text: Posted Online: Sept. 02, 2013, 11:07 pm Dale Owen named permanent Ascentra president and CEO Comment on this story BETTENDORF – Ascentra Credit Union's board of directors has named Dale Owen as president and CEO. He has been serving as interim CEO for 90 days. Before becoming interim CEO after the death of Paul Lensmeyer, Mr. Owen was executive vice president and chief lending officer since he joined the credit union in 1999. He has the certified chief executive (CCE) designation from the Credit Union Executive Society and graduated from the CUES CEO institutes at the Darden School of Business at the University of Virginia, the Johnson Graduate School of Management at Cornell University, and The Wharton School at the University of Pennsylvania. Mr. Owen has a master's of public administration from Drake University, Des Moines, and a bachelor's in public administration from Augustana College, Rock Island. He is president of the Iowa Credit Union Foundation, on the Quad City DollarWise board, the Augustana College Alumni board and is on the Federal Home Loan Bank MPF National Advisory Council. He has served on the CMGMI National Insight Panel, Iowa Credit Union League Payday Lending Task Force, is past president and board member of the Great River Chapter of Credit Unions, past treasurer and board member of the Davenport Neighborhood Housing Service and past board member for the Quad City Credit Association. He also has volunteered for The Children's Miracle Network, Junior Achievement, United Way, the Boys & Girls Club and Bethany for Children & Families. Founded in 1950, Ascentra Credit Union has more than $320 million in assets and 10 branches serving Bettendorf, Clinton, Davenport, Le Claire, Muscatine, and Moline. For more information, visit ascentra.org. Follow Ascentra on Facebook and Twitter @ascentra.


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Beginning Saturday, Microsoft will start pop-up notifications to remind users that as of April 8 it will cease providing any kind of patches or fixes to Windows XP.
The Tikker wristwatch on Kickstarter aims to be a constant reminder of how much time you have left in life.
Samsung teases its next smartphone while showing off a new tablet and streaming media box, and WebOS is alive -- in smart TVs.
Samsung teases its next smartphone while showing off a new tablet and streaming media box, and WebOS is alive -- in smart TVs.
Microsoft at the moment has just over 2,000 apps in the Windows Store, a month before Windows 8 and Windows RT are set to launch.
Apple has jettisoned half of its legal challenges in the courtroom war with Samsung.
Today marks 100 days before NASA's Mars Science Laboratory, carrying the Curiosity rover, is scheduled to touch down on the Red Planet. Here are some NASA images to tide you over.
Company will apparently make an announcement tomorrow regarding its line of Android-based smartphones.
It's a Special Valentine's edition just weeks away from the iPad 3's launch with all the latest details. Is an Apple TV really being tested? Plus, we'll show you how to express your love with the iPad.
Excitement is building around Motorola's forthcoming Droid Razr handset, and now there's a countdown to launch. Only 80-odd hours to go!

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"We owe it to ourselves to grow and learn together…" Jen Twigg on her experiences of sexism in the punk scene
(Andy's note: Thanks for all the support so far. The guest writers and myself are all extremely grateful and humbled by it. Without further ado, here's Jen.)
"A thousand tiny paper cuts." That's what my friend calls it when so many little injustices happen — you wouldn't make a big deal about one on its own, but a thousand of them together are a gaping wound. Add them up: some bro cheerfully telling a rape joke to a room full of laughing people, women-hating graffiti on the wall of the bathroom in the bar you're about to play, the door guy not believing you when you say you're in the band, any man in any situation talking over you to the guys in your band. That intangible, sinking, isolated, feeling of trying to be accomodating while retreating a little bit further into yourself for protection every time.
I came to punk through east coast hardcore, and everyone I knew was into it, so those were the shows I almost always went to at first. I remember standing in the back of a large room once and still getting punched hard in the face by a beefy dude. Afterward, I was talking with my ex-partner, just beginning to realize how much I was not into these displays of performative machismo, and he kept saying "this is just how it is, how it's always been. it's not going to change." I've written about violence at hardcore shows before so I won't rehash it all again, but I'll repeat this: privileging the antiquated notion that men will be men and need to blow off steam violently is not progressive, it's just mirroring mainstream values of entitlement and privilege. Allowing the posturing that happens in these cases to continue undermines any other progressive politics you might be supporting (commonly veganism and straight edge). There are ways to get stoked and go buck wild without being violent and creating an oppressive space; I've seen them at a million other shows.
Although I don't think we can ever talk too much about these things that happen to us since it never seems to sink in with those who need to hear it the most, and I have my fair share of stories, I want to address another side of it right now. If you think you don't do the things that we call out in pieces like these about gender (and race, and sexuality, etc. etc.) in punk, then prove it by being a good ally. The simple fact that you don't think you condescend to female musicians and don't tell rape jokes isn't enough; you need to be actively helping us have this conversation with folks who don't get it.
How can you be a good male ally to women (and everyone) in punk? It's really not that hard, I promise! All you have to do is listen to women and believe what they tell you about their own experiences. When someone comes to you with sexual assault allegations, don't pull the "he's always been cool to me" or "where's the proof?" cards (The only time punks love cops and believe the justice system works is when their friend has assaulted a lady in the scene.). When someone tells you they feel alienated, don't tell them they are taking it too seriously, or they should suck it up just because you have never felt alienated yourself, and don't think "Oh, well i know a woman who says she doesn't feel alienated, so that must mean you are wrong about how you feel!". Don't set up a system of competition where you privilege women who can hack it with the dudes, and look down on those who don't want to get in the pit. When you're standing around shooting the shit with a group of men and someone tells an off-color joke, speak up and let them know it's not okay, even if there are no women around. Will it be uncomfortable? Probably. Welcome to our world.
Don't condescend to women, even if you have more experience with gear and writing music than they do, because you may have been more supported in the past than they were. If they ask for your help, teach without judgment and value their input. At the same time, never assume that someone doesn't know lots about gear or writing music just because they are a woman! Educate yourself about consent. Most of all, don't expect to get patted on the back and heaped with praise every time you do something decent as an ally. Acting this way should be the norm, not the exception.
Punk is fiercely emotional for a lot of us, because we are tied so deeply to the community, and it can be hard not to take it personally and be defensive when something about it isn't perfect. We owe it to ourselves not to be defensive, though. We owe it to ourselves to grow and learn together in this community, to listen to each other, to actually be as progressive as we said we'd be when we left the mainstream to come here.
Jen Twigg is a writer, zine fest organizer, and enthusiastic girls rock camp volunteer living in Chicago. She will never give up on the Baltimore Orioles and has a guest column in this month's MRR. You can find out more about her band The Ambulars on facebook or ye olde myspace at theambulars.bandcamp.com and her personal blog is at jtwiggjtwigg.tumblr.com.
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When someone tells you they feel alienated, don't tell them they are taking it too seriously, or they should suck it up...
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I love this. As a mid 30's woman who spent all of her teenage years & most of her adulthood immersed in the hardcore...
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Great piece on gender and punk scenes. I think a lot of the advice on being a good ally holds in any scene / everywhere.
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Jen Twigg of DC/Chicago powerpop superband, The Ambulars, weighs in on the ongoing debate about sexism in punk rock....
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Filtering Posts By: "Super 55"
06.25.2014| By Cheryl Jennison DaProza|
Special Webinar Event: The History of the Shure Unidyne
Those of us who work with microphones day in and day out all have our favorites. Most singers swear by the SM58®. The SM7B is popular among broadcasters and recording engineers alike. And, any live sound engineer worth his salt will have several SM57s in his inventory. But when it comes …
03.07.2014| By Nelson Arreguin|
Juno Award Nominees Highlight – Breakthrough Artist of the Year
Breaking into anything is not easy. As it should be. It keeps bad guys out of places they shouldn't be, and it keeps us safe. It's not as convenient when we lock ourselves out of our own homes, but the benefits of security outweigh any inconvenience. It takes special talent …tagsBreakthrough Artist of the YearBrett KisselFlorence KJuno AwardsSuper 55Tim HicksTyler ShawUR2/SM58Wake Owl
10.10.2013| By Matt Dobschuetz|
Disney Sends Audio Signal Through Human Touch
At Shure, we talk a lot about the audio signal path when we refer to sound's journey from microphone to mixer to P.A. system. But we were blown away by a recent Disney Research Technology project called "Ishin-Den-Shin" which transmits an audio signal, in part, through the human body. The project's audio …
06.05.2013| By Brooke Giddens|
Microphone Makeovers: The Many Looks of Shure Mics
Coming into all of this, I knew nothing of microphones. I knew singers and bands used them to create sound, but that was about it. Already I've learned a lot about microphones and all the little things that make each Shure microphone unique; however I know there are still many …tagsAlex CubaBeta 58Chino Y Nachocustom micsfun.KSM9Mos DefSM58Summer InternSuper 55Talib KweliYasiin Bey
05.20.2013| By Ryan Smith|
What discontinued Shure Mic would you bring back and why?
