Obama in education or experience,Obama has more executive experience in running his campaign than Palin does in being a mayor and governor. Everyone gets on the mike and says the same things about Palin and McCain. Their plan is to steal the election not to earn the majority. Their mission is to divide the country and sqeak out a majority. It's been done twice before.
Posted by: janinej1 | September 2, 2008 11:13 PM | Report abuse
What a noob.
Posted by: ClariTee | September 2, 2008 11:13 PM | Report abuse
Joe Liberman is a fizzle. He's the spare tire that is flat, you carry around because everyone is supposed to have a spare tire.
Posted by: Uoughtano | September 2, 2008 11:10 PM | Report abuse
McCain and Lieberman put country first. Unfortunately that country is Israel.
Posted by: James | September 2, 2008 11:05 PM | Report abuse
Now we know their strategy, pretend they are not republicans.
Posted by: Anonymous | September 2, 2008 11:04 PM | Report abuse
Hey Holy Joe LIEbertoad! - Sarah Palin thinks you're going to Hell ... unless you come to her Republican Jesus.
As a Jew that kind of thinking bothers me. To say the least.
It's a disgrace that it apparently doesn't bother you, you little arse wipe
Posted by: Bush + Cheney = McCain | September 2, 2008 11:03 PM | Report abuse
I have been struggling in my decision to vote for Obama or McCain. Tonight Liererman made my decision for me. I could not bring myself to vote for anyone that would allow a turncoat to represent him.
Posted by: Mountaineer | September 2, 2008 11:02 PM | Report abuse
I'm listening to Lieberman live right now. Who is this guy? Really. I'm just shaking my head watching this weasel praise everything about John McCain and SARAH freakin' PALIN. Everything that he has believed in his entire career is directly opposed to these two, with the exception of the Iraq war. He's become a one-issue joke.
What are Lieberman's constituents thinking? He should have had the grace to simply stay out of the election.
Posted by: jyhume | September 2, 2008 11:00 PM | Report abuse
Hey Joe,
You're just bitter because you lost the Democratic primary in Connecticut.
Best Regards
just kidding, I hate you.
Posted by: Anonymous | September 2, 2008 10:59 PM | Report abuse
Will voters watch the Republicans with the same attention they gave Obama? Does Lieberman inspire anyone besides Republicans? And does the POW overload backfire for McCain? Questions...
Posted by: matt | September 2, 2008 10:50 PM | Report abuse
The comments to this entry are closed.


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| WMU News
Dawn M. Nelson, a former employee in the Grants and Contracts office at Western Michigan University, died Sept. 21 at home in Paw Paw, Michigan. She was 44.
Nelson joined the WMU staff in 1993 and left in August after 24 years of service to the University. She was a senior restricted fund accountant in Grants and Contracts, which primarily oversees the financial administration of externally funded grants and contracts received by WMU.
Although born in Kalamazoo, Nelson grew up in nearby Comstock and graduated from Comstock High School. She went on to double major at WMU, earning a bachelor's degree in social psychology and accounting.
Services have taken place.
The family obituary and message book are available at lifestorynet.com.
Memorial gifts may be made to the Breast Cancer Research Foundation.


text:
Drone technology provides agriculturists with a cost-effective method of infrastructure planning. In Nigeria it has accelerated the planning, design and construction of rice irrigation systems.
As the drone reappeared in the sky and lowered its altitude in an attempt to land, the research team's driver Richard, who had been volunteering to help out with the mission, ran towards the unpiloted plane in jubilation. 'You're welcome!' he said enthusiastically in both English and Hausa, the language that is spoken in northern Nigeria.
The growmoreX team of the London based company GMX Consultancy, which runs a drone-based farming application service, was in Nigeria to do a preliminary assessment for the development of a 3,000 hectares irrigated rice farm. The farm will be built on land that was acquired in a long term lease from the local government's irrigation authority. The aim of the project was to survey and map a total of 7,500 hectares in preparation of planning and building the irrigation infrastructure for the rice fields.
