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EPISD Begins Training for Safe Route To School Program
POSTED: Tuesday, May 24, 2011 - 11:16am
UPDATED: Tuesday, May 24, 2011 - 2:16pm
EL PASO- Teachers involved in the Safe Routes to Schools program will get a lesson in bike and pedestrian safety from the Texas Bike Coalition on Thursday.
The training, which is held at MacArthur Elementary/Intermediate School, is part of a $4.7 million grant program which begins this Fall for 3rd to 5th grade students at 47 elementary schools.
Students who participate and successfully complete the program are eligible for a free bicycle and helmet. Each of the 47 campuses will award 134 bicycles at the end of the school year for the next two years.
Funding will be used to educate students on bike and pedestrian safety and traffic education, a public awareness campaign, overtime costs for law enforcement, and the purchase of bicycles and safety gear.
Comments News Comments
You have got to be kidding!!! Our tax money is going to teach kids how to ride a bike? And to actually give them bikes? Now...how about guarding them from sexual perverts? Because no good parent would allow their child to walk or ride a bike to school for fear the child would be accosted by someone determined to expose themselves or some other heinous child involved crime.
This is beyond all comprehension.


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So, no need to berate me, I have already posted the "I EFFED up" thread. This is results. My "Forever will it Dominate Your Destiny" Stout began fermentation at 109F..... 12 lbs 2 row 1 lb chocolate 1 lb black patent 1 lb crystal 120 1 lb roasted 1 oz Fuggles 60 minutes dry ale yeast in fermenter Poured my 110F wort onto the yeast in my super chilled keg in my 0 F Keezer.... Within 60 minutes it was beginning to ferment add dropped a staggering 1 degree. I set the keezer to 34F and went to bed. Next morning fermentation was dormant. Warmed it up slowly to 60F. Fermentation restarted slow and low.Beer now kegged and carbed. Tastes Perfect. No Fusel or "Banana" I guess as a worst case:if you pitch too warm, get it cold as you can, way below ideal fermenting temps, slowly warm up.
I had an IPA that I was ready to throw out because I started the fermentation at about 82 and couldn't get it below 75 for about 48 hours. I was explaining to a friend that it could produce fusel alcohols so I decided to take the bung out of the top of my fermenter and smell the off gas. I almost fainted from the horrific harsh alcoholic smell. I left the beer on the giant yeast cake for and extra couple weeks before dry hopping and the beer came out great with a minimal bubblegum off flavor. I was so surprised. Some of the yeast strains we get to use are pretty good at cleanup. I am starting to love 1272 and 1056.
I am hoping that my experience proves that yeast can clean it up during primary fermentation. I have found IPAs to be very forgiving. The high bitterness masks some flavors.
While everything you did was a little extreme you actually followed best practice for properly pitching yeast. It is recommended that you pitch yeast slightly colder than desired fermentation temperature and then allow to self rise to the desired temp. This promotes a slow, controlled growth phase of the yeast and proves to be beneficial for overall fermentation. Good Job!
Allow me to berate you, you freaking dolt: That's too warm to pitch yeast. /berating
_________________________________Skal! Den Faaborg Bryggeri
I think above 95F, the yeast mortality rate is pretty high. You may not really have done much fermentation at those temps, and instead underpitched at low temps.
Say no to intolerance: love gluten and lactose.

subset: commoncrawl


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I am headed to my local Sherwin Williams paint store to grab a few strainer bags. They have 5 gallon size for a buck. Anything I need to be sure of before I buy some for brewing?
The paint strainer bags I got at my Ace hardware are coarser than the cheesecloth I used to use. Be careful of the amount of draff you get into your boil kettle. Too fine of a crush will cause astringency. I now just lauter in the bag, with the bag in my bottling bucket. It works fine because I can vorlauf that way.
To paraphrase Dr. England - "Off-flavors smooth with time. So do mountains. Brew it right from the start!" My blogsite: http://nateobrew.blogspot.com/
I get the same bags from Sherwin Williams too, I can usually fit about half of my grain bill in one bag(with a zip tie). I have used several crushes from several local and not so local brew shops with zero ill results. My only recommendation would be to remove the paper like tag sewn into it.