In our frequent travels as Shure Artist Relations representatives, we often hear from many engineers "You guys should really bring _____ mic back." If you look back in Shure's history, there are some amazing designs but also some wonderful sounding mics. Some people want them back because they just look …
08.24.2012| By Ryan Smith|
Jason Mraz & Christina Perri in Nashville
I knew I was in for a good show having listened to Jason's new CD Love Is A Four Letter Word. The Freedom Song starts off the CD and when the band kicks in, you can just imagine the live show. No more imagining, I was finally going to get to see a Jason Mraz show. First things first – I had to prepare questions for a short video interview with Jason. I like to research what other interviewers have recently asked an artist and then avoid those questions entirely…or at least rephrase them differently, …
11.18.2011| By Ryan Smith|
Mona !!!!!
November 12, 2011 The Vogue Theater- Indianapolis, IN Who? Mona. Never heard of them eh? Neither had I until a good friend of mine who does monitors for Ronnie Dunn introduced me to them. Mike Addams called me on the phone and said you HAVE TO work with these guys. They are about to explode. They have already taken off overseas and their record is going to release in the States early next year. Mike doesn't call me too often to tell me me about a group so I knew this was going somewhere. Mike …
10.18.2011| By Ryan Smith|
Casting Crowns delivers the message in Nashville
October 14, 2011 Bridgestone Arena – Nashville, TN The last time these wonderful Christian Artists were in Nashville, I had a prior commitment and was unable to stick around for the show. Tonight, I planned to spend the afternoon and evening with the band and crew. I meet up with Carter Hassebrook who is the bands FOH Engineer and stage manager. We went to the stage and I grabbed some video b-roll of the mics on stage. Shure UHF-R wireless mics are on all vocals with various capsules for each singer. For their acoustic set, …
09.26.2011| By Richard Sandrok|
Black Star::HoB Chicago::09/23/11
I don't know where Talib Kweli and Mos Def get their time. It has been said that in the current state of the music industry you have to be a shark – that you must always swim or drown – and if that's the case these two are makos. Talib Kweli has garnered a reputation as the MC's MC. At 35 years of age he has already released four solo albums, five with collaborators, be it Mos Def, Hi-Tek, or much-respected underground hip-hop producer Madlib, and a slew of mixtapes, singles, and guest appearances. Mos …
04.29.2009| By Shure Notes|
The Super 55
A Shure Educational Podcast In this episode of our podcast, we introduce the the Super 55. Our guest, Product Specialist Rob Klegon, takes us on a tour of the new product and how it compares to its predecessor, the 55SH Series II.


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Chequered Flag International is pleased to offer this 1963 1/2 Original 260 V8 4 speed Ranchero in Rangoon Red. 6315 Rancheros were built in... More
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Partially restored running and driving Falcon Ranchero. Recent paint and a great starting point for a project - Or just drive as-is.... More
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FORD: 1979 Ranchero, auto, V8, a/c, mint condition, gagrage kept, 34K miles, original, new condition, $9,000. 972-979-2348, TX.... More


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Additional ReadingThe Complete Works of St. Thomas More (1963 ), is being published by Yale University Press; the best edition of his golden little book is Utopia, ed. by George M. Logan, Robert M. Adams, and Clarence H. Miller (1995). Separate collections of his letters include Elizabeth Frances Rogers (ed.), The Correspondence of Sir Thomas More (1947), and Selected Letters (1961). Bibliographies are R.W. Gibson and J. Max Patrick (compilers), St. Thomas More: A Preliminary Bibliography of His Works and of Moreana to the Year 1750 (1961); Constance Smith, An Updating of R.W. Gibson's St. Thomas More: A Preliminary Bibliography (1981); Frank Sullivan and Majie Padberg Sullivan, Moreana: Materials for the Study of Saint Thomas More, 5 vol. (196471), and supplements; and Moreana (quarterly), a forum for ongoing research and updated bibliography begun in 1963. Studies of his life and works include R.W. Chambers, Thomas More (1935, reissued 1976), although it has been criticized for toning down the starkness of More's asceticism, the radicalism of his Utopian vision, his hatred of schism and heresy, and the dogmatic motives of his martyrdom; E.E. Reynolds, The Field Is Won: The Life and Death of Saint Thomas More (1968, reissued as The Life and Death of St. Thomas More, 1978); R.S. Sylvester and G.P. Marc'hadour (eds.), Essential Articles for the Study of Sir Thomas More (1977); J.A. Guy, The Public Career of Sir Thomas More (1980), emphasizing More's role as government administrator; Alistair Fox, Thomas More: History and Providence (1982); Anthony Kenny, Thomas More (1983); George M. Logan, The Meaning of More's Utopia (1983); Ruth Norrington, In the Shadow of a Saint: Lady Alice More (1983); Richard Marius, Thomas More (1984); Louis L. Martz, Thomas More: The Search for the Inner Man (1990); Dominic Baker-Smith, More's Utopia (1991); Peter Ackroyd, The Life of Thomas More (1998).
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CHILTON, WI (WTAQ) - A man from Calumet County faces life in prison for allegedly assaulting a four-year-old child.
72-year-old Norbert DeGroot has been charged with one count of sexual assault. Because DeGroot was already convicted in 1989 for sexually assaulting another child he faces life in prison without the possibility of parole.
According to the criminal complaint, DeGroot assaulted the child in 2011.
DeGroot is scheduled to be back in court next month.


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Applying nitrogen—corn's most important and trickiest nutrient—doesn't do a lick of good if it escapes into the air or, even worse, into water sources before plants can use it.
Products that slow the loss of nitrogen can help you hold it in place, to feed hungry corn and boost yield—but only if you understand them well enough to choose the right one, says Farm Journal Field Agronomist Ken Ferrie.
To select the right product, consult the table on page 32. But first, decide what type of nitrogen loss—denitrification, leaching or volatilization—you need to prevent.
Denitrification and leaching occur only with the portion of your soil nitrogen that is in the nitrate form. Denitrification occurs when soil is saturated with water, especially when standing water is combined with warm temperatures and a carbon source. In those con- ditions, denitrifying bacteria increase. They strip oxygen away from nitrate-nitrogen (NO3-N) molecules, releasing nitrogen as a gas.
Sandy and well drained soils, where water percolates through, can leach NO3-N. "Stabilizers that reduce denitrification and leaching do it by slowing the nitrification process, in which ammonium is converted to nitrate," Ferrie says. "They either kill some of the nitrifying bacteria or slow down their activity."
Since you can't protect nitrate, you have to keep it in the stable ammonium form, by preventing nitrification, until the crop is ready to use it, Ferrie summarizes. Soils that stay wet and sandy soils that are subject to leaching are situations where nitrification inhi-bitors can help.
Volatilization affects surface-applied urea, which must break down, or hydrolyze, into ammonium. The ammonium then goes through the nitrification process to produce nitrate. Both forms can be used by corn plants, but until it is either tilled into the soil or washed in by rainfall or irrigation,
ammonia can be created during the hydrolysis process and be lost as a gas.
The urease enzyme. Bio-inhibitor products reduce the risk of volatilization of urea prior to tillage or rainfall by blocking the urease enzyme. That enzyme, found in crop residue and in the upper few inches of soil, triggers the breakdown process that creates the risk of volatilization.
Managing urease enzymes is very important if urea is applied on the surface and not incorporated—which is becoming more common as farmers adopt no-till, strip-till and pre-emergence weed and feed programs. That's because urea hydrolysis causes soil pH to go up around the nitrogen molecule; high pH speeds the conversion of stable ammonium molecules to ammonia gas, which can be lost by volatilization.
The same concern arises in soils with a naturally high pH and where lime is applied without incorporation, also a no-till/strip-till situation.
Another approach to nitrogen stabilization involves controlled-release products, which slow the breakdown of urea by encapsulating the particles. They can reduce both volatilization and denitrification.
So far, so good, but it gets more complicated in the field because you have to understand your nitrogen source. "For example, 28% and 32% UAN [urea-ammonium nitrate] solutions contain 50% urea, 25% ammonium and 25% nitrate," Ferrie says. "So you may need two products: one to help prevent the urea from volatilizing—unless you incorporate immediately after application—and one to slow the conversion of stable ammonium into nitrate, which is easily lost."
Picking the correct stabilizer depends on various factors, including your nitrogen product and whether you plan to incorporate. "Say you're in no-till or strip-till and you apply 28% UAN solution on the soil surface and you added a stabilizer that protects only against denitrification and leaching," Ferrie says. "Late in the growing season, you'll be wondering why your crop ran out of nitrogen.
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A new year, a new way to travel. Southwest Airlines has announced some changes to their boarding process.
Starting January 22, the airline will offer customers the opportunity to purchase one of the earliest boarding positions at the gate for $40 per flight, when available.
The option will only be offered at the gate on the day of travel, beginning 45 minutes before the flight departs.
Video: FOX 10's Syleste Rodriguez reports.
