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As we noted yesterday, Bill Frist's offer of a compromise on judicial nominations was a statesmanlike effort. It was scrupulously fair to both parties, constrained the majority just as it would the minority–more, really, since absent the compromise, the majority, Republican or Democrat, would always have the Constitutional option at its disposal–and effectuated the principle, endorsed by an overwhelming majority of Americans of both parties, that all judicial nominees should be voted on.
Harry Reid's response, as reported by the Washington Times, was childish and incoherent. Unable to deny the fairness or the logic or Frist's offer, Reid descended into babble:
[T]he Senate's top Democrat immediately expressed doubt about the proposal, calling it "a big wet kiss to the far right."
"I don't really like the proposal given, but I'm not going to throw it away," Mr. Reid said. "I'm going to work on it."
In his floor speech, Mr. Reid called Mr. Frist's proposal a "slow-motion nuclear option." "After 100 hours, the rights of the minority are extinguished," he said, acknowledging that the purpose of the filibusters hasn't been to continue debate on nominees, but simply to stop them.
"I say to everyone within the sound of my voice: 'Test us,' "he said. "Let's see how we can do in the future. I can't say there won't be any filibusters, but I think we're going to have a much better situation."
Reid's incoherence couldn't conceal what he didn't dare say out loud: obstruction is the heart and soul of the Democratic Party.
The Times also reads Frist's speech as confirming our prediction that Priscilla Owen will be the nominee whose case is used to break the filibuster.

subset: commoncrawl


text:
"Miss Guided" (KOAT-7 7 p.m.) Screwed out of her first big TV appearance as Miss Golden Globes on the Golden Globes (canceled due to the Writers strike), Rumer Willis gets revenge with a sitcom cameo. Watch this Teenage Frankenstein act and try to guess which leftover parts her parents (Demi Moore, Bruce Willis) used to construct her.
"Battlestar Galactica: Revisited" (Sci-Fi 8 p.m.) The producers and writers of "Battlestar Galactica" jog our memories of what's happened over the last few seasons just in time to get us all psyched about the final season (starting April 4). Followed at 8:30 p.m. by "Battlestar Galactica: The Phenomenon," which hopefully will prove we're not a bunch of geeks for liking the show.
"21st Annual Kids Choice Awards" (Nickelodeon 6 p.m.) Jack Black hosts and it's a pretty good bet that the Jonas Brothers will be involved in some way. ... On a sobering sidenote: If you actually voted in the very first Kids Choice Awards 21 years ago, you can no longer be classified as a kid.
"Who Cares About Girls: Sex Workers or Victims?" (Oxygen 8 p.m.) Lisa Ling investigates the modern world of prostitution. This sounds like a serious journalistic report, but it could just be a cheap excuse to interview celebrity hooker du jour Ashely Dupre.
Crash N' Burn (Spike 8 p.m.) Erik Palladino ("ER") and Michael Madsen (Reservoir Dogs) star in this made-for-TV version of The Fast and the Furious about an FBI agent who goes undercover to nab a ring of high-speed car thieves. Nice cars, though.
"The Bachelor" (KOAT-7 8:36 p.m.) Do you love "The Bachelor"? Do you find that one hour simply isn't enough to luxuriate in the brilliance of this TV dating show? Are 90-minute specials six minutes too long for your attention span? Boy have I got a solution for you: tonight's 84-minute long episode of "The Bachelor"!
"Hell's Kitchen" (KASA-2 8 p.m) Gordon Ramsay returns to berate, scream at and cuss out a new crop of wannabe chefs.
"Live to be 150 ... Can You Do It?" (KOAT-7 9:02 p.m.) You? ... Oh, hell, no.
"The Real World Awards Bash" (MTV 11 p.m.) Cast members from all the past seasons get together to give each other awards. Like what? Best Drunk? Best Hot Tub Hookup? ... Actually, those are probably real categories.
"DEA" (Spike 9 p.m.) Spike rides shotgun with the DEA with this new reality series, which at least sounds more exciting than A&E's meter maid reality series "Parking Wars."


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Since you have selected a LeTourneau University educator preparation program, the certification office is here to support you as you work toward obtaining Texas educator credentials.
During your educational journey, there will be times when you have questions or need additional information. If you are enrolled in TRAD, TEACH, or MEd, your Advisor is your primary source of information.
When necessary, your Advisor will refer you to the certification office. Become familiar with the content of the certification office web pages since you will need this information in order to earn your educator credentials.
The School of Education is a dynamic place to be. The staff in the Office of Educator Certification looks forward to supporting your efforts in earning Texas educator credentials.
May God richly bless your endeavors!
If you believe you have a complaint concerning the Educator Preparation Program, please contact your academic advisor. Complaints will be heard and resolved informally, if possible. If the complaint is not resolved, you may appeal to the program chair. For issues or complaints about this educator preparation program that cannot be resolved by the program chair, you may contact the Texas Education Agency, Complaints (located in the blue banner at the bottom of the home page), or mail to Correspondence Management, Communications Division, Texas Education Agency, 1701 North Congress, Austin, TX 79701-1494.