Although a manned aircraft could have done the job, it also would have cost a fortune. The alternative is unmanned aerial vehicle (UAV) technology. The project site was in a sparsely populated area, located approximately 75 kilometres from the town New Bussa, some 500 kilometres away from the capital Abuja with limited access to roads, electricity, clean water, and other amenities. Local livelihoods here are mainly based on small-scale agriculture. Crops are grown annually during the rainy season, and include sorghum, rice and beans. Tomatoes are grown during the dry season using pump-fed irrigation.
First flight
A fixed-wing UAV, which was imported directly from the US with assistance from a local project partner, was used for the first flight. It took a day to assemble it. That gave the team time to sort out technical hiccups and figure out how to use its automatic mission planning function. The activity attracted attention from local villagers, who had already been informed about the forthcoming agribusiness development.
When all the checks were completed, the team set the UAV's navigation system to 'automatic'. Then the UAV's propeller was turning and it was launched into the air, witnessed by a crowd of people who had gathered to watch the first flight. The mission had begun.
Although the UAV had made it into the air, it suddenly began to fly away instead of starting its pre-programmed mission – likely due to the direction of the wind. The team lost telemetry communication with the drone, and it was thought that the UAV had crashed.
Suddenly, the radio established a connection with the UAV again, and it finally began its automatic mapping mission. It took the UAV only a few minutes to reach the optimal surveying altitude of 150 metres above ground level. Once at this altitude, it began to fly in a specific pattern, shooting images automatically as it went.
Advance planning
After the UAV landed safely the camera was checked immediately. The photos looked sharp and beautiful. There were a lot of them: during the 55-minute flight, the drone took overlapping photos of nearly 300 hectares of land.
The UAV was able to fly for roughly four hours a day when the sun cast the fewest shadows. This meant that the team was able to map about 1,000 hectares in a single day. That is fast, especially if the harsh terrain and working conditions with high temperatures are considered. Estimations assume that it would have taken a professional surveyor working on foot about twenty days to cover the same area.
To operate an UAV requires advance planning. The researchers made sure no specific regulations barred the team from using the UAV. The local Emir, the village chief and a military airport located about 100 kilometres from the project site were informed of the plans to make use of an UAV. Fortunately, the local authorities welcomed the new technology. There was only one condition: the Emir insisted that we do a flyover of his village, so that his people could see both the drone and the pictures it would take.
The village flyover had an unexpected result. For the first time the team could establish exactly how many houses and dwellings there are in the village, thus enabling researchers to make a much better estimation of its population. This information will be very useful, because the research team is planning to hire local labour to build the rice farm and to run it.
The hypothesis was proved wrong
Wonderful as the village flyover was, the main objective was to begin planning the rice farm's irrigation infrastructure. For the preliminary investigation, the researchers needed to create a map at a scale of 1:2,000 (1 centimetre on the map represents 20 metres). With such a map the research team could make informed decisions on the best layout of the paddy fields, the irrigation and drainage systems.
Based on the limited information from previous visits to the site, it was hypothesised that it would have been able to lay out the rice fields as large, rectangular basins. Large earth moving and farming machinery would have been needed to build and cultivate those basins. Paddy fields for rice cultivation need careful water management as water levels impact weed and nutrient distribution. This meant that for every 100 metres, half a metre of soil at the top of the field had to be removed to raise its lower end during the levelling process.
However, the drone survey proved the hypothesis wrong. Although it was certainly true that parts of the project site were flat, most of the terrain was an undulating landscape.
The sloping terrain combined with a thin top soil layer led the team of researchers to radically change their designed hypothesis, away from large rectangular basins and towards long, narrow fields that would follow the terrain. But this change also meant that a very different irrigation system design was necessary.
Avoiding unnecessary costs
By using data required from UAV technology, agricultural planners can now easier avoid incorrect infrastructural planning. This information also makes it easier to organise the right procurement of machinery, avoiding unnecessary large upfront investments that can break a project if they are improperly planned.
Water is the deciding factor in Africa's rice self-sufficiency. Most rice cultivation is rain-fed in Africa. The lack of irrigation infrastructure is a major obstacle to increase rice production on the continent. Most of the existing systems are poorly designed, built, and maintained.