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In honor of the International Day of Families, May 15, a video illustrating Article 16 of the Universal Declaration of Human Rights which reads:(1) Men and women of full age, without any limitation due to race, nationality or religion, have the right to marry and to found a family. They are entitled to equal rights as to marriage, during marriage and at its dissolution. (2) Marriage shall be entered into only with the free and full consent of the intending spouses. (3) The family is … [Read more...]
The Church of Scientology Greater Cincinnati celebrated the opening of its expansive new home Saturday, February 25. The inauguration was attended by more than a thousand Scientologists and state, county and city officials. Located in Florence, Kentucky, within the Cincinnati-Northern Kentucky metropolitan area, the Church's new home stands at the intersection of Dixie Highway and Union Pike, the junction that inspired Florence's original name of "Crossroads." The new facility will not only prov … [Read more...]
Scientology settles into historic home
A photo story on the Church of Scientology Tampa appears on the Tampa Bay Fox TV affiliate."Chances are you've driven by or even been to Ybor Square, the home of Creative Loafing, Spaghetti Warehouse and the Church of Scientology. But you might not know exactly how much history it holds..."Tampa's first brick cigar factory, built in 1886 and located at Ybor Square, now is home to the Church of Scientology. (Photos Kerry Klecic/MyFoxTampaBay.com) … [Read more...]
The Church of Scientology is formed into an ecclesiastical structure which unifies and aligns a multitude of diverse religious activities, including not only ministering Scientology religious services and practices, but proselytization, ecclesiastical management, relay of communication, production of dissemination materials and many other functions. Thus the Scientology religious community is united both by common beliefs and practices and an organizational form uniquely suited to its religious … [Read more...]
Scientology Offers Open House For New Downtown Sacramento Building
SACRAMENTO (CBS13) — After its grand opening over the weekend, the Church of Scientology offered the media a closer look at its new Sacramento location on Monday after a transition that took eight years to complete.They took great care to preserve the exterior facade of the historic building on J and 6th streets. Inside it's completely different, gutted and renovated from top to bottom.The building, formerly the Ramona Hotel, is six stories and 58,000 square feet."It's a big diffe … [Read more...]
Scientologists have a new church
By Jennifer Garza firstname.lastname@example.com: Tuesday, Jan. 31, 2012 - 12:00 am | Page 2BIt took two years and about $10 million, but the 12,000 local members of the Church of Scientology in Sacramento have a new house of worship.The new Church of Scientology of Sacramento is a seven-story building at the site of the Old Ramona Hotel on the corner of Sixth and J streets. Inside can be found a chapel, multiple seminar rooms, classrooms, dozens of rooms for auditing or spir … [Read more...]


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Oct 06, 2013 Bob Buskirk All News, Gaming News, Hardware News 0
AMD Radeon R9 280X, Radeon R9 270X, and many different AMD R7 series card from several different AMD AIC partners have been unveiled. Many of these cards feature a non-reference cooling design from the manufacturer and some of the more notable cards are ASUS's DirectCU, MSI's Gaming Series and XFX double dissipation.
Oct 04, 2013 Bob Buskirk All News, Gaming News, Hardware News 1
Photos of ASUS's top of the line Republic of Gamers Matrix R9 280X Platinum graphics card have been leaked! Looking at the photos you can see that this card is pretty much a rebrand of the Radeon HD 7970 ROG Matrix card. The only real difference is that this card has overclocked memory. The clocks for core clock, boost clock and memory are 950 MHz, 1100 MHz and 1600 MHz respectively.
Sep 27, 2013 Ahmed Humayun All News, Hardware News 0
The RT-AC68U Wireless-AC1900 Dual-band router has finally been announced by ASUS and its availability at pre order is now possible with the help of selected resellers. ASUS has always been a popular name in this industry and it is one of the leading brand names in technology and this much awaited release has finally been communicated to the onlookers by ASUS in an official announcement.
Sep 16, 2013 Ahmed Humayun All News, Gaming News, Hardware News 0
ASUS has publicized its new graphics card; the GeForce GTX 760 DirectCU Mini which has been built and designed exclusively for the mini ITX motherboards which are mostly found in the small form factor PCs. Yet the performance of the graphics card is outstanding with the NVIDIA GTX 760 GPU and is perfect to be used for all sorts of tasks. The tiny card is a dual slot and is a mere 17 inches. With a PCB of 170 mm in height, it can be best suited to all space restricted cases. The 8-pin PCIe power connector provides the necessary power to the 5-phase VRM which forms part of this PCB.