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Contact: Jerry Horn
Staten Island, NY – Fr. Frank Pavone, National Director of Priests for Life, today encouraged participation in a 40-day prayer vigil at a newly constructed Planned Parenthood clinic outside Chicago. The facility, dubbed an "abortion fortress" because of its high brick walls and numerous security features, is scheduled to open September 18.
"It doesn't matter how much money or how many facilities Planned Parenthood has." When the people of a community come together in prayer and protest to stop the killing, they are more powerful than the abortion industry. I urge the participation of everyone in this 40-day vigil," Fr. Pavone said.
Priests for Life is the nation's largest Catholic pro-life organization dedicated to ending abortion and euthanasia. For more information, visit www.priestsforlife.org.


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Welcome to Prices and Performance. Here you can find prices and performance for the CI investment products of your choice. To assist you in your search, you can select products by asset class or fund family from the charts below. Or to view all the products in particular section - click on the view all button. To view the daily price for all CI fund classes
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Finding the Right Read
Please Note: By clicking a link to any resource listed on this page, you will be leaving the KidsHealth site.
Parent and afterschool resources.
Association for Library Service to Children
A division of the American Library Association. Our members are dedicated to creating a better future for children through libraries.
Reading Is Fundamental
Founded in 1966, RIF is the oldest and largest children's and family nonprofit literacy organization in the United States.
Nemours BrightStart!
This literacy program prepares young children to read. Take the reading readiness screener to see if your preschooler's pre-reading skills are on track. Get tips and ideas for reading aloud, and learn about games, songs, and other tools that can help your child on the path to success.