The good news is that UAV technology can potentially accelerate the planning, design and construction of Africa's irrigation infrastructure. As this project has shown, UAV technology could provide agriculturists with a cost-effective method of irrigation infrastructure planning.
And that is not all. After the farm planning stage, UAVs could be useful for farmers to estimate more accurately how much fertilizer and planting materials they will need during the growing season. Once crops have been planted, UAVs equipped with special sensors can monitor their growth.
With the help of agricultural UAVs, Africa can leapfrog into the quickly-advancing area of precision agriculture – just as African mobile phone companies bypassed traditional fixed line infrastructure to create an innovative mobile finance system.


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Friday, March 11 | 2:00 pm | SH 2509
On Friday, March 11, Transcriptions will host a seminar with HUMlab director, Patrik Svensson. He is pre-circulating a draft version of an article forthcoming in Digital Humanities Quarterly as the basis for the seminar; please email email@example.com for a copy of the article. Patrik has suggested that a general theme for the discussion might be "how to build/create/design for different kinds of institutional contexts/projects/programs."
Wine and light refreshments to follow.


text:
- Methodology article
- Open Access
Directed PCR-free engineering of highly repetitive DNA sequences
© Scior et al; licensee BioMed Central Ltd. 2011
- Received: 26 May 2011
- Accepted: 23 September 2011
- Published: 23 September 2011
Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products.
For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays.
Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.
- Repetitive Sequence
- Polyglutamine
- Magnesium Chloride
- Potassium Acetate
- Sumo Protease
Expansions of DNA repeat sequences are associated with many inherited neurodegenerative diseases [1–3]. One of the best-studied examples for a trinucleotide expansion disease is the neurological disorder Huntington's chorea, where the accumulation of CAG triplets within the first exon of the gene encoding the Huntingtin (Htt) protein leads to an elongated polyglutamine (Poly-Q) stretch in the polypeptide. It has been shown that more than 36 consecutive glutamine residues are pathogenic as they promote Htt aggregation into amyloid-like fibrils [4]. The translation product of the first exon of the htt gene was previously used to study the aggregation behavior of Poly-Q proteins in vitro [5]. In order to investigate the influence of the length of the Poly-Q stretch on aggregation kinetics, we wanted to clone reporter constructs containing defined numbers of glutamine residues. We developed a PCR-free cloning strategy allowing us to create repetitive DNA sequences encoding glutamine stretches of defined length. These sequences were used to generate improved constructs for filter retardation assays to study Huntingtin aggregation in vitro.
Several methods have been described for cloning long DNA repeat tracts. However, most of them include PCR-based amplification steps, generate imperfect repeats, or result in a pool of clones that differ in the number of repeats [6–13]. As a consequence, additional effort is required to identify and isolate clones with the desired length of nucleotide repeats. Therefore, we developed a simple multi-cycle cloning strategy using synthetic oligonucleotides and Type IIS restriction endonucleases to engineer highly repetitive DNA fragments. Our approach is PCR-free and generates exclusively clones carrying the desired length of repeat sequences in each step. Moreover, it is not only suitable to multiply the length of existing repetitive sequences but also to vary the number of inserted repeats in every elongation cycle. Finally, we can easily recombine constructs of the same or different repeat lengths to accelerate the construction of the desired number of repeats.
Results and discussion
The antiparallel Q-block oligonucleotides described above were commercially synthesized, annealed, and subcloned via the BsaI and SacI restriction sites into a vector containing a unique BsaI site (Figure 1B and 1C). The following elongation steps of the Poly-Q encoding sequence only require the double-stranded oligonucleotides treated with the appropriate enzymes as described above and a cloning vector without BsmBI sites. Hence, we performed the elongation cycles in the plasmid pMK1 from which all BsmBI sites were removed by site-directed mutagenesis. The initial introduction of the subcloned Q-block fragment into pMK1 resulted in the plasmid pMK1-Q11 (Figure 1C, upper panel). As a consequence, this vector now contains a single BsmBI site downstream of the glutamine-encoding triplets, which could be used for the further elongation of the Q-block (Figure 1C). Next, pMK1-Q11 was digested with the enzymes BsmBI and SacI. The resulting cohesive ends were compatible with the overhangs of the double-stranded Q-block oligonucleotides cut by BsaI and SacI (Figure 1C). Ligation of the insert with pMK1-Q11 gave rise to pMK1-Q20. Thereby a unique BsmBI site was re-introduced downstream of the elongated Poly-Q encoding region (Figure 1C). As the cohesive ends of the annealed and digested oligonucleotides are only compatible with the doubly-cut plasmid, multiple insertions were efficiently prevented. Using this strategy the directed elongation of the Q-block was achieved by repeated cycles of digestion and ligation.