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Tai Chi for Arthritis - Barlow Library
Wed, September 24, 2014 @ 10:00A
The ancient discipline of Tai Chi combines agile steps, joint safe exercise, and mental strength to improve relaxation, concentration, memory, balance, strength, stamina, and more. Join Pat Davis with the O'Neill Center Volunteer Program on Wednesdays at 10 a.m. for this free class. To register, call Pat at 740-678-2839. The Barlow Library is located at 45 White Oak Professional Center, Vincent, OH. Questions? Call the Barlow Library at 740-678-0103.
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43 U.S. Code § 619b - Reimbursement of funds advanced by non-Federal purchasers; uprating program; repayment requirement; visitor facilities program
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Reimbursement of funds advanced by non-Federal purchasers for the uprating program shall be a repayment requirement of the Boulder Canyon project beginning with the first day of the month following completion of each segment thereof. The cost of the visitor facilities program as defined in section 619 (a) of this title shall become a repayment requirement beginning June 1, 1987, or when substantially completed, as determined by the Secretary of the Interior, if later.
Source(Pub. L. 98–381, title I, § 106,Aug. 17, 1984, 98 Stat. 1339.)
The table below lists the classification updates, since Jan. 3, 2012, for this section. Updates to a broader range of sections may be found at the update page for containing chapter, title, etc.
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fingolimod, Gilenya
Pharmacy Author:
Omudhome Ogbru, PharmD
Omudhome Ogbru, PharmD
Dr. Ogbru received his Doctorate in Pharmacy from the University of the Pacific School of Pharmacy in 1995. He completed a Pharmacy Practice Residency at the University of Arizona/University Medical Center in 1996. He was a Professor of Pharmacy Practice and a Regional Clerkship Coordinator for the University of the Pacific School of Pharmacy from 1996-99.
Medical and Pharmacy Editor:
Jay W. Marks, MD
Jay W. Marks, MD
Jay W. Marks, MD, is a board-certified internist and gastroenterologist. He graduated from Yale University School of Medicine and trained in internal medicine and gastroenterology at UCLA/Cedars-Sinai Medical Center in Los Angeles.
GENERIC NAME: fingolimod
BRAND NAME: Gilenya
DRUG CLASS AND MECHANISM: Fingolimod is an oral medication used for treating multiple sclerosis (MS). Its mechanism of action is unknown, although it may work by reducing the number of circulating lymphocytes (a type of white blood cell), leading to reduced migration of white blood cells into the central nervous system. White blood cells cause inflammation and destruction of nerves in patients with MS. Fingolimod does not cure MS. It decreases the number of MS flares and slows down the development of physical disability caused by MS. The FDA approved fingolimod in September 2010.
PRESCRIPTION: Yes
GENERIC AVAILABLE: No
PREPARATIONS: Capsules: 0.5 mg.
STORAGE: Fingolimod should be stored at room temperature, 15-30 C (59-86 F).
PRESCRIBED FOR: Fingolimod is used for reducing the frequency of relapses and for delaying the occurrence of physical disability in patients with relapsing forms of multiple sclerosis.
DOSING: The recommended dose is 0.5 mg orally once daily, with or without food. Doses higher than 0.5 mg cause more adverse reactions without providing additional benefit.
DRUG INTERACTIONS: The blood levels of fingolimod are increased when combined with ketoconazole (Nizoral, Extina, Xolegel, Kuric). This may increase the incidence of side effects.
Vaccines may be less effective during and up to 2 months after discontinuation of fingolimod treatment. Live attenuated vaccines should not be administered during and for 2 months after fingolimod treatment because of the risk of infection.
When combined with drugs that decrease heart rate (for example, atenolol [Tenormin]) there is an additional 15% reduction of heart rate.
PREGNANCY: Fingolimod has not been adequately studied in pregnant women.
NURSING MOTHERS: It is not known whether fingolimod is secreted in breast milk.
SIDE EFFECTS: The most common side effects are headache, flu, diarrhea, back pain, elevations of liver enzymes and cough. Fingolimod may decrease heart rate, especially after the first dose. Patients should be observed for signs and symptoms of low heart rate for 6 hours after the first dose. Fingolimod may increase the risk of infections. Signs and symptoms of infection should be monitored during treatment and for two months after discontinuation of treatment. Fingolimod should not be administered to patients who have an infection. Fingolimod may cause inflammation of the eye (uveitis) and other eye problems. Therefore, visual acuity should be checked prior to starting therapy, 3 to 4 months after initiation of therapy, and during routine patient evaluation. Fingolimod has also been associated with difficulty breathing. Fingolimod reduces the white blood cell count, and this effect may last for 2 months after treatment is discontinued.
Reference: FDA Prescribing Information for Gilenya (fingolimod)
Last Editorial Review: 10/26/2010 1:38:23 PM
Report Problems to the Food and Drug Administration
You are encouraged to report negative side effects of prescription drugs to the FDA. Visit the FDA MedWatch website or call 1-800-FDA-1088.
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Back to Medications Index


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Don't-Miss MP3 player accessory Stories
Expo: Tune Blocker saves you from yourself
Matias introduces Tune Blocker, a cable that lets you sync your iPod or iPhone without fear.
iLuv dual-dock alarm clock offers either port in a storm
The iMM183 Hi-Fi Dual Dock Alarm Clock with Weather Brand Radio features docks for charging two devices at once, including any iPod with a dock connector and the iPhone 3G.
Mouse-maker Logitech to cut 525 salaried employees
Logitech said Tuesday it will cut 525 salaried employees as it expects weaker consumer demand for its computer peripherals.
Vuzix intros Wrap 920AV video glasses
Vuzix has introduced new video glasses that look like sunglasses, and they work with the iPod and iPhone.
Audioengine W2 wireless adapter debuts for iPod
Audioengine's W2 enables you to stream your music wirelessly from an iPod to a nearby stereo system.
Marantz offers Bluetooth dock for iPod
Marantz's new IS301 lets iPods communicate with home entertainment systems wirelessly, using Bluetooth.
Our favorite iPod accessories of 2008
Even with all the focus on the App Store and its software, 2008 was still a great year for iPod and iPhone accessories. Here are 11 add-ons that caught our attention in the past year.
ReviveLite scares away the iPhone killers
Need a nightlight? Need a way to charge your iPod or iPhone? The ReviveLite fulfills both needs.
Gear Guide, part two: Gear for your iPod
In the second part of our four-part Winter Gear Guide, we continue to spotlight the accessories that will help you enjoy your Apple products to the utmost. We turn our attention to nine iPod add-ons and accessories that will have your music player singing your praises.
Griffin offers iPod component, composite video cables
Griffin is offering component and composite video cables complete with USB connectors and an AC charging block, to connect your iPod to a home entertainment system.
Gear Guide, part one: Gear for your iPhone
We kick off our holiday Gear Guide with 10 accessories, add-ons, and applications sure to add shine to your iPhone.
DLO finally releases hi-def iPod video dock
11 months after it was introduced, the DLO HomeDock HD is finally available for purchase.
Alpine HD car radio offers iTunes tagging
Alpine has introduced a new accessory for receiving HD Radio signals on Alpine head units that lets you 'tag' interesting songs you hear.


text: Gores Pavilion. 1959.Gores Pavilion. 1960. Located in Irwin Park, Weed Street. One of scores of modern buildings in New Canaan, this pavilion was designed by Landis Gores, of the famous Harvard Five group of modern architects. His clients were Jack Irwin, U.S. ambassador to France and an Undersecretary of State, and his wife, Jane Watson, whose father was the founder of IBM. Designed as a pool house and personal lodge for the Irwins, it is set about a hundred yards from the Irwins' more conventional house, in New Canaan's Irwin Park. When it was threatened with demolition in 2006, supporters of modern architecture, led by the Historical Society, rallied to protect it.


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Home > 2006 News Releases > News Release
News Release
Sergeant Michael B. Webb
Kentucky State Police
Public Affairs Branch
Office (502) 782-1780
Thirteen Die On Kentucky Roadways During Memorial Day Holiday Period
Date of News Release: 05/30/2006
(FRANKFORT, Ky.) - Preliminary statistics* indicate that 13 people died in 11 separate crashes on Kentucky's roadways during the Memorial Day holiday period from 6 p.m. Friday, May 26, 2006, through 11:59 p.m. Monday, May 29, 2006.
Preliminary data* indicates alcohol was a suspected factor in three of the 11 crashes. Nine of the crashes involved motor vehicles, one involved a motorcycle and one involved an all-terrain vehicle (ATV). Six of the 11 motor vehicle victims were not wearing seat belts, four of the victims were wearing seat belts, and the seat belt status of one of the victims is not known. The motorcycle and ATV victims were not wearing helmets.
A double-fatality motor vehicle crash occurred in Elliott County and one in Marshall County. Two single-fatality motor vehicle crashes occurred in Pendleton.County. The remaining five single-fatality motor vehicle crashes occurred in Allen, Casey, Jefferson, Johnson and Livingston counties. The motorcycle fatality occurred in Owsley County and the ATV fatality occurred in Perry County.