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These two maps appear in an atlas of Hampshire County, Massachusetts, published by FW Beers and Co. The Beers maps of this time period are informative because the show and label nearly every building in town. The map of Granby is actually of the town center only. It is a typical New England town center with a town hall, school and Congregational church. The entire town of Huntington, in the western part of Hampshire County, is shown here. Incorporated as a town in 1855, Huntington included two branches of the Westfield River and the Boston and Albany Railroad. As in many New England towns in the nineteenth century, the combination of rivers and railroads promoted industrial development. A close look at the map reveals a number of factory buildings.
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Joined: 20 Feb 2007
Posts: 3431
Location: GERMANY
Posted: Wed May 07, 2008 5:46 pm
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Here, for my friends here at this board. Taken from Hellpike #14 (currently not available). New plan is to release issue 16 (120 pages) together with the LD Picture Disc (just at the same time), and make the older issues available again - if fukkin' time allows...
Teitanblood comes from Spain and plays brutal deathblack metal the Beheritian-way, so you should now what´s the law here...
All Hails! Teitanblood is a pretty young band, please introduce the unholy trinity to us...
The Triad was formed in mid 03, even though each of the 3 members have their own projects aside since quite long ago... nevertheless, TEITANBLOOD is a single act created from the collision of 3 paths who crossed when and how it had to happen. No side activity of anyone, whose personalities behind have no relevance at all.
Genocide Chants to Apolokian Dawn was recently released, what can you tell us about that dark demo which is in the same unholy direction which once was created by older forces like Von, the Lord Diabolus/Beherit, Blasphemy etc.!
Needless to say that we praise the bands you mentioned, therefore we had no doubt about how TEITANBLOOD should honour the Satanic Black/Death tradition: in the most abominable, primitive, obscure, morbid, dark, sick but also in a solemn and ritualistic way... a scornful bath of excrement, a dense sip of warm blood... the disturbing breath of the Devil
The lyrics are quite long and written well-done (even if they´re hard to read sometimes, due to the small letter)! What´s the source of your inspiration, do you follow any concepts with the lyrics and the band?
Thanks for your compliments. Yeah, it is a pain in the ass that we had to use such a small font for the lyrics, but they play a fundamental role in TEITANBLOOD (as lyrics should in any band). Source of inspiration comes forth from Awareness, Faith, Discipline, Sacrifice, the Evolution/Degeneration/Destroying Path growing backwards, the Unsewing of Flesh and Spirit, the Infernal Crusade!!! Visions resulting from self experience and the spiritual research, are of extreme importance when it comes to write the lyrics, always embodied in Leviathanic Psalms of Massacrament and Punishment.
Still about the lyrics: On Necrosemen you did a little tribute to holy Maria (or Sancta Maria)! I guess this is pure blasphemy! Don´t you fear to get troubles with some christian institutions? Is it some kind of satisfaction to write such evil lyrics? Or is it more like a pain in the ass to write lyrics?
I hardly think we ever could have a problem with any Christian organization. They seem to have realized that it is more fruitful to abuse of children instead of burning infidels/heretics.
All the lyrics have been written before the music, and are not a pain in the ass at all even if the ones of the demo are not as good as the future ones...
OK, you are also using pseudonyms in the pure Blasphemy vein! Why do you use them? Don´t you think, that someone could take them only as funnie/joking-thing and then telling that Teitanblood isn´t really serious? What does such pseudonyms mean to you?
Its our personal tribute to bands like Blasphemy, Archgoat or Beherit, and their meanings are an extension of our way to express ourselves through a ritualistic way, with specific meanings of course, as lyrics, image or lay out do, but in a far less important level for sure. We are maybe falling in the needing of expressing ourselves as much as we can, for being a debut release. They are not stated by chance, either to look grim or any sort of necrogoatnuklearcommandoattack666, although I agree that, the usage of Blasphemy-like nicknames can turn really funny/stupid in some cases, anyway... I have more important things to think about than how the Internet forums could judge us for such details.
Teitanblood is a christcrushing band from Spain, so what can you tell us about any scene in your country/city? There´s little to know about that scene, so what about worthy bands, labels, etc.?
Yes, contrary to other places, the best moment for Black Metal in Spain is up to come. Proclamation is the best exponent of the Traditional Merciless and Bestial Black Death Cult. Other names to keep in mind are Nazgul/UrukHai, Final Solution and Panoptikon zines.
Is there any goal you want to follow with Teitanblood?
Not exactly, we 3 have our personal goals, and we are not even in regular contact. TEITANBLOOD is summoned spontaneously.
You´ve announced a split EP with another band from Spain, namely Proclamation! It should be unleashed upon mankind soon and by the label of the Goatvomit guy (Kiss of Shame)! Can you tell us already some details about?
It has been agreed, and KoS showed a huge interest on this release,
but we do not really know the priority that KoS has with us, so, I cannot tell you too much about this... another split EP is planned with a new label, but I cannot unveil more details by now.
It would be stupid to ask about any Teitanblood live appearances in the past, but do you plan some for the future? What are your imaginations of a perfect Teitanblood show? Any comradeship bands you would like to play together, to desecrate the golden halls?
To be honest, I do not think Teitanblood will ever play live, never say never anyway.
Maybe a more stupid question, but I want to know your opinion about! Spain was recently victim of terroristic actions! I guess everybody into misanthropic black metal do appreciate such activities, but most of them are living far away from the real terror, in sweet little cities etc! So, what are the opinion of someone who is living in the near area of the last terror-acts? Do you live in fear know, what´s the mood? Do you avoid to go to bigger places, train-stations, etc.? Please your honest opinion about!
Would´t it be a damn pity to die an unworthy death without the own choice of your death?
Correct, in fact 2 band members do not live near, yet in the very same place that Terror happened, as well as all our families are in Madrid too. There have been terror attacks in Spain since dictatorship, and its already a showbusiness, and a great ace card for political brainwashing achievements, therefore, why should they want to throw it away and end with terror? We are aware of whats going on and who plays this game... there is no fear to worry about.
Its not a surprise either, and of course, doesnt make us react in any way, not even sadness or hatred. This so called Evilness is everywhere, it happens in many forms, it only depends the point of view you want to see/call it, and which side you want to choose. The Adversary has Won, and that is what every Black Metal band should Glorify!! When you are dealing with these concepts in a metaphysical or universal way, things like homeland, or any topic related with these acts like race, politics, or history are meaningless, so why should things change when these acts take place in our country? We all have one moment and one place to die.
When mine will come, then be it. On the other hand, I think this act has fortunately revealed the totally fake behaviour of many Black Metal bands from Spain, demostrating that all that hatred for human life and all that death worship is a mere black metal cliché, and total unseriousness, hail to that!
In the inlay of your demo there´s a latin sentence to read (aside of the desecrated picture of Maria), what´s the meaning behind, why do you use it? Also an unholy sigil is to see in the same booklet, which could be taken from the Necronomicon (as it looks similar the these sigils), what´s to tell about this (I mean this sigil under the lines of the line-up)?
Pray for us sinners, now and in the hour of our death. You can notice that Necrosemen is just a slightly changed version of Ave Maria prayer. The usage of Latin gives a more extolling, liturgic and solemn feeling, fitting with our ritualistic idea. This element has become obligatory for us. The Belial sigil you refer to is not taken from Necronomicon, yet the Lesser Key of Solomon... one of the hundred sources of the literature we devour (including philosophic, esoteric/occultism, religion, and mythologic texts).
A little trademark are those unholy souding guitar solos, will you keep them for future, too? It seems, that everybody can handle his instrument very well, since when do you play on your weapons?
We can play our instruments the way we want to, which is still enough. Agree. Those guitarworks are fucking great and we will keep them in the future for sure.
Is Teitanblood available for everybody or only for chosen ones? Will be your stuff limited or do you want to reach every fukker outthere with your music?
We cannot label who are the chosen ones or not. We do not know everyone who gets our stuff, and we are not interested either, as well as their pathetic lives, their bands, what black metal means for them, their music collection or what they wear... As long as people want to corrupt themselves, we will always be there to handle the razor, theyre permanently invited.
The future plans are:
We will go with slow, firm yet fanatical step. In June 04 we will return to the Krematorium Chapel to record the stuff for the EP, I started to build the skeleton of the full lenght, whose musical, lyrical and visual will be developed at the same time, but, an LP is an ultimate release. There are already many demos to release till that moment...
Ok, that´s all, until the next time, last words are yours...
Thanks to you Hurricane, for the interview.
Genocide Chants to Apolokian Dawn, demo 1 is available for 5 (p.p):
Ignacio Muñoz
Avda Libertad 3 - 6ºC
30009 Murcia, SPAIN
AAAArg, whilst editing this interview here I really feel ashamed due to my bad questions. Nevermind, that was 3 or 4 years ago. Issue 15 was lots better, haha...
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Joined: 11 Dec 2006
Posts: 2996
Location: London
Posted: Thu May 08, 2008 1:33 am
Post subject:
Cheers for that! Also will look out for the other zines too!
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Joined: 26 May 2007
Posts: 10679
Posted: Thu May 08, 2008 4:37 am
Post subject:
the demo isnt available anymore right? @masters throne
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Joined: 20 Jul 2007
Posts: 691
Posted: Thu May 08, 2008 7:09 am
Post subject:
CR99 wrote:
the demo isnt available anymore right? @masters throne
no, it's not. Long sold out
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Joined: 26 May 2007
Posts: 10679
Posted: Thu May 08, 2008 7:22 am
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Joined: 20 Aug 2006
Posts: 137
Location: IL
Posted: Thu May 08, 2008 1:30 pm
Post subject:
There's 2 versions of the demo, the original had red paper with lyrics and the other edition (second edition I guess) has a yellowish/tan paper and has no lyrics.
I believe the 2nd edition is still available from some Spanish distros.
"What's with the girlie screams? We're not the Beatles!"
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Joined: 20 Jul 2007
Posts: 691
Posted: Thu May 08, 2008 1:37 pm
Post subject:
Black Seed prod made them only for their own distro as far as I know.
EDIT: and are sold out
Last edited by tom245 on Thu May 08, 2008 1:41 pm; edited 1 time in total
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Posts: 10679
Posted: Thu May 08, 2008 1:41 pm
Post subject:
i thought the yellow paper thing is a bootleg
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Posts: 691
Posted: Thu May 08, 2008 1:44 pm
Post subject:
Read the Metal Archives page now. i changed the info there about a week or two back.
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Abomination of Desolation
Posted: Fri May 09, 2008 1:03 am
Post subject:
there exist:
2 sided RED version made by the band
1 sided RED version made by the band
that yellow-whatever version: only made by BSP with approval of the band
all of them sold out.
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Joined: 27 Jul 2007
Posts: 249
Location: Slovenija
Posted: Fri May 09, 2008 6:15 am
Post subject:
Abomination of Desolation wrote:
that yellow-whatever version: only made by BSP with approval of the band
that's right, the yellow version is
a bootleg. BSP did 20 - 30 copies, when the red version was sold out.
the demo is excellent of corpse, I'm still dreaming about a Teitanblood/Proclamation Split demo LP...
14 WORDS: "We Must Secure Total Annihilation Of Humanity And End All Life On This Planet"
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Posts: 10679
Posted: Fri May 09, 2008 6:20 am
Post subject:
rahhh soldoutsoldoutsoldOUT
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Bringer of Terror
Joined: 26 May 2006
Posts: 207
Location: Chile
Posted: Fri May 09, 2008 11:01 am
Post subject:
Abomination of Desolation wrote:
there exist:
2 sided RED version made by the band
1 sided RED version made by the band
that yellow-whatever version: only made by BSP with approval of the band
all of them sold out.
Whats te difference between the red versions that the band released???
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Joined: 26 May 2007
Posts: 10679
Posted: Fri May 09, 2008 11:09 am
Post subject:
Someone has a spare? Give my Terror Squad demo for it
EDIT: What's BSP?
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Joined: 20 Jul 2007
Posts: 691
Posted: Fri May 09, 2008 11:35 am
Post subject:
Bringer of Terror wrote:
Whats te difference between the red versions that the band released???
Red, thick card, lyrics on back.
pale yellow/marble, paper, no lyrics.
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More instructive than the speechlessness of the Democratic Party, unable to react coherently to the bloody impasse in Iraq, is the debate among progressive writers about the justice of the invasion. To penetrate the thinking of the prowar liberals, whose zeal for toppling a malignant dictatorship split the left and therefore eased the slog to disaster, we need to cast our eyes back to the 1990s. Written by thoughtful observers of the current crisis, two new highly personal books help us understand the gestation of liberal hawks in the dozen years between the fall of the wall and the fall of the towers. Images of Rwanda and Kosovo were not especially poignant for the principal Bush Administration insiders who made the decision to invade Iraq. At the outset, for them, humanitarianism was not even a pretext for war. But the appalling failures and modest successes of humanitarian intervention during the 1990s did shape the thinking of certain sparkling liberal intellects. Their heady support for war played little or no role in the decision to invade Iraq. But it did diminish and isolate voices of dissent, helping insure that Bush's ill-fated war was set afoot with little national debate, even in the high-circulation liberal press.
The tenor of Paul Berman's new book, Power and the Idealists, is suggested by the word "tragedy" in the title to his concluding chapter, "The '68ers and the Tragedy of Iraq." He freely acknowledges "the scale and gravity of America's blunders in Iraq." But he can find nothing especially critical to say about the handful of former '68ers who, invoking humanitarian commitments, clambered aboard the wagons of war. They should be judged for their good intentions alone, he implies.
The seeds of this forgiving and self-forgiving attitude are sown in chapter one. The book opens with a lengthy essay on the tribulations of Joschka Fischer, the popular Green politician and German foreign minister from 1998 to 2005, who had been embarrassed by the publication of some old photos that showed him as a young man beating a policeman. Originally published in the September 3, 2001, edition of The New Republic, Berman's reflections on Fischer open a window onto his own pre-9/11 mindset. His preoccupation, at that time, was to show "the evolution of the leading '68ers from revolutionary leftism to liberal internationalism." He traced the path by which Fischer, the young antibourgeois street fighter in bluejeans who flirted discreetly with lethal vandalism, came to endorse German participation in the Kosovo war. Berman's theme was "how someone with an extremely radical New Left orientation could have ended up, in the fullness of time, a friend of NATO."
Foreign-policy realists would never have backed the Kosovo intervention, Berman contends, because "realism is never genocide's enemy." The lofty principles that inspired the Kosovo action were kept alive, he speculates, by "the veterans of the student uprisings circa 1968." These idealists hated genocide and "put matters of conscience at the heart of their thinking." Thus, Berman concludes, "NATO's intervention could just as easily be described as the '68ers' War."
The shift from skirmishing with the police to bombing the génocidaires was "a generational trajectory," not limited to Fischer's swapping of bluejeans for a three-piece suit. It was the story, according to Berman, of how the New Left, beginning in France in the 1970s, shed its antimilitarist, anticapitalist, antibourgeois and anticolonialist stances and became, instead, fiercely antitotalitarian. The Cambodian genocide had been an earsplitting wake-up call, forcing open-minded leftists to admit that cruelty and oppression do not stem exclusively from Western imperialism. By the mid-1990s some had come to believe that American power could be a, even the, force for good in the world.