Our approach is also suitable to increase the length of repetitive sequences by multiple insertions as described previously [13]. Thereby, simultaneous digestion of the annealed oligonucleotides or pMK1-Qn plasmids with both Type IIS restriction enzymes would result in Q-block fragments with compatible overhangs (Figure 1A). This would allow for ligation of multiple Q-blocks in a single step.
To study the aggregation kinetics of Poly-Q proteins in vitro, we applied a widely used filter retardation assay, which allows the detection and quantification of small amounts of Poly-Q containing aggregates [5]. This assay is based on the characteristic of Poly-Q fibrils to be insoluble in solutions containing 2% sodium dodecyl sulfate (SDS). Such fibrils are specifically retained on a cellulose-acetate filter, whereas the soluble monomeric species are denatured by SDS and filtered through the membrane. The captured aggregates can then be visualized by immunodetection. For such experiments fusion constructs were commonly used carrying a N-terminal glutathione S-transferase (GST) domain fused to the first exon of htt via a factor Xa cleavage site [5]. In these constructs the globular GST domain prevents aggregation of the monomers. However, aggregation can be induced by release of the GST moiety upon proteolytic factor Xa cleavage. Based on this principle we designed a new construct in which the first exon of the htt gene was fused to a SUMO domain (Saccharomyces cerevisiae Smt3) carrying an N-terminal His6-tag for affinity purification (Figure 6A). Importantly, the His6-SUMO domain keeps the purified Poly-Q containing fusion proteins soluble and allows for the controlled induction of aggregation by treatment with the Ulp1 protease (SUMO protease from S. cerevisiae), which cleaves behind the two conserved C-terminal glycine residues of folded SUMO [21].
Using the His6-SUMO fusion strategy the Poly-Q proteins can be easily affinity purified under denaturing conditions using silica-based Ni2+-matrices (Figure 6B). As Poly-Q proteins often tend to form insoluble inclusions when expressed in E. coli, we purified our constructs under denaturing conditions using 6 M guanidine hydrochloride and refolded them on the affinity matrix by slowly decreasing the concentration of the denaturant. The efficiency of the refolding process was monitored by digestion of the purified and soluble Poly-Q fusion proteins with Ulp1 (Figure 6C). Importantly, this protease does not recognize a specific amino acid sequence but rather the native structure of the SUMO-domain. Under our experimental conditions the purified fusion proteins were almost completely digested by Ulp1 within one minute, indicating that refolding of the constructs was successful. Therefore, we performed the filter retardation assay with the Poly-Q fusion proteins and induced aggregation by Ulp1 mediated cleavage. Figure 6D exemplifies the assay with our construct containing 47 glutamine residues. In the control reaction, where Ulp1 was omitted, no aggregation was observed over six hours beyond marginal background signals. By contrast, significant amounts of SDS-resistant Poly-Q fibrils were formed after one hour upon induction of SUMO cleavage. Accordingly, release of His6-SUMO from the Poly-Q constructs occurs much faster than fibril formation (Figure 6C and 6D). Thus, our His6-SUMO fusion strategy provides a useful tool to study the kinetics of Poly-Q aggregation in vitro.