Preliminary statistics show that from Monday, May 22, 2006, through Sunday, May 28, 2006, 23 people died in 21 separate crashes on Kentucky's roadways. Preliminary data indicates alcohol was a suspected factor in six of those crashes. Eighteen of the victims were involved in motor vehicle crashes, three of the victims were involved in motorcycle crashes and one victim was riding an ATV. One of the victims was a pedestrian.
Of the 23 victims for the week, ten of the 18 motor vehicle victims were not wearing seat belts, five of the victims were wearing seat belts, and the seat belt status of three of the victims is not known. Two of the three motorcycle victims and the ATV victim were not wearing helmets. The helmet status of the third motorcycle victim is not known.
Through May 29, 2006, a total of 325 people have lost their lives on Kentucky roadways. Of the 282 motor vehicle fatalities, 182 victims were not wearing seat belts. Of the 22 motorcycle victims, 13 were not wearing helmets. There have been 21 pedestrian fatalities.
Through the same period in 2005, 357 people had lost their lives on Kentucky roadways.
The "Buckle-Up Kentucky -- It's the Law and It's Enforced" campaign runs through midnight on Sunday, June 4, 2006. Traffic safety checkpoints as well as saturation patrols will continue throughout this period with special emphasis on seat belt violations, speeding and impaired driving.
Citizens can contribute to highway safety by reporting erratic drivers to the Kentucky State Police toll-free at 1-800-222-5555 (in the state of Kentucky only, does not work out of state). Callers will remain anonymous and should give a description of the vehicle, location, direction of travel and license number if possible.
*These statistics are still preliminary as KSP waits for all local law enforcement agencies throughout the state to report any crashes and fatalities that may have occurred in their areas.


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Across Age ™ HD
- Publisher: FDG Entertainment
- Genre: Adventure
- Released: 30 Sep, 2010
- Size: 183.7 MB
- Price: $4.99
- App Store Info
DescriptionEpic journey through time, recreated for the iPad HD screen. "close enough to Hyrule to make Zelda fans feel right at home" (www.appletell.com)
Exclusive HD features:
✓ Pixel Perfect HD Graphics
++ first 2D JRPG in iPad resolution!
✓ FLING support
++ compatible with the Fling controller
✓ New Dungeon, New Boss
++ more playtime, more fun!
✓ Much Improved Battle System
++ whole new gameplay experience!
✓ HD Anime Cut-Scenes
Press Reviews:
RPGFan: "Across Age is a throwback to classic Action RPGs of the Super Nintendo and Genesis era... see how gorgeous the game looks"
Touch Arcade: "A great combination of fun combat and good puzzle variation allows Across Age to swiftly avoid the problems that many RPGs run into"
GamePro Magazine: "Across Age is unique and well-crafted, making it an easy contender for the title of "best action RPG on the App Store."
Kotaku.com: "retro charm and intriguing time-travel puzzles"
Across Age rekindles the golden 16-bit era.Travel back through time and experience an all new oldschool 2D Action RPG! "Across Age" is the first Zelda like Action RPG to include real environmental puzzles, with the look of famous SNES and Genesis cartridge games.
The game offers 15 hours of classic 2D Action RPG gameplay, exciting story, anime cutscenes, a sweeping soundtrack and tons of diversified locations, monsters and riddles.
Step into the role of 2 switchable characters: swordsman Ales and magician Ceska, each with own fighting and special skills that are needed to solve tricky puzzles and successfully battle fierce monsters. This cooperative element is one of the key features of the game. Another key feature is the possibility to travel through time and revisit places, enabling exciting events and riddles.
Find spike shoes to walk on ice, burn bushes to open new paths, find friends who help you on your journey.
Let the adventure begin!
What's New in Version 1.6.3- Fixed an issue that prevented the game to launch on iPads with iOS below 4.2
- Other minor bug fixes
Please rate the game with 5 stars after every update to keep the free updates coming.
Thanks for your support!
Note: If you experience any problems running the updated app, please restart your device
Join us at the following social networks to stay up to date:


text:
Sign Up
Your AMS membership supports our vital mission and includes valuable opportunities and benefits.
View more benefits of membership in AMS »
You asked—and we listened!
Recordings of select American Montessori Society webinars are now available for you to rent and view online. Each webcast provides essential tips for your classroom or program—and can earn you hours of continuing professional development (CPDs) that qualify for the AMS professional development requirement. AMS webcasts let you fit professional development into your schedule at the time that works best for you.
$55 individual (CPD certificate for 1 person)
$200 group (CPD certificates for up to 20 persons)
All webcast rentals are valid for 30 days.
"A Shared Humanity: Geography Lessons that Go Beyond Borders"
Presenter: Angie Meadows
For: Early Childhood & Lower Elementary teachers
Focusing on the concept of the Blue Marble, that there are no borders visible from outer space, this webinar provide a plethora of resources to help you enhance your early childhood geography lessons. Angie Meadows will cover topics, materials, and resources related to physical geography—including landforms and globes—as well as ways to enrich students' understanding of the world through cultural geography,
sharing a variety of traditions from around the world.
Individual Rental
Group Rental
"Building a Simple Yet Rich Birth to 3 Environment Indoors, Outdoors, and at Home"
Presenters: Eva Parucci & M. Loreto Wille
For: Teachers, parents, and teacher educators of infants and toddlers
This webinar will show you how to create a well-prepared Montessori environment with children's stages of development in mind, whether at school or at home. The presentation will cover exceptional indoor environments providing space for movement, exploration, and the use of materials, as well as exemplary outdoor environments where children can explore, discover, and interact with nature.
"Children's House Extension for Geography and Science"
Presenters: Shanna Honan and Tricia Smith
For: Early childhood teachers, administrators, and teacher educators
Have you ever wondered how to extend work with the puzzle maps and the botany cabinet? This webinar will look at basic presentation in geography and science and explore ways to think beyond ordinary extensions. You'll leave with fresh ideas and activities to help children move from concrete to abstract in geography and science.
"Children's 'Misbehavior'−and How to Respond Effectively"
Presenter: Yvonne Nass
For: Everybody!
This webinar will help you recognize common goals of misbehavior and explore non-punitive techniques for redirecting that behavior so that children can react to their experiences, and ask for what they need, in ways that are positive, appropriate, and self-empowering.
"Healthy Classrooms"
Presenter: Angie Meadows
For: Teachers and administrators of children ages 2.5 – 12
This webinar will introduce participants to the Healthy Classroom Checklist− addressing social interactions, work choices, independence, and classroom management techniques. You'll come away with an understanding of how you can create a healthy classroom checklist of your own.
"Helping Children Experience the Joy and Wonder of Summer"
Presenter: Mariah Bruehl
For: Educators and parents of children ages 2.5 – 9
Most children love the freedom that summer provides but we can sometimes feel overwhelmed with keeping them occupied, engaged, and happy. Are you ready for the challenge? Join Mariah as she shares her philosophy, ideas, and activities. You will walk away with enticing activities for your children, from backyard and urban adventures, photography explorations, natural crafts, writing investigations, and more to keep everyone happy all summer long!
"Let's Make Music!"
Presenter: Frank Leto
For: Parents and teachers of children ages 2.5 – 6
Be prepared to tap your feet and sway to the beat as Frank Leto, one of Montessori's favorite musicians, demonstrates the many benefits of music education in the classroom or home− from enhancing language and spatial intelligence to promoting teamwork, craftsmanship, relaxation, creative thinking, and more.
"Lighting a Child's Inborn Sense of Wonder: Mobilizing Your School to Bring Nature Back to Children"
Presenter: Geoffrey Bishop
For: Teachers of children ages 2.5 – 8
This webinar presents strategies for reconnecting children to nature. You'll experience activities and curriculum ideas that engage students with the natural world through art, creative writing, science observation, and alternatives to traditional play concepts, all using available natural space.
"Nurturing Nature Inside and Outside the Early Childhood Classroom"
Presenter: Amanda Sanderson
For: Early childhood teachers, heads of schools, and teacher educators
This webinar will offer practical ways to help children connect to nature using botany and zoology materials that emphasize your local plants and animals. You'll learn techniques for fostering a relationship with the natural world both indoors and outdoors through weekly nature lessons focused on developing observation skills.
"Using Gardening and Art to Preserve Wonder and Weave Learning Across Montessori Curricula"
Presenter: Kelly Johnson
For: Teachers of Early Childhood & Elementary students
Learn how gardening preserves the delicate childhood sense of wonder. This webinar will explore how to keep gardening journals, while integrating art and poetry into gardening to enhance class focus, promote nature connection, and assess learning across curricula. We will also address challenges within school garden programs and brainstorm ways to enhance student, teacher, and administrative support for gardening.
"Connections Matter: Skills for Creating, Strengthening, & Maintaining Meaningful Interpersonal Connections"
Presenter: Candace Proctor
For: Everybody
This webinar will outline Dr. Edward Hallowell's research on the importance of interpersonal connection, which he believes is vital to joy and personal satisfaction. Learn techniques to help you and your students initiate, maintain, and strengthen connections in all areas of your lives.