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Gramicidin A ion channel
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Life in Christian County, Kentucky... The Rural Life...
Our Mennonite neighbor, Willis, is good at watching the weather. Most farmers are, but Willis doesn't have a radio or television to get the latest weather reports. He does have a telephone number that he can call to hear the forecast. He supplements that by asking everyone he sees if they've heard what the weather is supposed to do.
Willis keeps a journal of weather observations. He writes down the amount of rainfall, the depth of the snow, the dates of the last frost and the first freeze, and the details about any unusual weather, from a bad storm to a heavy dew. He uses his records to track microtrends for his farm that would never be seen by a general weather analysis of the region.
Willis also watches the weather throughout each day. He's aware of how the clouds look and where they are, whether the temperatures are going up or down, the strength of the wind, and most importantly, the direction in which the wind (thus the weather) is moving. He makes comments that remind me of the things my own dad and mom used to say. "I don't like how the wind has switched to the southeast." Or, "It will clear off now that the wind's changed." Or, "I'm afraid the wind's going to get in the southwest and stay there."
From all of this, plus frequent readings of his barometer, Willis knows more than most of us what the weather will be like. Last week, he spread manure on the field in the photo, and yesterday he plowed it. Today it rained. That's not a coincidence. Willis can't control the weather, but he can work around it.