As mentioned above, several other seamless cloning strategies were described previously, indicating the demand for such methods. However, our approach has several advantages compared to existing methods. Here we used synthetic oligonucleotides to overcome the limitations of PCR-based amplification of repetitive DNA sequences and combined this strategy with seamless cloning using Type IIS restriction enzymes. The usefulness of this method was demonstrated by means of two frequently occurring problems: the cloning of defined stretches of Poly-Q encoding sequences and the generation of polyadenine sequences for in vitro transcription of polyadenylated mRNA. We chose the latter application because we wanted to demonstrate that the technique is suitable for generating fully homogenous nucleotide sequences of defined length. Another important advantage is that our method is directed. This means that in each elongation cycle, the number of nucleotides added to the repetitive sequence can be determined precisely by simply designing the oligonucleotides accordingly. Importantly, a single product is obtained in each step. Other methods often generate a mixture of products due to multiple insertions. Consequently, products of the right length or sequence have to be selected or purified via agarose gels, which makes the procedure more complicated, time-consuming, and error-prone. As we did not observe multiple insertions and only rarely religations of the vector, this is not necessary using our technique. In addition, the protocol presented here is straightforward. One round of elongation can be done within one day, including restriction digestion of the vector, ligation, and transformation. Bacterial growth and sequencing requires approximately two further days. We obtained approximately 100-300 clones per transformation and in most cases it was sufficient to sequence a single clone. Moreover, combining pre-existing constructs can rapidly increase the length of the repetitive sequence (Figure 2 and 3). For example, once the method was established, the pMK1-constructs could be generated within three weeks including sequencing (Figure 3A). We did not encounter a limitation of the number of repeats for our purposes.
In general, our method is cheap because only the synthetic oligonucleotides have to be purchased for each application. We usually ordered 0.02 μmol of commercially synthesized, HPLC-purified oligonucleotides, respectively. The costs depend therefore on the list price per nucleotide, purity, scale, and length of the oligonucleotides. Once annealed and digested, the oligonucleotides can be used for all subsequent elongation rounds and do not have to be re-ordered. All other materials (e.g. restriction enzymes, agarose gels, competent cells, DNA ligase) are standard equipment for molecular cloning. It should be mentioned that in principle the strategy could be used also to assemble non-repetitive sequences. In this case, the DNA fragments could be amplified even by PCR with primers carrying the Type IIS and Type IIP restriction sites in the 5' overhang regions. Finally, it should be also possible to fuse different repetitive sequences in a directed and seamless manner (Figure 5).
In summary, our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning techniques. Besides their applications in basic research, as exemplified here, repetitive nucleotide sequences become increasingly important in biopolymer technology. As artificial proteins with unique physical properties such as elastomeric polypeptides and synthetic silk fibers often contain multiple amino acid repeats, new strategies are required that improve the cloning of synthetic genes for the production of protein-based polymeric materials in biological systems [22, 23]. The method presented here is both, cheap and fast, and can be easily adapted to produce any desired DNA sequences for a wide range of applications.
Cloning Methods