Sep 15, 2013 Ahmed Humayun All News, Hardware News 0
One of the world's very first NFC enabled desktop has been launched by ASUS. The multinational computer hardware and electronics company has announced the M70 PC which will be equipped with the latest fourth generation Intel core processor for a remarkable performance and to make increased support for all sorts of demanding tasks, the best […]
Sep 13, 2013 Ahmed Humayun All News, Hardware News 0
Five overclocking world records were broken by the ASUS Republic of Gamers (ROG) Rampage IV Black Edition. ASUS, the leading multinational of computer hardware and other electronics, hosted an event in San Francisco along with Corsair and Intel where the Rampage IV black Edition was paired with other high end components and was given the highest scores in five different areas.
Sep 12, 2013 Bob Buskirk All News, Hardware News 0
ASUS has just unveiled their latest Republic of Gamers motherboard the Rampage IV Black Edition at IDF13. The Rampage IV Black Edition features the X79 chipset and LGA 2011 socket which supports Intel's latest Ivy Bridge-E Processors.
Sep 06, 2013 Bob Buskirk All News, Hardware News 0
Last week we unveiled ASUS's latest X79 Motherboard for Intel's Ivy Bridge-E processors the X79 Deluxe. When we posted that article not much was know about the board, we just had to go on the photos that were leaked. Well our friends over at The Tech Report got their hands on the board for a full review so we have much more detail on this high-end board!
Sep 05, 2013 Ahmed Humayun All News, Hardware News 0
The Transformer Book Trio, based on the two world famous operating systems of Windows and Android, has finally been announced by ASUS and is expected to enter the markets soon enough. The machine providing super portability can be used as a tablet and a laptop at the same time by more than one person. This has been made possible with the two part dock-able design which brings together the portable tablet and the laptop. The dock of the Transformer book trio can be used as a normal PC station once a monitor has been attached to its head while the other part can be used as a tablet.
Aug 28, 2013 Bob Buskirk All News, Hardware News 1
ASUS's latest upcoming X79 motherboard has been unveiled, it is the X79 Deluxe. It will feature improved components and support for the upcoming Intel Ivy Bridge-E processors. Ivy Bridge-E processors are backwards compatible with all socket 2011 motherboards via a BIOS update, but that does not mean companies will not come out with new boards.
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HOLLYWOOD HOOKUP (FOX44) – After more than two decades of marriage former California Governor Arnold Schwarzenegger and his wife Maria Shriver have separated. The couple issued a statement Monday night saying they have decided to live apart while they think things through. They have four children and they just marked their 25th wedding anniversary a few weeks ago.
– Oprah Winfrey is seriously thinking about pursuing an acting career as soon as her show ends at the end of the month. The 57-year-old says she's going through a stack of Broadway plays and she was just in New York for meeting with producers.
– The Jersey Shore crew is back at it, we're told they'll be fist-pumping in Italy for season four. Word was production had to be delayed because the city of Florence didn't want them to film at several historic landmarks. But it appears that its all been worked out.
– Gisele Bundchen, Heidi Klum, and Kate Moss are the highest paid supermodels according to Forbes Magazine. Gisele rakes in about 45-million dollars a year, which makes her the top breadwinner in the Tom Brady household. The patriots quarterback reportedly makes about half that much.
Tune in every night at 9:00 p.m. on FOX44 News Baton Rouge for more Hollywood Hookup.


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Car Rear View Mirror GPS navigation system with touch screen DVR in car audio player 1080P video recorder Bluetooth with Hands f
50 Sets (Min. Order)
Place of Origin: CN;GUA
Combination: Bluetooth-Enabled;Built-in GPS;MP3 / MP4 Players;Radio Tuner;Touch Screen;TV;iPod, Disc play,DVR. Camera
Brand Name: KOVAN
Warranty: 1 Years
Car Make: All
Model Number: KC-6000
Verified Supplier - Dongguan Kovan Electronics & Technology Co., Ltd.