text: Treasures in Vogue, Inc. is a fashion house launched by Manisha Dhuper. Exuding considerable passion for style with a keen sense for detail, Manisha brings to the "modern woman of today" a collection that boasts class and style! Her creative and passionate mind, always racing to find that different look, took her back home to India to find and bring to the U.S. this handmade and traditionally-chic, timeless collection of Jewelry!


text:
Keychain Screwdriver Set
Miniature Phillips head and flathead screwdrivers for your keyring.
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The Keychain Screwdriver Set includes a pair of miniature flathead and Phillips-head screwdrivers designed to be carried on your keychain. At 2.5" long, they won't get in your way, and their flattened handles provide enough torque to make them useful for simple household chores.
Made from heat treated blackened steel. Includes a keyring (because it wouldn't be a Keyring Screwdriver Set without one!). Does not include the key to my house.
Features & specs
- Dimensions: 2.5" x 0.625" x 0.25"
- Material: Heat treated blackened steel
- Includes one (1) Phillips head and one (1) flathead screwdriver
- Keyring is included
- Both screwdrivers are #2 size (most common for household jobs)
As the Boy Scouts say, "Be Prepared"
Simply put, screwdrivers are handy tools. Having a small pair on your keychain makes them easily accessible during life's little emergencies.
This miniature pair includes a flathead and a Phillips-head screwdriver made from heat treated blackened steel. That means this Keyring Screwdriver Set is tough and looks cool.
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  Fig. 3 Legend

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Lock and Key

Experimental Methods

Challenges Faced

The Search for Locks and Keys

 After designing our DNA origami box, we started looking for a suitable "lock and key" system. With a suitable lock and key, we would be able to open a closed origami box, as shown in Fig. 1.

Fig. 1(a) Front view of a closed origami box

Fig. 1(b) Top view of an open origami box. Box opens when the key binds to the lock.

Fig. 1 When the lock and key interact, the origami box opens.

 For our purposes, we adapted the lock and key system based on the specific binding of PDGF (Platelet Derived Growth Factor) to an aptamer strand. Such a system has been successfully used for a similar application (Douglas et al., Science 17 Feb 2012, Vol 335: 831-834). Aptamers are artificial specific oligonucleotides, DNA or RNA, with the ability to bind to non-nucleic acid target molecules, such as peptides, proteins, drugs, organic and inorganic molecules or even whole cells, with high affinity and specificity (Mairal et al., Anal Bioanal Chem 2008, 390: 989–1007). PDGF is one of the numerous proteins regulating cell growth and division. It is considered a potent activator for the cell types essential for tissue repair and wound healing (GF. Pierce et al., Biochem Apr 1991, 45 (4): 319-26). In our system, we used a PDGF-specific aptamer based locking system, as described by Douglas et al. Each lock is essentially composed of two complementary oligonuleotidic strands - an aptamer strand specific to PDGF and a strand complementary to it.