All cloning steps were performed according to standard protocols [24]. Restriction enzymes were named according to [25]. HPLC-purified synthetic Q-block (5'-ccagtgGGTCTCaCAGCAACAGCAACAGCAGCAGCAACAGCAGcagagacgGAG CTCgatc-3' and 5'-gatcGAGCTCcgtctctgCTGCTGTTGCTGCTGCTGTTGCTGTT GCTGtGAGACCcactgg-3') and poly(A) oligonucleotides A31 (5'-gccgAAGCTT aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaGAGACCcactgg-3' and 5'-ccagtgGGTCTCtttttttttttttttttttttttttt tttttAAGCTTcggc-3') and A32 (5'-ccagtgGGTCTCAaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaAGAGACG acagCTCGAGgccg-3' and 5'-cggcCTCGAGctgtCGTCTCTttttttttttttttttttttttttttttttTGAGACCcactgg-3') were obtained from Thermo Fisher Scientific. For annealing 10 pmol of the antiparallel oligonucleotides were mixed in a final volume of 100 μl. The reaction mixture was heated up to 95°C for 5 min, incubated for 10 min at 55°C, and finally cooled down to room temperature. The annealed oligonucleotides were digested with the respective restriction endonucleases (NEB) according to the manufacturers protocol. Next, the DNA was precipitated by addition of 1/10 volume 3 M sodium acetate pH 5.2, 2.5 volumes of 100% (v/v) ethanol (p.a.) and incubation at -20°C for at least 12 h. The DNA was recovered by centrifugation at 16000 × g for 30 min at 4°C and the DNA pellets were washed with 70% (v/v) ethanol. The DNA pellets were dried at room temperature and subsequently resuspended in 100 μl sterile water. Aliquots of the double-stranded digested oligonucleotides were stored at -20°C and used for the subsequent cloning cycles to elongate the repetitive sequences. Between 0.1 μl and 5 μl of the digested oligonucleotides were ligated with 30-70 ng linearized and dephosphorylated plasmid DNA and T4 DNA ligase (Fermentas) in a 20 μl reaction mixture according the manufacturers instructions. The ligations were carried out at 23°C for 2 h and transformed into 100 μl RbCl2-competent E. coli cells (strain: DH5αZ1). Plasmid preparation, gel extraction, and DNA purification kits were obtained from Qiagen. All DNA fragments originating from plasmid DNA were separated by agarose gel electrophoresis and extracted using the QIAquick Gel Extraction Kit (Qiagen). All plasmids generated during this study were verified by DNA sequencing (GATC, Germany).
To generate the poly(A) constructs the protocol was adapted due to the lower melting temperature of the poly(A) oligonucleotides. The antiparallel oligonucleotides were mixed as described above, heated to 95°C, and annealed by a continuous temperature gradient of 0.1°C per second to 4°C. To avoid any denaturation of poly(A) stretches the annealed oligonucleotides as well as vectors containing poly(A) regions were digested with the Type IIS restriction enzymes overnight at 37°C. Restriction enzymes were removed by phenol/chloroform extraction according to standard protocols and the DNA was recovered by ethanol precipitation as described above. The resulting poly(A) constructs were analyzed by restriction digestions and the lengths of the poly(A) stretches up to 100 base pairs could be determined precisely by sequencing (GATC, Germany).
Expression and purification of Poly-Q proteins
The pLANA-Qn constructs (Figure 6A) were transformed into the E. coli strain BL21(DE3). A two liter LB-culture was inoculated with 20 ml of a stationary overnight culture and grown to an OD600 nm of 0.6-0.8 at 30°C. Expression was induced by the addition of 1 mM isopropylthio β-D-1-galactopyranoside (IPTG). After 5 h cells were harvested by centrifugation and flash-frozen in liquid nitrogen. The cell pellets were resuspended in lysis buffer (30 mM HEPES-KOH pH7.4, 500 mM potassium acetate, 5 mM magnesium chloride 5% (v/v) glycerol, 1 mM ß-mercaptoethanol, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 × protease inhibitor cocktail (Roche)) and lysed by French press. After centrifugation at 30000 × g for 30 min at 4°C the pellet was resuspended in denaturation buffer 1 (6 M guanidine hydrochloride (GdnHCl), 30 mM HEPES-KOH pH 7.4) and incubated with 2.5 g Ni-IDA silica matrix (Protino; Macherey-Nagel) for 30 min at 4°C while rotating. The matrix was washed twice with 40 ml denaturation buffer 1, twice with 40 ml denaturation buffer 2 (4 M GdnHCl, 30 mM HEPES-KOH pH 7.4), twice with 40 ml denaturation buffer 3 (2 M GdnHCl, 30 mM HEPES-KOH pH 7.4), twice with 40 ml high salt buffer (30 mM HEPES-KOH pH 7.4, 1 M potassium acetate, 5 mM magnesium chloride, 5% (v/v) glycerol, 1 mM ß-mercaptoethanol), and finally four times with with 40 ml low salt buffer (30 mM HEPES-KOH pH 7.4, 50 mM potassium acetate, 5 mM magnesium chloride, 5% (v/v) glycerol, 1 mM ß-mercaptoethanol). Proteins were eluted with four times 10 ml elution buffer (30 mM HEPES-KOH pH 7,4, 50 mM potassium acetate, 5 mM magnesium chloride, 5% (v/v) glycerol, 1 mM ß-mercaptoethanol, 250 mM imidazole-HCl pH 8.0). The purest fractions were dialyzed against 5 l low salt buffer for 3 h. To avoid aggregation the purified proteins were diluted, frozen in liquid nitrogen, and stored at -80°C. The purified proteins were analyzed by SDS-PAGE and Coomassie staining as well as immunoblotting using FLAG antibodies (ANTI-FLAG, Sigma).