"School Accreditation 101"
Presenter: Sara Wilson
For: Administrators
If you are contemplating AMS accreditation for your Montessori school, or if you are just beginning the process, participation in AMS's School Accreditation 101 will provide you with vital information about protocol, goals, and more.
This webcast is free if you have purchased the School Accreditation Information Packet. If you have questions, or would like to purchase the information packet, contact Marcy Rice, school accreditation coordinator. Otherwise, click on the appropriate rental link below.
"Technology for the Digital Immigrant"
Presenter: Michelle D'Antonio
For: Teachers & school administrators
This webinar will focus on cross-platform computer programs that can be used to improve school administration and classroom management. Focusing on Dropbox and Google forms, participants will learn how to set up shared folders and access them across devices. The presentation will also address best practices for sharing documents with your school's many communities.
Each webcast rental qualifies the viewer for a CPD certificate of attendance, and earns the renter or renting group 1.5 hours of AMS continuing professional development. Individual renters receive 1 CPD. Groups can receive up to 20. All CPD requests must be made within the 30-day rental period. To receive your CPD(s), send an e-mail to firstname.lastname@example.com within the 30-day rental period. Include the name(s) of the viewer(s), the title of the webcast, the date of rental and confirmation number from Vimeo. A completed CPD will be emailed back to you within 2 business days.
Renters will be required to create a Vimeo account to rent and view a webcast. Rented webcasts will be added to the "My Library" section of your Vimeo account. Click "Rent" on a webcast page and follow the prompts to login or create an account. This login information will be required to re-watch a webcast during the rental period. Issues with Vimeo, including video playback, should be directed to Vimeo's Help Center.
You can view other AMS videos on our Vimeo Channels.
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Did you know?
May 22nd, 2012 by Jan in Uncategorized
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If you are in search of Homes for sale in Montana you are making a very good decision because for sure you will love this so-called "Land of Shining Mountains". There are many reasons to fall in love with this state. Montana is famous for its Rocky Mountains, Great Plains and picturesque lakes. It is free of high-rises and buildings that is why it is also called "Big Sky Country".
When checking out the properties listed in a Montana Real Estate Company of your choice, there are some factors you need to consider to ensure that you will make the right decision:
Make sure to check with the state officials what the zoning regulations in MT are. This is to see if the type of structure you desire, if you are buying a land, can be constructed there. You also have to find out about future zoning to find out if there will be airports, shopping malls, hospitals, schools, etc. to be put up that may affect the value of your property.
If the property you are getting, especially if it is a land is situated on the hills you have to make sure that the land is stable because if it's not, then you could be taking the risk of your foundation breaking down. Stay away from areas close to the paths of potential landslides.
Not all Homes for sale in MT have a water table that is deep enough for making a well. So before buying a land, you may want to check the depth of the water table so you can start making digging plans if digging is necessary.


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text: Clearance Red Geometric Print Cork Board Item #: 121898 $29.99 $14.97 Ship to Home Keeping your notes organized has never looked more stylish! This Red Geometric Print Cork Board is a great way to show off photos or mementos in your home or dorm room. Board measures 26.5L x 1W x 32.75H in. Crafted of polystyrene Black frame finish Cork board features red geometric pattern Back of frame opens for the replacement of the cork board Comes ready for wall mount; no hanging hardware required This item is no longer available for purchase.


text:
Princess No More: No Sympathy Second Time Around
This pregnancy? Fogeddaboutit. Just for the record, with Abbey and Jonah, I experienced nausea but never barfed. This one has sent me to the porcelain god with great regularity. It was so regular, that Scott thought it was funny, but more on my darling spouse in a second. I was so nauseated that I couldn't stand to be near almost all types of food. But being a stay-at-home parent with toddlers, it was my job to make them three meals a day, limiting my vomiting to in between recipe steps. At night, I'd hold off serving dinner for as long as possible, praying Scott would get home from work so I could barricade myself in the bedroom and burn rose scented candles after spraying orange room deodorizer everywhere.
One night at dinner, I was determined to sit at the table with everyone, a vain attempt to achieve some semblance of normalcy. Then I saw Abbey cramming chicken and rice into her mouth. It was all over. As I made a dash for the bathroom, I could hear Scott chuckling. When I came back ashen-faced, he said sarcastically, "Oh, so you've decided to join us? Isn't that nice guys?"
Then there was my brother. At Christmas – during which I ate virtually nothing and held my breath for nearly 48 hours for fear of being nauseated and blowing chunks all over the Christmas tree and my grandmothers – my brother thought it would be funny to thrust platters of appetizers in my face. "Hey Mere, want some shrimp? How 'bout some kielbasa? Chipped beef and onion spread?"
About a month ago, I kept feeling as though my heart was beating outside my chest. I'd read in my handy dandy "What to Expect When You're Expecting" book that this was typical for my stage of pregnancy. When I told Scott about it, his only suggestion was to stop reading that book, insinuating that I was fabricating symptoms based on what I'd read, given that I was overly emotional and hormonal. This overly emotional, hormonal chick used everything in her power not to use that book to pummel him until he cried. Instead, I grabbed his hand and pressed it to my neck so he could feel my pulse. "It's fast," he admitted. The last time I was pregnant, had I expressed any teensy-weensy whimper, he would've come running with the first aid kit and the phone dialed to 911.
Perhaps there should be a special hot line for those of us coping with second (or more) pregnancies whose husbands and male relatives have lost all capacity for compassion. Either that, or they should be forced to wear a 30-pound fanny pack bound very tightly to their guts so that they can barely breathe, get lower back aches, suffer non-stop heartburn and feel fatigued at 4 p.m. On top of that, we'll make them run after kids in diapers all day, drink 15 glasses of water (as my ob/gyn has told me) and conduct mini-art classes with the tots, trying to comfortably bend over the burgeoning belly to wipe the finger-paint and Play dough off the rug.
When they complain, we can say, "It's all in your head."
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How to load google ads in widget
This article does not meet the wiki quality standards: Please make it more useful by adding links or additional information as discussed in the quality standards. When you have done so you can delete the {{NeedsMoreWork}} template from the article to remove this warning.
Reaons: - hamishwillee (10 Oct 2012) The article is incomplete and does not include enough information for developers to create a full solution.
Now a days google provides google ads for mobile.
Mobile Webpage Markup Languages:
wml (WAP 1.x.) xhtml (WAP 2.0) chtml (imode, etc.)
Website Requirements:
Webpages must be mobile-compliant and developed with a server-side scripting language such as PHP; AdSense for mobile ad code will not display ads on standard webpages.
Ad Placement and Behaviour
Only one AdSense for mobile ad unit may be displayed per mobile webpage. A "double" ad unit can only be placed on the bottom of the page, but may appear above the page's footer (e.g. navigational links, copyright messages, etc.). Google ads displayed on a publisher's mobile-compliant webpage may not be modified or obscured in any way. After a user click, display of the advertiser's landing page may not be interrupted or prevented in any way. For instance, publishers may not display their own interstitial pages before taking users to the advertiser's website.


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I sat on the mettle stool, pushing against the wooden workbench as I twirled in circles. My eyes caught my grandfather's wink as he entered the little workshop and I grabbed the bench to a sudden stop, eager to jump to his side for a shared embrace. I melted into his belly and he tossed his brown felt cap to the chair. At ten, I could now reach my face to his soft-shaven cheek and brush him a kiss. I watched as he and my father tinkered their grease-slicked screwdrivers on old radio parts.
I sat at the Sunday table, a teenage mind distant in thought and bored with the adult conversation passing to each end. Suddenly, my eyes reached across the table to gaze at my grandfather's face. His thinned hair now gray and his bald spot broader to the sides, I imagined few men as handsome as he. A life void of higher eduction, the pitch in his voice sparkled with interest as I discussed my college goals.
I was called home from college, my grandfather abruptly scheduled for heart surgery. I leaned against the flat gurney as he lay, turning his face to smile and give a charm-filled wink. I brushed my hand across his and he clasped hold, still sweet and kind and beautiful.
And that was my last memory of a gentle, simple man.
I new my grandfather labored years in the Oklahoma oilfields; a hard-toiled job with rough-talking men and sweat-drenched days. I presumed he was a roughneck; an uneducated sort, but with a heart and mind that gave competition to the best.
I was content with that; left with a memory of the kindest grandfather God made.
And then I searched his 1930 Census record. Ancestry.com's latest beta device provides clear pictures of your grandparent's records, highlighting fascinating details. I clicked the zoom to read my grandfather's employer:
"Oil company."
And then occupation:
My heart contracted as I realized my simple grandfather quietly spent a career in charge of the Reno Oil Company in Nowata, Oklahoma.
Or perhaps I was told, but failed to give his history my attention.
Become reacquinted with your grandparents and give their life the attention you give to ancestors you never knew. Find them on the 1930 US Census and discover if they had a radio in their home or served in the military.
Or lived a life you never knew.
Keep searching for answers,
(Only 36 days until the 1940 US Census is searchable for free!)


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Ries GroupPublications
The yeast kinetochore - structural insights from optical microscopy.