<big>Aptamer strand:  5'TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTA3'</big>

<big>Aptamer locking strand: 3'ATGAGTCCCGACACGTTCGTTAACACCAGGGTTACCCGACTCAT5'</big>

When Human PDGF-BB interacts with such a hybrid, it associates with the aptamer strand and the two strands of the lock dissociate. In other words, the complementary strand is displaced by PDGF because it has a higher affinity to the aptamer (Kd = 0.129±0.011 nM) (Green et al., Biochemistry 1996, 35: 14413-14424). To enhance the efficiency of the system, the aptamer locking strand is designed to be partially complementary to the aptamer strand. Such a design with shorter complementary sequences (24 bp) combined with a stretch of 16 mismatches between the two strands, facilitates strand displacemnet and hence, opening of the lock by PDGF. However, the lock is still stable enough when the Origami box is closed. The locks were hence designed as described in Fig. 2(a). The two strands of the lock are attached to the the origami box by means of origami attachment sequences, complementary to the origami scaffold.

In order to see how efficiently this lock and key system works we had to come up with an assay to characterize its functioning. To actualize this, Black Hole Quencher (BHQ) labeled aptamer strands and Cyanine 3 (Cy3) labeled aptamer locking strands were used to form the lock. In principle, when the DNA origami box is closed, the fluorescence of the Cy3 fluorophore is quenched due to its proximity to the BHQ (Fig. 2 (a)). In the presence of PDGF, When the box is open, the distance between the BHQ and Cy3 is large enough to observe a strong Cy3 fluorescence (Fig. 2 (c)). Consequently, opening of the structure can be detected by an increase in the Cy3 fluorescence signal.

(a) Labeled aptamer lock

(b) Legend

(c) The lock opens when PDGF binds

Fig. 2 Lock Sequences.

Spectrophotometric Measurements

 In all our experiments, the lock is a hybrid of the complete aptamer strand and the complete aptamer locking strand. These strands will heneceforth be refered to as the aptamer strand and the aptamer locking strand respectively. Unless otherwise specified, the mention of a "lock" refers to a hydrid of the complete aptamer and aptamer locking strands, labeled with BHQ and Cy3 respectively. Whenever, labeled strands were used, the aptamer strand was labeled with BHQ and the aptamer locking strand was labeled with Cy3. All the samples were prepared as described under (Lab Book -> Protocols).

 The legend below (Fig. 3) shows all the components mentioned here.

 Fig. 3 Legend

Fig. 4 Linear increase in fluorescence with increase in Cy3 labeled lock concentration. No BHQ.

To begin with, the experiments for characterization of the lock were performed independent of the origami. In these experiments, the aptamer strand of the lock was not labeled with BHQ.
Only the aptamer locking strand was labeled with Cy3.

We performed experiments to obtain an optimal fluorophore (Cy3) labeled lock concentration with an optimal signal-to-noise ratio. In all such measurements,
samples containing the fluorophore were excited at 510 nm and emission was recorded at 564 nm, using a spectrophotometer. Fluorescence was measured for 1 nM, 10 nM and 100 nM Cy3 labeled lock concentrations. It was observed that the intensity of fluorescence increased linearly with the increase in Cy3 concentration (Fig. 4). A 10 nM Cy3 labeled lock concentration proved optimal, based on the fluorescence spectra obtained.

Fig. 5 Fluorescence measurements with closed lock

Solutions with 10 nM lock concentrations were measured for fluorescence. A control experiment was also designed, in which the aptamer strand of the lock hybrid was unlabeled. In such a control, since the Cy3 fluorescence from the fluorophore on the aptamer locking strand, does not get quenched, a high fluorescence was obtained. In comparison, the lock hybrid with both labels, showed a considerably lower fluorescence signal (Fig. 5). Hence, it was clear that the closed state of the lock is clearly discernible.

Fig. 6 Fluorescence measurements with the lock

We now had the lock and had to work on opening it with its key. Samples of the lock were incubated with PDGF for 24 Hrs. PDGF was always used in a 10 times excess concentration than that of the lock. Control experiments were also designed. The negative control, consisted of the lock. Hence, very minimal signal would be expected with such a control. The positive control sample had the lock with only the aptamer locking strand labeled. In such a control, since the BHQ is absent, a high fluorescence signal would be expected. After repeated trials of such experiments, no significant increase in fluorescence intensity was observed in the presence of PDGF (Fig. 6).

 Since, the use of PDGF to open the lock did not seem to work, the question remained; why did the PDGF key not open the aptamer lock?

- What would you do if you cannot open a lock with your key?

- You would probably try another key!

That is exactly what we did. We used two oligonucleotidic single strands, with sequences complementary to the aptamer strand and the aptamer locking strand, which we call blockers (Fig. 7). Blocker 1 and blocker 2 are complementary to the aptamer strand and the aptamer locking strand respectively. One may consider blockers as better keys because their interaction with the lock is guaranteed due to their sequence complementarity.

(a) Use of blocker 1 complementary to the aptamer strand, opens the lock

(b) Legend

(c) Use of blocker 2, complementary to the aptamer locking strand, opens the lock

Fig. 7 Blocker sequences can open the lock.

Experiments were hence also designed, including the blockers. The following samples were prepared, to serve as the experiments and the controls.