Filter retardation assay
The Q-containing fusion proteins were thawed on ice and diluted with low salt buffer (30 mM HEPES-KOH pH 7.4, 50 mM potassium acetate, 5 mM magnesium chloride, 1 mM ß- mercaptoethanol, 10% (v/v) glycerol) to a final concentration of 0.02 mg/ml. Insoluble material was removed by centrifugation at 16000 × g for 5 min at 4°C. The supernatant was divided into two samples, which were directly used for the filter retardation assay. The SUMO protease was added to one of the samples to a final concentration of 10 μg per mg substrate protein. The control reaction contained BSA instead of protease. The reactions were incubated at 22°C in a final volume of 350 μl. At the indicated time points 50 μl samples (1 μg fusion protein) were taken and mixed 1:1 with stop solution (4% (w/v) SDS, 100 mM dithiothreitol) followed by incubation at 95°C for 5 min. Under these conditions monomeric Poly-Q proteins are denatured whereas Poly-Q aggregates remain intact. Subsequently, the samples were applied to a Dot-blot filtration unit and filtered through a cellulose-acetate membrane (0.2 μm pore size) equilibrated with 2% (w/v) SDS in TBS (100 mM Tris-HCl pH 8.0, 150 mM sodium chloride). After washing with 0.1% (w/v) SDS in TBS the membrane was blocked in TBS-T (100 mM Tris-HCl pH 8.0, 150 mM sodium chloride, 0.05% (v/v) Tween-20) containing 5% (w/v) non-fat dried milk. The aggregates were detected by immunoblotting using primary FLAG-antibodies and alkaline-phosphatase coupled to anti-rabbit IgG (1:10000, Sigma).
We thank Janine Kirstein-Miles, Rainer Nikolay, and Christina Schlatterer for proofreading the manuscript. This work was supported by fellowships of the Konstanz Research School Chemical Biology to M. K., A. S. and S. P. and a grant of the DFG (DE-783) to E. D.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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McCormick, Robert Rutherford
McCormick, Robert Rutherford,1880–1955, American journalist, b. Chicago. He held local public offices, was admitted (1907) to the bar, and practiced law in Chicago. He worked with his brother, Joseph Medill McCormick, in the management of the Chicago Tribune, and, after serving in World War I, he became sole owner of the newspaper. He rapidly extended his journalistic holdings and soon was dominant in the midwestern newspaper world. The Chicago Tribune steadily and vehemently maintained an extreme right-wing position on various issues—it condemned labor unions and attacked the participation of the United States in world affairs. McCormick's works include The American Revolution and Its Influence on World Civilization (1945) and The War without Grant (1950).
See biography by R. N. Smith (1997).


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- 2 Yellow & 2 Red peppers
- Large stick of celery
- 1 Onion
- 2 cloves of garlic
- 150g Grated breadcrumbs
- 1 tablespoon of capers
- Splash of red wine vinegar
- Salt & pepper to taste
- Extra virgin olive oil
Wash the peppers, dry and cut them into large square chunks.
2. Wash the celery stalks and chop finely with the onion.
3. Add the olive oil to a pan on a medium heat and sauté the celery and onion. Once the onion has started to turn transparent, add the garlic and fry gently.
4. Add the chopped peppers and continue cooking for a few minutes, add the splash of red wine vinegar and a glass of water and continue to cook.
5. Add more water, during cooking, if the peppers become too dry (Cooking time about 20 minutes).
6. When the peppers are softened and well cooked, add salt to taste and the breadcrumbs. Mix well and add capers
7. Your Peperonata is now ready.
Buon Appetito!
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