Cieslinski, K. & Ries, J.
Curr Opin Chem Biol. 2014 Jun;20:1-8. doi: 10.1016/j.cbpa.2014.03.020. Epub 2014Apr 22.
In eukaryotic organisms each chromosome is captured by spindle microtubules. This interaction is mediated by an evolutionarily conserved, multi-protein complex called kinetochore. The main function of the kinetochore is to maintain correct chromosome segregation during cell division by transporting each sister chromatid to the spindle pole bodies localized at the opposite sites of the yeast nucleus. The kinetochore of budding yeast (Saccharomyces cerevisiae) is often used as a model system due to its simple composition compared to higher eukaryotes. This review highlights results obtained using optical imaging that revealed relative positions and stoichiometry of the major components of the budding yeast kinetochore.
Europe PMC
Tracking single serotonin transporter molecules at the endoplasmic reticulum and plasma membrane.
Anderluh, A., Klotzsch, E., Ries, J., Reismann, A.W., Weber, S., Folser, M., Koban, F., Freissmuth, M., Sitte, H.H. & Schutz, G.J.
Biophys J. 2014 May 6;106(9):L33-5. doi: 10.1016/j.bpj.2014.03.019.
Transmembrane proteins are synthesized and folded in the endoplasmic reticulum (ER), an interconnected network of flattened sacs or tubes. Up to now, this organelle has eluded a detailed analysis of the dynamics of its constituents, mainly due to the complex three-dimensional morphology within the cellular cytosol, which precluded high-resolution, single-molecule microscopy approaches. Recent evidences, however, pointed out that there are multiple interaction sites between ER and the plasma membrane, rendering total internal reflection microscopy of plasma membrane proximal ER regions feasible. Here we used single-molecule fluorescence microscopy to study the diffusion of the human serotonin transporter at the ER and the plasma membrane. We exploited the single-molecule trajectories to map out the structure of the ER close to the plasma membrane at subdiffractive resolution. Furthermore, our study provides a comparative picture of the diffusional behavior in both environments. Under unperturbed conditions, the majority of proteins showed similar mobility in the two compartments; at the ER, however, we found an additional 15% fraction of molecules moving with 25-fold faster mobility. Upon degradation of the actin skeleton, the diffusional behavior in the plasma membrane was strongly influenced, whereas it remained unchanged in the ER.
Europe PMC
Localization microscopy in yeast.
Mund, M., Kaplan, C. & Ries, J.
Methods Cell Biol. 2014;123C:253-271. doi: 10.1016/B978-0-12-420138-5.00014-8.
Conventional light and fluorescence microscopy techniques have offered tremendous insight into cellular processes and structures. Their resolution is however intrinsically limited by diffraction. Superresolution techniques achieve an order of magnitude higher resolution. Among these, localization microscopy relies on the position determination of single emitters with nanometer accuracy, which allows the subsequent reconstruction of an image of the target structure. In this chapter, we provide general guidelines for localization microscopy with a focus on Saccharomyces cerevisiae. Its different cellular architecture complicates efforts to directly transfer protocols established in mammalian cells to yeast. We compare different methodologies to label structures of interest and provide protocols for the respective sample preparation, which are not limited to yeast. Using these guidelines, nanoscopic subcellular structures in yeast can be investigated by localization microscopy, which perfectly complements live-cell fluorescence and electron microscopy.
Europe PMC
A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of beta-amyloid production.
Udayar, V., Buggia-Prevot, V., Guerreiro, R.L., Siegel, G., Rambabu, N., Soohoo, A.L., Ponnusamy, M., Siegenthaler, B., Bali, J., Simons, M., Ries, J., Puthenveedu, M.A., Hardy, J., Thinakaran, G. & Rajendran, L.
Cell Rep. 2013 Dec 26;5(6):1536-51. doi: 10.1016/j.celrep.2013.12.005.
Alzheimer's disease (AD) is characterized by cerebral deposition of beta-amyloid (Abeta) peptides, which are generated from amyloid precursor protein (APP) by beta- and gamma-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Abeta levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Abeta production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled beta-secretase endosomal recycling to the plasma membrane and thus affected Abeta production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Abeta levels and show how systems biology approaches can unravel the molecular complexity underlying AD.
Europe PMC
Superresolution imaging of amyloid fibrils with binding-activated probes.
Ries, J., Udayar, V., Soragni, A., Hornemann, S., Nilsson, K.P., Riek, R., Hock, C., Ewers, H., Aguzzi, A.A. & Rajendran, L.
ACS Chem Neurosci. 2013 Jul 17;4(7):1057-61. doi: 10.1021/cn400091m. Epub 2013Apr 22.
Protein misfolding into amyloid-like aggregates underlies many neurodegenerative diseases. Thus, insights into the structure and function of these amyloids will provide valuable information on the pathological mechanisms involved and aid in the design of improved drugs for treating amyloid-based disorders. However, determining the structure of endogenous amyloids at high resolution has been difficult. Here we employ binding-activated localization microscopy (BALM) to acquire superresolution images of alpha-synuclein amyloid fibrils with unprecedented optical resolution. We propose that BALM imaging can be extended to study the structure of other amyloids, for differential diagnosis of amyloid-related diseases and for discovery of drugs that perturb amyloid structure for therapy.
Europe PMC
The bacterial SMC complex displays two distinct modes of interaction with the chromosome.
Kleine Borgmann, L.A., Ries, J., Ewers, H., Ulbrich, M.H. & Graumann, P.L.
Cell Rep. 2013 May 30;3(5):1483-92. doi: 10.1016/j.celrep.2013.04.005. Epub 2013May 9.
The bacterial SMC (structural maintenance of chromosomes) complex binds nonspecifically to DNA in vitro and forms two discrete subcellular centers in vivo, one in each cell half. How this distribution is maintained is unclear. We show by time-lapse imaging of single molecules that the localization is achieved through limited, yet rapid movement of the SMC subunits through the nucleoid. Accessory ScpAB subunits mediate the arrest of 20% of SMC molecules at the center of a cell half and do not move together with the 80% mobile SMC molecules. Only free SMC, but not the preformed SMC/ScpAB complex, was able to bind to DNA in vitro, revealing distinct functions of SMC fractions. Thus, whereas SMC alone dynamically interacts with many sites on the chromosome, it forms static assemblies together with ScpAB complex partners. Our findings reveal two distinct modes of interaction of SMC with the chromosome and indicate that limited diffusion within a confined space and transient arrest may be a general mechanism for positioning proteins within a chromosome and within a noncompartmentalized cell.
Europe PMC
Tuning the "roadblock" effect in Kinesin-based transport.
Schmidt, C., Kim, B., Grabner, H., Ries, J., Kulomaa, M. & Vogel, V.
Nano Lett. 2012 Jul 11;12(7):3466-71. Epub 2012 Jun 14.
Major efforts are underway to harness motor proteins for technical applications. Yet how to best attach cargo to microtubules that serve as kinesin-driven "molecular shuttles" without compromising transport performance remains challenging. Furthermore, microtubule-associated proteins (MAPs) can block motor protein-powered transport in neurons, which can lead to neurodegenerative diseases. Again it is unclear how different physical roadblock parameters interfere with the stepping motion of kinesins. Here, we employ a series of MAPs, tailored (strept)avidins, and DNA as model roadblocks and determine how their geometrical, nanomechanical, and electrochemical properties can reduce kinesin-mediated transport. Our results provide insights into kinesin transport regulation and might facilitate the choice of appropriate cargo linkers for motor protein-driven transport devices.
Europe PMC
A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.
Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H.
Nat Methods. 2012 Jun;9(6):582-4. doi: 10.1038/nmeth.1991. Epub 2012 Apr 29.
We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.
Europe PMC
Fluorescence correlation spectroscopy.
Ries, J. & Schwille, P.
Bioessays. 2012 May;34(5):361-8. doi: 10.1002/bies.201100111. Epub 2012 Mar 13.
Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure concentrations, mobilities, and interactions of fluorescent biomolecules. It can be applied to various biological systems such as simple homogeneous solutions, cells, artificial, or cellular membranes and whole organisms. Here, we introduce the basic principle of FCS, discuss its application to biological questions as well as its limitations and challenges, present an overview of novel technical developments to overcome those challenges, and conclude with speculations about the future applications of fluorescence fluctuation spectroscopy.
Europe PMC
Fluorescence Correlation Spectroscopy (FCS).
Ries, J., Weidemann, T. & Schwille, P.
Comprehensive Biophysics 2, Biophysical Techniques for Characterization of Cells, 210-245 (2012).
Binding-activated localization microscopy of DNA structures.
Schoen, I., Ries, J., Klotzsch, E., Ewers, H. & Vogel, V.
Nano Lett. 2011 Sep 14;11(9):4008-11. Epub 2011 Aug 18.