China (Mainland)
Contact Details
China (Mainland)
Contact Details


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List Price: $41.99Publisher: Random House Information GroupBinding: PaperbackPages: 496Size: 8.50" wide x 11.00" long x 1.25" tall Weight: 2.002Language: English
If you need to know it, it's in this book.Cracking the GRE with DVD, 2013 Editionhas been completely revised and updated for the changes made to the GRE in late 2011. It includes: • Access to 4 full-length practice tests (2 online and 2 in the book) • Practice questions with detailed explanations for every question • Key strategies for solving Text Completions, Sentence Equivalents, Numeric Entry, Quantitative Comparisons, and more • Thorough review of all GRE topics, including everything on the revised GRE • DVD supplement with tutorials and helpful information from The Princeton Review's top GRE course instructor
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Harry's Rules (Harry being Harry S. Lodge, M.D.) are only seven, but they completely reverse the typical path of aging. Exercise six days a week for the rest of your life. Quit eating crap. Connect and commit. And to prove it is his star patient, the no-punches-pulled Chris Crowley, a 70-year-old who left the slippery slope of retirement and turned his life around. Harry tells you what to do. Chris tells you how. And their argument is irresistible.
You're a guy. You have responsibilities. You think about the future, the 401K, the kids' schools. There's one other thing you can't afford not to think about: yourself. Marrying science and reality, Younger Next Year is a convincing and passionate argument that if you train for the Next Third of life, you'll have a ball. Follow its simple rules and you'll find yourself in perhaps the best shape, in mind, body, and spirit, of your life.
©2004 Chris Crowley and Henry S. Lodge, M.D.; (P)2004 HighBridge Company
"Brain-rattling, irresistible, hilarious. If you're up for it...it could change your life." (The Washington Post)
"Younger Next Year is a wiscracking but scientifically serious guide to health." (Newsweek)
"Excellent information"
I am a 43 year old physician and I found this book to be very informative, educational and motivational. I am recommending it to all of my friends and some patients. I am only giving it 3 stars because of the painful over the top reading of the parts for Chris Crowley. This book has co-authors and the person reading the Chris Crowley parts is so painful to listen to that I bought the book so I could read his portions of the book. The reader for Mr. Crowley is overly dramatic and ruined HIS portions of this book for me. However if you can get through the book, you will do yourself a big favor. The results are amazing.
I enjoy reading reviews & listening to audio books
"Great ideas but written for MEN"
Disappointment. Some of the info and ideas could have easily applied to both men and women but the book was really written to appeal to and specifically for men. The tone and manner of the authors and narrators were 100% old boy. That should have been made clear in the review.
"Don't wait until next year to start!"
Aimed at men, it was a good mix of motivation and the "whys" of how the program works. The authors don't tell you specifically what to do to exercise (there are plenty of other books for that), but they damn well tell you why you shouldn't wait another day to start. If you're over 50, 60 or 70 it's not too late and in fact it's aimed at you. If you're under 50 (but getting close) you'll see the long term benifits of beginning a long-term exercise program and looking at your life now to prepare for the "last third" and making that last third as much fun as the middle third!
And Funny too! I am a 54 year old woman. I've been working out about 6 days a week .. every week since I was 21....so as the book says I don't look 54. But I have been suffering from burn out the last few years. Over 30 years of going to the gym...can't I catch a break? This book got me excited again. It provided me with reasons to keep going and I loved his naration. I found it well written, funny, and I intend to listen to it again as soon as I finish.
"Entertaining and Motivational"
I'm a 40-something woman--not the intended audience for this book--but I loved it. Complex physiological aging processes are explained in a clear and motivational way. So are exercise benefits. I loved the focus on health rather than appearance, and the straightforward, you-can-take-it-you're-a-grownup style. There was plenty of substance for me AND it was HILARIOUS. I find it easier now to do the things I know I should do (eat right, exercise regularly). If I can get my husband to listen to it, I'm sure he will like it.
"I can't wait to get to the gym!"
I'm a 51 year old woman and found it extremely motivational. I have gone to the gym for most of my life and this book helped me understand why heart rate, nutrition and good relationships are so important.
I have also changed up my routine since reading the book and I am having better results for my age.
Chris's language is a bit gritty, but I'm sure you men will appreciate that. I do wish there was more for women in it.
"Outstanding Motivational Book"
This book has got me off my butt and back into exercise mode. Chris speaks to you as if he is your best buddy. Henry gives you the details of how your body works and why he is recommending particular types of activities. Most of the book focuses on exercise stating that it is the most difficult aspect of the program. This is a great book for anyone who needs a little push to get started exercising. It's written to appeal to men but women can get the same benifits.