Fig 8. Fluorescence measurements to study lock function

Samples

Lock

Lock - aptamer strand without BHQ, aptamer locking strand with Cy3

Lock - aptamer strand without BHQ, aptamer locking strand with Cy3; with PDGF

Lock; with PDGF

Lock; with blocker 1

Lock; with blocker 2

Lock; with both blockers, blocker1:blocker2 = 10:1

Lock; with both blockers, blocker2:blocker1 = 10:1

Lock; with both blockers, blocker1:blocker2 = 10:1, sample annealed before incubation

Lock; with both blockers, blocker2:blocker1 = 10:1, sample annealed before incubation

PDGF was always used in a 10 times excess concentration than that of the lock. All the samples were incubated at room temperature for 24 Hrs. The results of this experiment are presented in Fig. 8. The results shown were obtained from averaging the measurements from three sets of experiments. The error bars show the standard deviation with is reasonably low. Hence, the results were consistent over multiple experiments

 From the results obtained, it was observed hat the blockers work better than PDGF at opening the lock hybrid. Blocker 1 was found to be more efficient than blocker 2 as expected, since Blocker 1 is complementary to the aptamer strand over a longer length than Blocker 2 to the aptamer locking strand.

Gel Shift Assays

Our lock and key system was adapted from previously published results (Douglas et al., Science 17 Feb 2012, Vol 335: 831-834), the only variable being that both the aptamer strand and the aptamer locking strand were labeled. Hence, when our spectrophotometric measurements did not give us affirmative results, our first concern was whether the presence of the fluorophores was causing steric hinderences that prevented the key from binding to the lock. To clarify this concern, we ran a gel shift experiment (Fig. 9). With this experiment, we wanted to observe the differences in the functioning of the lock and key system with and without modification with fluorophores. The samples for each lane of the gel are as described below.

Lanes:

Lock

Lock; PDGF

Aptamer strand with BHQ

Aptamer strand with BHQ; with PDGF

Aptamer locking strand with Cy3

Ladder

Lock - aptamer strand without BHQ, aptamer locking strand without Cy3

Lock - aptamer strand without BHQ, aptamer locking strand without Cy3; PDGF

Aptamer strand without BHQ

Aptamer strand without BHQ; PDGF

Fig 9. Gel shift assay to study the functioning of the locks - 1

All samples were incubated at room temperature for 24 hours, before being run on the gel. A 20 bp DNA ladder was used. A shift was clearly observed everytime PDGF was present with the aptamer strand or the lock hybrid. More importantly, it was observed that labeling the strands did not affect the binding of PDGF to the aptamer, since similar shifts in bands were observed with and without the fluorophores.

As the next step, to reaffirm the specificity of PDGF to the aptamer strand, another gel experiment was run. The gel is shown in Fig. 10. The samples for each lane of the gel are as described below.

Fig. 10 Gel shift assay to study the functioning of the locks-2

Lanes:

PDGF

Aptamer locking strand with Cy3; PDGF

Aptamer locking strand with Cy3

Aptamer locking strand

Aptamer strand with BHQ

Aptamer strand

Lock

Lock - aptamer strand without BHQ, aptamer locking strand with Cy3

Lock - aptamer strand without BHQ, aptamer locking strand with Cy3; with PDGF

Aptamer strand without BHQ, incubated with PDGF. Aptamer Locking strand with Cy3 added after incubation period.

Ladder

Lock; PDGF

Unspecific ds-DNA, one strand labeled with Cy3

Unspecific short ds-DNA, one strand labeled with Cy3; PDGF

Lock,aptamer strand without BHQ, aptamer locking strand with Cy3; blocker 1 and blocker 2

All samples were incubated at room temperature for 24 hours, before being run on the gel. A 20 bp DNA ladder was used. The gel was scanned in a gel scanner for the presence of bands rich in Cy3. A Cy3 band is seen in every lane that has DNA labeled with Cy3. In all the wells with PDGF – 2,9, 10,12 and 14, binding is observed, because Cy3 is observed in the wells. In well 9, no separate Cy3 band is observed except in the well. This suggests that the two strands of the lock hybrid do not separate when PDGF binds to the aptamer. In lanes 2 and10, a separate Cy3 band is observed, way below. This is easily explained for well 10, because, in this sample, the aptamer and PDGF were incubated for 24 hours and the complementary Cy3 labeled strand was added later. Ideally in lane 2, we should have seen just the Cy3 band in the gel with no Cy3 remanants in the well, since we expect PDGF to bind specifically only with the aptamer strand. This is however not the case. Similarly, in lane 14, we would expect just the Cy3 band in the gel with no Cy3 remanants in the well. However, we again notice unspecific binding here. These observations suggest that PDGF do not specifically bind only to the aptamer. However, in the last lane with the blockers, we observe to faint bands. One of these bands corresponds to the single stranded complementary strand. The other band corresponds to the complementary strand-blocker2 hybrid. This suggests that when the blockers are used, the lock does open.