Many nucleic acid stains show a strong fluorescence enhancement upon binding to double-stranded DNA. Here we exploit this property to perform superresolution microscopy based on the localization of individual binding events. The dynamic labeling scheme and the optimization of fluorophore brightness yielded a resolution of approximately 14 nm (fwhm) and a spatial sampling of 1/nm. We illustrate our approach with two different DNA-binding dyes and apply it to visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy (BALM) can be extended to other dyes and targets such as protein structures.
Europe PMC
Cxcl12 evolution--subfunctionalization of a ligand through altered interaction with the chemokine receptor.
Boldajipour, B., Doitsidou, M., Tarbashevich, K., Laguri, C., Yu, S.R., Ries, J., Dumstrei, K., Thelen, S., Dorries, J., Messerschmidt, E.M., Thelen, M., Schwille, P., Brand, M., Lortat-Jacob, H. & Raz
Development. 2011 Jul;138(14):2909-14.
The active migration of primordial germ cells (PGCs) from their site of specification towards their target is a valuable model for investigating directed cell migration within the complex environment of the developing embryo. In several vertebrates, PGC migration is guided by Cxcl12, a member of the chemokine superfamily. Interestingly, two distinct Cxcl12 paralogs are expressed in zebrafish embryos and contribute to the chemotattractive landscape. Although this offers versatility in the use of chemokine signals, it also requires a mechanism through which migrating cells prioritize the relevant cues that they encounter. Here, we show that PGCs respond preferentially to one of the paralogs and define the molecular basis for this biased behavior. We find that a single amino acid exchange switches the relative affinity of the Cxcl12 ligands for one of the duplicated Cxcr4 receptors, thereby determining the functional specialization of each chemokine that elicits a distinct function in a distinct process. This scenario represents an example of protein subfunctionalization--the specialization of two gene copies to perform complementary functions following gene duplication--which in this case is based on receptor-ligand interaction. Such specialization increases the complexity and flexibility of chemokine signaling in controlling concurrent developmental processes.
Europe PMC
Principles and Applications of Fluorescence Correlation Spectroscopy (FCS).
Schwille, P. & Ries, J.
6385 (Biophotonics: Dordrecht, 2010).doi:10.1007/978-90-481-9977-8_4
Fluorescence correlation spectroscopy (FCS) is one of the various modern optical techniques that allow access to single fluorescently labeled biomolecules in aqueous solution. In contrast to other fluorescence measurements, however, the parameter of primary interest is not the emission intensity itself, but rather spontaneous intensity fluctuations caused by the minute deviations of a small ensemble of molecules from thermal equilibrium. In general, all physical parameters that give rise to fluctuations in the fluorescence signal are accessible by FCS. It is, for example, rather straightforward to determine local concentrations, mobility coefficients or characteristic rate constants of inter- or intramolecular reactions of fluorescently labeled biomolecules at nanomolar to micromolar concentrations.
DNA damage regulates the mobility of Brca2 within the nucleoplasm of living cells.
Jeyasekharan, A.D., Ayoub, N., Mahen, R., Ries, J., Esposito, A., Rajendra, E., Hattori, H., Kulkarni, R.P. & Venkitaraman, A.R.
Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21937-42. Epub 2010 Nov 22.
How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus.
Europe PMC
Automated suppression of sample-related artifacts in Fluorescence Correlation Spectroscopy.
Ries, J., Bayer, M., Csucs, G., Dirkx, R., Solimena, M., Ewers, H. & Schwille, P.
Opt Express. 2010 May 24;18(11):11073-82. doi: 10.1364/OE.18.011073.
Fluorescence Correlation Spectroscopy (FCS) in cells often suffers from artifacts caused by bright aggregates or vesicles, depletion of fluorophores or bleaching of a fluorescent background. The common practice of manually discarding distorted curves is time consuming and subjective. Here we demonstrate the feasibility of automated FCS data analysis with efficient rejection of corrupted parts of the signal. As test systems we use a solution of fluorescent molecules, contaminated with bright fluorescent beads, as well as cells expressing a fluorescent protein (ICA512-EGFP), which partitions into bright secretory granules. This approach improves the accuracy of FCS measurements in biological samples, extends its applicability to especially challenging systems and greatly simplifies and accelerates the data analysis.
Europe PMC
Scanning FCS for the characterization of protein dynamics in live cells.
Petrasek, Z., Ries, J. & Schwille, P.
Methods Enzymol. 2010;472:317-43.
Scanning fluorescence correlation spectroscopy (sFCS) is the generic term for a group of fluorescence correlation techniques where the measurement volume is moved across the sample in a defined way. The introduction of scanning is motivated by its ability to alleviate or remove several distinct problems often encountered in standard FCS, and thus, to extend the range of applicability of fluorescence correlation methods in biological systems. These problems include poor statistical accuracy in measurements with slowly moving molecules, photobleaching, optical distortions affecting the calibration of the measurement volume, membrane instabilities, etc. Here, we present an overview of sFCS methods, explaining their benefits, implementation details, requirements, and limitations, as well as relations to each other. Further, we give examples of different sFCS implementations as applied to cellular systems, namely large-circle sFCS to measure protein dynamics in embryo cortex and line sFCS to measure protein diffusion and interactions in unstable membranes.
Europe PMC
A Comprehensive Framework for Fluorescence Cross-Correlation Spectroscopy.
J. Ries, Z. Petrasek, A. Garcia-Saez, and P. Schwille.
New Journal of Physics 12, 113009 (2010).
Membrane promotes tBID interaction with BCL(XL).
Garcia-Saez, A.J., Ries, J., Orzaez, M., Perez-Paya, E. & Schwille, P.
Nat Struct Mol Biol. 2009 Nov;16(11):1178-85. Epub 2009 Oct 11.
Two important questions on the molecular mechanism of the B cell CLL/lymphoma 2 (BCL2) proteins involve the interaction network between pro- and antiapoptotic members and the role of their translocation to the mitochondrial membrane during apoptosis. We used fluorescence correlation spectroscopy to quantify the molecular interactions of BH3-interacting domain death agonist (BID) and its truncated form tBID with the B cell lymphoma extra-large protein truncated at the C terminus (BCL(XL)DeltaCt) in solution and in membranes, and we found that (i) only the active form tBID binds to BCL(XL)DeltaCt and (ii) that the membrane strongly promotes binding between them. Particularly, a BH3 peptide from BID disrupts the tBID-BCL(XL) complex in solution, but only partially in lipid bilay
Europe PMC
Detergent-activated BAX protein is a monomer.
Ivashyna, O., Garcia-Saez, A.J., Ries, J., Christenson, E.T., Schwille, P. & Schlesinger, P.H.
J Biol Chem. 2009 Sep 4;284(36):23935-46. Epub 2009 Jun 29.
BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation with detergents, such as n-octyl glucoside. During BAX activation with n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.
Europe PMC
Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.
Yu, S.R., Burkhardt, M., Nowak, M., Ries, J., Petrasek, Z., Scholpp, S., Schwille, P. & Brand, M.
Nature. 2009 Sep 24;461(7263):533-6. Epub 2009 Sep 9.
It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.
Europe PMC
Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.
Ries, J., Yu, S.R., Burkhardt, M., Brand, M. & Schwille, P.
Nat Methods. 2009 Sep;6(9):643-5. Epub 2009 Aug 2.
Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.
Europe PMC
Accurate determination of membrane dynamics with line-scan FCS.
Ries, J., Chiantia, S. & Schwille, P.
Biophys J. 2009 Mar 4;96(5):1999-2008.
Here we present an efficient implementation of line-scan fluorescence correlation spectroscopy (i.e., one-dimensional spatio-temporal image correlation spectroscopy) using a commercial laser scanning microscope, which allows the accurate measurement of diffusion coefficients and concentrations in biological lipid membranes within seconds. Line-scan fluorescence correlation spectroscopy is a calibration-free technique. Therefore, it is insensitive to optical artifacts, saturation, or incorrect positioning of the laser focus. In addition, it is virtually unaffected by photobleaching. Correction schemes for residual inhomogeneities and depletion of fluorophores due to photobleaching extend the applicability of line-scan fluorescence correlation spectroscopy to more demanding systems. This technique enabled us to measure accurate diffusion coefficients and partition coefficients of fluorescent lipids in phase-separating supported bilayers of three commonly used raft-mimicking compositions. Furthermore, we probed the temperature dependence of the diffusion coefficient in several model membranes, and in human embryonic kidney cell membranes not affected by temperature-induced optical aberrations.
Europe PMC
Fluorescence correlation spectroscopy in membrane structure elucidation.
Chiantia, S., Ries, J. & Schwille, P.
Biochim Biophys Acta. 2009 Jan;1788(1):225-33. Epub 2008 Aug 29.
This review describes the application of fluorescence correlation spectroscopy (FCS) for the study of biological membranes. Monitoring the fluorescence signal fluctuations, it is possible to obtain diffusion constants and concentrations for several membrane components. Focusing the attention on lipid bilayers, we explain the technical difficulties and the new FCS-based methodologies introduced to overcome them. Finally, we report several examples of studies which apply FCS on both model and biological membranes to obtain interesting insight in the topic of lateral membrane organization.
Europe PMC
Plasma membranes are poised for activation of raft phase coalescence at physiological temperature.