"My next adventure"
I laughed, I learned and I shared. This book tells it like it is for the last third of your life, in a very funny way. My wife is now listening to the edition for women and we are having fun discussing what she is learning each time she takes a listen. It is bringing us closer by presenting dialog to us that we would have trouble discussing otherwise.
"Simply the Best"
This is my favorite out of about 50 non-fiction titles I have listened to in the last few years. I have actually started exercising everyday for the past four months as a result of listening. It may be a cliche, but this book has changed my life. I disagree with other reviewers that don't like the narration, the Chris Crowley narrator holds my interest and is fun for me to listen to. I am a clinical psychologist and have recommended the audio version to several patients.
"Practical Science - Very Motivating!"
I am grateful to Crowley and Lodge for undertaking this project ... I learned a lot, and it has changed my life.
The explanations of why our bodies go into "winter-famine" mode on the typical American diet (and what to do about it); and how to signal your cells to grow rather than decay (thru 45 mins exercise 6x week for the rest of your life!) are fascinating. And motivating. Armed with this knowledge I now have no problem carving out an hour a day for aero or weights; before I wondered why I needed to do it. Harry is the kind doctor I wish I had. And Chris, well he's a character: self-absorbed, a little neurotic, amusing part of the time, but less than he thinks he is; in other words, a lot like me!
Highly recommended for anyone age 45+ who would prefer to stay young right up to age 100.
"Horrible audiobook"
Both narrators sound like used-car salesmen and come across as smug, insincere, conniving and with an agenda. There are a few golden nuggets hidden in the book, but really only enough to fill a few pages and the rest seems to be a waste. I would not recommend this audiobook at all
The narration is annoying. The two voices sound like they are from beer or toothpaste commercials. Totally artificial and disingenuous
There are some nuggets up to the point I couldn't take any more. Exercise more regularly, eat more healthy food, be bold and join groups. Hmmm
There are no listener reviews for this title yet.


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Abandon your Sunday plans: Return to Dark Castle ships
On the one hand, I'm very excited to revisit this diversion from my childhood; on the other, I wonder what my jaded offspring will make of the throwback fun of pitching rocks at bats and jumping off of ropes. $30 says they'll enjoy it as much as I did back in the day.
Full feature list after the break.
Thanks Mark!
Return to Dark Castle features --
- Over 50 brand-new levels, including:
* All 30 classic, re-mastered levels
from Dark Castle and Beyond Dark
Castle integrated into a new, extensive
* New Secret Rooms and Mini-games.
- 25 orbs to collect (5 times that of BDC).
- All of the classic foes plus new monsters and bosses.
- New weapons, power ups, and secret items.
- Random labyrinths and mazes provide unique puzzles every time.
- Game recording and playback so users can share replays of their accomplishments.
- Customizable costumes to give each player a unique look.
- Support for most gamepads and joysticks.
- Please note that the game does not include a level editor as previously announced.
Software Updates
more updates- Apple Remote Desktop updated with Yosemite support
- OS X Yosemite 10.10.2, iOS 8.1.3 updates now available
- Sports Illustrated 120 SPORTS channel comes to Apple TV
- Logic Pro X update brings AirDrop support, new effects, tools, and more
- Parallels Access 2.5 released, adds file manager, computer-to-computer remote access
- The Google Translate iOS app is about to get a lot smarter


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Coalition plays underdog card
PUBLISHED: 19 Feb 2013 00:05:21 | UPDATED: 19 Feb 2013 04:51:10PUBLISHED: 19 Feb 2013 PRINT EDITION: 19 Feb 2013The federal Coalition is laying claim to underdog status, despite the latest Nielsen poll putting Tony Abbott's team on track for a landslide victory at September 14 election.
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People | Tony Abbott, Julia Gillard, Joe Hockey |
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There is your problem. youSubmitted by Guest (not verified) on Fri, 01/21/2011 - 8:15am.
There is your problem. you don't have kids. you will eat alot of words when you do. don't say what you will do until you are put in that position. for someone that doesn't watch this show you sure have a big opinion of it. »
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Search for any place in the USA:
Overview, People, Health, Economy, Housing, Rankings, Climate, Crime Education, Comments, Transportation, Cost of Living, Religion, Voting
The 2014 Grand Junction, CO, population is 58,867. There are 1,540 people per square mile (population density).
The median age is 37.3. The US median is 37.2. 50.88% of people in Grand Junction, CO, are married. 14.18% are divorced.