To confer specificity to the opening of our DNA Origami Box, we wanted to use an aptamer based lock and key system. The specificity of aptamer-protein binding reactions, opens up numerous possibilities for using the system in biological scenarios. Our lock and key mechanism was adapted from previously established results. However, the system did not work as well as expected. It was hard to ascertain that the lock was definitely open. Multiple spectroscopic measurements were run with different parametric conditions and different lock and key concentrations. The end result was however, not satisfactory. To rule out the possibility of hinderances in aptamer-protein binding due to the presence of the fluorophores, gel shift assays were run. It was seen that the presence of the fluorophores did not show results different from when fluorophores were present. To confirm the specificity of PDGF to the aptamer, gel shift assays were run. It was observed that PDGF bound to the unspecific sequences too. Overall, the system did not perform as expected. However, the use of blockers
to open the lock was our temporary fix to the problem.


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Dividend Yield
Intel Corp.
Nat'l Bank of Canada
Teva Pharmac. ADR
Baxter Int'l Inc.
Brit. Amer Tobac. ADR
CA, Inc.
Chevron Corp.
Enbridge Inc.
Lockheed Martin
Magellan Midstream
Mattel, Inc.
McDonald's Corp.
Microsoft Corp.
NextEra Energy
Northeast Utilities
Occidental Petroleum
Owens & Minor
South Jersey Inds.
Sysco Corp.
Target Corp.
TELUS Corporation
Village Super Market
Wisconsin Energy
At the time of this article's writing, the author did not have positions in any of the companies mentioned.


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From stock to custom !!!
dodge 2500 SLT This truck has been pretty well built and a lot of it is billet .Lets start with the machined aluminum 0-Ringed head to Handel a lot more that a stock head might not, Head studs(ARP0,over sized fuel injectors, BD twin turbo that will run 55 POUNDS of boost all day long and to make all this stuff sound really cool , Fass fuel pickup, Edge stage five comp box,Down from there it has a A billet triple disk torque converter, billet input and output shaft, with a performance billet tranny.custom rims and tires,after market lights all around ,mirrors front grill Teflon coated rims [img][img][img][/img][/img][/img]
Hello, this is my 1966 Ford 427 Shelby Cobra (replica)
I actually bought the engine before I bought the replica kit. I contacted Shelby Motors, and at that time, he was starting up the "continuation series" of Cobra's. I asked if he had any parts from the past for sale (I thought, ya, ,...right) !
I was able to talk with the legend, Mr. Carroll Shelby himself. Being such a great man and a icon of racing, I felt and always will, honored ! He asked what I wanted to purchase, I replied jokingly, "a engine". He said he had 5 from the "old days" and said, "pick one". I about fainted. He raced these engines in 1967. I asked to speak with one of his engine builders. He emailed me the stats/specs and brief history, picked one and had it shipped home. The only parts missing was the sidewinder intake, Cobra oil pan and balancer.
I left it stock for 3 years. The automotive touring group, "Americruise" came to Lincoln with apx. 3,000 muscle car's and Hot Rods. A bunch of us car nut guys !!!, had breakfast, pulled onto 27th street and boom,...I thought damn ! I shut her down, pulled onto a side street, saw nothing wrong and went to the show. The next morning, I took off the valve covers and the rocker shaft at # 1 cylinder was broke in half ! The reason,......Ford for some reason did not have support stands on the ends, just the middle ???
To my amazement, both intake and exhaust valves were frozen shut, and the exhaust pushrod was bent 90% inside the manifold ! I asked the engine builder that machines the sprint car engine, what he thought happened, he was dumbfounded too. I could see one valve guide either splitting or had shrank therefore freezing shut but, not both at the same time ?
Anyway, these engines were the top dogs in racing (along with the Heim's) but the pushrods went through the intake manifold which made them all most straight on, not angled. It made for a better design. So, since I had to take apart the whole top end, I rebuilt it in 2003.
I was able to do all of the car except the engine machining and a great friend helped me assemble it.
I custom mixed the blue and added pearl to the white stripes. I also made several aluminum parts on the car. (see photo's)
I've built other cars over the years but, my Cobra is just a kick in the pants fun ! Like a skate board on wheels.
I love to go to shows with family, friends and talk to other owners of their cars. I let most people, including children, sit in the car and sometimes, let them fire up the engine. That's what shows are all about, the sharing and comradely with others. Trophy's are fine but I enjoy the time spent with car enthusiasts the most.
A friend and I are planning on starting car shows with no awards, any vehicle can enter, free to the public and just have a great time ! Hopefully we can accomplish this.
I have a long,.... story from my first show. It's about the "Jerry Lewis Telethon for Muscle Dystrophy". I took a young man that is the "Ambassador for MS" for a ride in the Car. "My favorite of all time", he said. Sadly, he succumbed to the disease 4 months later. All of what happened is a great memory I'll have forever, and no trophy in the world can make me feel better than sharing the love of car's and time spent with, with Brian.
Thank you,
Cummins 2500 4wd , horse power between between 540 and 600 horse have not put on a dino in a little while.
No specifications listed.
leather seats,all disk breaks and a few other perks .
No factory options listed.
This truck has been pretty well built and a lot of it is billet .Lets start with the machined aluminum 0-Ringed head to Handel a lot more that stock head might not, Head studs(ARP0,over sized fuel injectors, BD twin turbo that will run 55pounds of boost all day long and to make all this stuff sound really cool , Fass feul pickup, Edge stage five comp box,Down from there its has a A billet torque converter, input and output shaft, with a performance billet tranny.custom rims and tires,after market lights all around ,mirrors front grill and few things that i have forgotten.
No modifications listed.
"427HISS...... Power" has no photo albums.
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