Lingwood, D., Ries, J., Schwille, P. & Simons, K.
Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10005-10. Epub 2008 Jul 9.
Cell membranes are not randomly organized, but rather are populated by fluctuating nanoassemblies of increased translational order termed lipid rafts. This lateral heterogeneity can be biophysically extended because cooling formaldehyde-isolated plasma membrane preparations results in separation into phases similar to the liquid-ordered (Lo) and liquid-disordered (Ld) states seen in model membrane systems [Baumgart T, et al. (2007) Proc Natl Acad Sci USA 104:3165-3170]. In this work we demonstrate that raft clustering, i.e., amplifying underlying raft-based connectivity to a larger scale, makes an analogous capacity accessible at 37 degrees C. In plasma membranes at this temperature, cholera toxin-mediated cross-linking of the raft ganglioside GM1 induced the sterol-dependent emergence of a slower diffusing micrometer-scale phase that was enriched in cholesterol and selectively reorganized the lateral distribution of membrane proteins. Although parallels can be drawn, we argue that this raft coalescence in a complex biological matrix cannot be explained by only those interactions that define Lo formation in model membranes. Under this light, our induction of raft-phase separation suggests that plasma membrane composition is poised for selective and functional raft clustering at physiologically relevant temperature.
Europe PMC
Total internal reflection fluorescence correlation spectroscopy: effects of lateral diffusion and surface-generated fluorescence.
Ries, J., Petrov, E.P. & Schwille, P.
Biophys J. 2008 Jul;95(1):390-9. Epub 2008 Mar 13.
Fluorescence correlation spectroscopy with total internal reflection excitation (TIR-FCS) is a promising method with emerging biological applications for measuring binding dynamics of fluorescent molecules to a planar substrate as well as diffusion coefficients and concentrations at the interface. Models for correlation functions proposed so far are rather approximate for most conditions, since they neglect lateral diffusion of fluorophores. Here we propose accurate extensions of previously published models for axial correlation functions taking into account lateral diffusion through detection profiles realized in typical experiments. In addition, we consider the effects of surface-generated emission in objective-based TIR-FCS. The expressions for correlation functions presented here will facilitate quantitative and accurate measurements with TIR-FCS.
Europe PMC
New concepts for fluorescence correlation spectroscopy on membranes.
Ries, J. & Schwille, P.
Phys Chem Chem Phys. 2008 Jun 28;10(24):3487-97. Epub 2008 Mar 27.
Fluorescence correlation spectroscopy (FCS) is a powerful tool to measure useful physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in model and cell membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we assess the potential and limitations of standard confocal FCS on lipid membranes and present recent developments which facilitate accurate and quantitative measurements on such systems. In particular, we discuss calibration-free diffusion and concentration measurements using z-scan FCS and two focus FCS and present several approaches using scanning FCS to accurately measure slow dynamics. We also show how surface confined FCS enables the study of membrane dynamics even in presence of a strong cytosolic background and how FCS with a variable detection area can reveal submicroscopic heterogeneities in cell membranes.
Europe PMC
Spatial regulators for bacterial cell division self-organize into surface waves in vitro.
Loose, M., Fischer-Friedrich, E., Ries, J., Kruse, K. & Schwille, P.
Science. 2008 May 9;320(5877):789-92.
In the bacterium Escherichia coli, the Min proteins oscillate between the cell poles to select the cell center as division site. This dynamic pattern has been proposed to arise by self-organization of these proteins, and several models have suggested a reaction-diffusion type mechanism. Here, we found that the Min proteins spontaneously formed planar surface waves on a flat membrane in vitro. The formation and maintenance of these patterns, which extended for hundreds of micrometers, required adenosine 5'-triphosphate (ATP), and they persisted for hours. We present a reaction-diffusion model of the MinD and MinE dynamics that accounts for our experimental observations and also captures the in vivo oscillations.
Europe PMC
Role of ceramide in membrane protein organization investigated by combined AFM and FCS.
Chiantia, S., Ries, J., Chwastek, G., Carrer, D., Li, Z., Bittman, R. & Schwille, P.
Biochim Biophys Acta. 2008 May;1778(5):1356-64. Epub 2008 Feb 29.
Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.
Europe PMC
Supercritical angle fluorescence correlation spectroscopy.
Ries, J., Ruckstuhl, T., Verdes, D. & Schwille, P.
Biophys J. 2008 Jan 1;94(1):221-9. Epub 2007 Sep 7.
We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.
Europe PMC
Rho regulates membrane transport in the endocytic pathway to control plasma membrane specialization in oligodendroglial cells.
Kippert, A., Trajkovic, K., Rajendran, L., Ries, J. & Simons, M.
J Neurosci. 2007 Mar 28;27(13):3560-70.
Differentiation of oligodendrocytes is associated with dramatic changes in plasma membrane structure, culminating in the formation of myelin membrane sheaths. Previous results have provided evidence that regulation of endocytosis may represent a mechanism to control myelin membrane growth. Immature oligodendrocytes have a high rate of clathrin-independent endocytosis for the transport of membrane to late endosomes/lysosomes (LE/Ls). After maturation and receiving signals from neurons, endocytosis is reduced and transport of membrane from LE/Ls to the plasma membrane is triggered. Here, we show that changes in Rho GTPase activity are responsible for switching between these two modes of me
Europe PMC
Combined AFM and two-focus SFCS study of raft-exhibiting model membranes.
Chiantia, S., Ries, J., Kahya, N. & Schwille, P.
Chemphyschem. 2006 Nov 13;7(11):2409-18.
Dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/cholesterol) model membranes exhibit liquid-liquid phase separation and therefore provide a physical model for the putative liquid-ordered domains present in cells. Here we present a combination of atomic force microscopy (AFM) imaging, force measurements, confocal fluorescence imaging and two-focus scanning fluorescence correlation spectroscopy (two-focus SFCS) to obtain structural and dynamical information about this model membrane system. Partition coefficients and diffusion coefficients in the different phases were measured with two-focus SFCS for numerous fluorescent lipid analogues and proteins, while being directly related to the lateral organization of the membrane and its mechanical properties probed by AFM. Moreover we show how the combination of these different approaches is effective in reducing artifacts resulting from the use of a single technique.
Europe PMC
Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy.
Meacci, G., Ries, J., Fischer-Friedrich, E., Kahya, N., Schwille, P. & Kruse, K.
Phys Biol. 2006 Nov 28;3(4):255-63.
In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spectroscopy. Two distinct timescales are clearly visible in the correlation curves. While the faster timescale can be attributed to cytoplasmic diffusion, the slower timescale could result from diffusion of membrane-bound proteins or from protein exchange between the cytoplasm and the membrane. We determine the diffusion constant of cytoplasmic MinD to be approximately 16 microm(2) s(-1), while for MinE we find about 10 microm(2) s(-1), independently of the processes responsible for the slower time-scale. The implications of the measured values for the oscillation mechanism are discussed.
Europe PMC
Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy.
Ries, J. & Schwille, P.
Biophys J. 2006 Sep 1;91(5):1915-24. Epub 2006 Jun 16.
Here we discuss the application of scanning fluorescence correlation spectroscopy (SFCS) using continuous wave excitation to analyze membrane dynamics. The high count rate per molecule enables the study of very slow diffusion in model and cell membranes, as well as the application of two-foci fluorescence cross-correlation spectroscopy for parameter-free determination of diffusion constants. The combination with dual-color fluorescence cross-correlation spectroscopy with continuous or pulsed interleaved excitation allows binding studies on membranes. Reduction of photobleaching, higher reproducibility, and stability compared to traditional FCS on membranes, and the simple implementation in a commercial microscopy setup make SFCS a valuable addition to the pool of fluorescence fluctuation techniques.
Europe PMC
Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS.
Chiantia, S., Kahya, N., Ries, J. & Schwille, P.
Biophys J. 2006 Jun 15;90(12
The sphingolipid ceramides are known to influence lipid lateral organization in biological membranes. In particular, ceramide-induced alterations of microdomains can be involved in several cell functions, ranging from apoptosis to immune response. We used a combined approach of atomic force microscopy, fluorescence correlation spectroscopy, and confocal fluorescence imaging to investigate the effects of ceramides in model membranes of biological relevance. Our results show that physiological quantities of ceramide in sphingomyelin/dioleoylphosphatidylcholine/cholesterol supported bilayers lead to a significant rearrangement of lipid lateral organization. Our experimental setup allowed a simultaneous characterization of both structural and dynamic modification of membrane microdomains, induced by the presence of ceramide. Formation of similar ceramide-enriched domains and, more general, alterations of lipid-lipid interactions can be of crucial importance for the biological function of cell membranes.
Europe PMC
Experimental vacuum squeezing in rubidium vapor via self-rotation.
Ries, J., Brezger, B. & Lvovsky, A.
Phys Rev a 68, (2003)
Quantum scissors: Teleportation of single-mode optical states by means of a nonlocal single photon
Ries, J. & Lvovsky, A.
EPL (Europhysics Letters) (2003).