The average household size is 2.28 people. 31.67% of people are married, with children. 15.78% have children, but are single.
89.15% of people are white, 0.91% are black, 0.98% are asian, 0.87% are native american, and 4.98% claim 'Other'.
13.10% of the people in Grand Junction, CO, claim hispanic ethnicity (meaning 86.90% are non-hispanic).
meth : i saw no signs of meth use in g.j. co. i liked the city my xwife and oldest son live there i did see a little or few signed the v.a. doctor was very rude i said i was... (read more)Grand Junction versus the eastern slope. : Grand junction rules. I lived there from about 2000 to 2008, left and moved to Boulder and Longmont, and am now moving back (building a home in the Redlands). I... (read more)re: Meth use - 6/3/2010: sadly that's true, I have been directly effected by the "meth" problem in Grand Junction, and have seen first hand just how big of a problem it is, people should only... (read more)Have an opinion about Grand Junction? Leave a commentTo See All SperlingViews for Grand Junction Click Here


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These authors contributed equally to this study.
Liver Injury/Regeneration
In vivo phosphoenolpyruvate carboxykinase promoter mapping identifies disrupted hormonal synergism as a target of inflammation during sepsis in mice†
Article first published online: 4 AUG 2009
DOI: 10.1002/hep.23194
Copyright © 2009 American Association for the Study of Liver Diseases
Additional Information
How to Cite
Chichelnitskiy, E., Vegiopoulos, A., Diaz, M. B., Ziegler, A., Kleiman, A., Rauch, A., Tuckermann, J. and Herzig, S. (2009), In vivo phosphoenolpyruvate carboxykinase promoter mapping identifies disrupted hormonal synergism as a target of inflammation during sepsis in mice. Hepatology, 50: 1963–1971. doi: 10.1002/hep.23194
- †
Potential conflict of interest: Nothing to report.
Publication History
- Issue published online: 20 NOV 2009
- Article first published online: 4 AUG 2009
- Accepted manuscript online: 4 AUG 2009 12:00AM EST
- Manuscript Accepted: 18 JUL 2009
- Manuscript Received: 5 MAR 2009
Funded by
- Deutsche Forschungsgemeinschaft. Grant Numbers: He3260/2-1, Tu220/3
- European Foundation for the Study of Diabetes
- Marie Curie Excellence Grant (European Commission)
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
In mammals, proper maintenance of blood glucose levels within narrow limits is one of the most critical prerequisites for healthy energy homeostasis and body function. Consequently, hyper- and hypoglycemia represent hallmarks of severe metabolic pathologies, including type II diabetes and acute sepsis, respectively. Although the liver plays a crucial role in the control of systemic glucose homeostasis, the molecular mechanisms of aberrant hepatic glucose regulation under metabolic stress conditions remain largely unknown. Here we report the development of a liver-specific adenoviral in vivo system for monitoring promoter activity of the key gluconeogenic enzyme gene phosphoenolpyruvate carboxykinase (PEPCK) in mice. By employing in vivo promoter deletion technology, the glucocorticoid response unit (GRU) and the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) were identified as critical cis-regulatory targets of proinflammatory signaling under septic conditions. In particular, both elements were found to be required for inhibition of PEPCK transcription during sepsis, thereby mediating endotoxic hypoglycemia. Indeed, expression of nuclear receptor cofactor peroxisome proliferator-activator receptor coactivator 1α (PGC-1α), the molecular mediator of GRU/CRE synergism on the PEPCK promoter, was found to be specifically repressed in septic liver, and restoration of PGC-1α in cytokine-exposed hepatocytes blunted the inhibitory effect of proinflammatory signaling on PEPCK gene expression. Conclusion: The dysregulation of hormonal synergism through the repression of PGC-1α as identified by in vivo promoter monitoring may provide a molecular rationale for hypoglycemia during sepsis, thereby highlighting the importance of hepatic glucose homeostasis for metabolic dysfunction in these patients. (HEPATOLOGY 2009.)
One of the prominent features of liver metabolism is the ability for de novo glucose synthesis, gluconeogenesis, during periods of food deprivation in order to provide glucose for extrahepatic tissues such as erythrocytes, renal medulla, and particularly the brain.1 The main regulatory enzymes are pyruvate carboxylase (PC), which converts pyruvate into oxaloacetate, phosphoenolpyruvate carboxykinase (PEPCK) promoting the decarboxylation of oxaloacetate to phosphoenolpyruvate, and, finally, glucose-6-phosphatase (G6Pase), hydrolyzing glucose-6-phosphate into free glucose and inorganic phosphate.2, 3
PEPCK is considered to be the rate-limiting step in the gluconeogenic pathway. During fasting, expression of the cytosolic form of PEPCK is induced synergistically by glucagon and glucocorticoid hormones, whereas a carbohydrate-rich meal and the concomitant increase in plasma insulin levels acutely inhibit its synthesis rate. The hormonal counterregulation of PEPCK gene transcription by glucagon, acting through the intracellular second-messenger cyclic adenosine monophosphate (cAMP), and glucocorticoids, on the one hand, and insulin on the other has been established as the major regulatory axis for PEPCK activity in response to the fasting to feeding transition.2
In this regard, several cis-acting elements of the PEPCK promoter have been shown to mediate cAMP-responsiveness, in particular the so-called cAMP-responsive element (CRE), which is a direct target sequence for the cAMP-responsive element binding transcription factor CREB and is located at around −100 basepairs (bp) relative to the PEPCK transcription start site.2, 4–6 Upon binding to this site, CREB mediates cAMP-dependent as well as basal promoter activity.6–9 Importantly, a functional CRE site is also required for the full glucocorticoid response of the PEPCK promoter which is conferred by a glucocorticoid-response unit (GRU) located between bp −467 and −349 within the PEPCK proximal promoter region. The GRU represents a binding site for multiple transcriptional activators including the glucocorticoid receptor (GR), nuclear receptor hepatocyte nuclear factor (HNF) 4, and Foxo1.10, 11
Importantly, PEPCK messenger RNA (mRNA) levels are elevated in animal models of obesity and type II diabetes,12, 13 and transgenic overexpression of PEPCK is sufficient to induce hyperglycemia and type II diabetes.14, 15 In contrast, the inhibition of PEPCK mRNA expression and activity through proinflammatory cytokines following exposure to endotoxin has been identified as a critical event in the manifestation of endotoxic hypoglycemia as associated with impaired survival rates of experimental animals and septic patients.16–18
Given the critical importance of PEPCK regulation for systemic energy homeostasis, this study aimed at the establishment of an adenovirus-based in vivo PEPCK promoter reporter system in mice, allowing monitoring of the response of distinct PEPCK cis-regulatory elements to pathophysiologic liver metabolism. Using this technology, we here identify the interference with cAMP/glucocorticoid synergism and nuclear receptor cofactor peroxisome-proliferator-activated receptor coactivator (PGC)-1α through proinflammatory signaling as the critical event underlying endotoxic repression of gluconeogenesis during sepsis.
Materials and Methods
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Recombinant Adenoviruses.
Adenoviruses expressing PGC-1α or dominant-negative CREB (ACREB) were generated through homologous recombination between a linearized transfer vector pAD-Track and the adenoviral backbone vector pAD-Easy as described.12, 19, 20 To construct adenoviral reporter gene vectors, the firefly luciferase gene from pGL3 basic (Promega, Mannheim, Germany) vector was introduced into the multiple cloning site of the adenoviral shuttle vector pAD-Track. To avoid crosstalk between PEPCK promoter fragments and the cytomegalovirus (CMV) promoter directing the expression of GFP in the original pAD Track vector, the CMV promoter was excised by restriction digest. Subsequently, the following 5′-regulatory flanking regions of the mouse PEPCK gene were cloned upstream of luciferase: −1330 bp, −490 bp, −355bp, −490 bp carrying a point mutation within the PEPCK CRE (−490 CREmut),21 and −490 bp carrying a point mutation within the GR binding site (−490 GREmut).22 As a negative control, a reporter virus lacking any promoter element upstream of luciferase was used in all experiments. Homologous recombination between the modified pAD-Track vectors and adenoviral pAD-Easy backbones was performed as described.20 All viruses were purified by cesium chloride gradients as described.23
Animal Experiments.
Male 8-12-week-old C57Bl/6J mice were obtained from Charles River Laboratories (Brussels, Belgium) and maintained on a 12-hour light-dark cycle with unrestricted access to food. For fasting experiments, mice were fasted for 12 hours or fasted for 12 hours and then refed for 6 hours. For sepsis experiments, animals were fasted for 12 hours with free access to water and subsequently injected intraperitoneally (i.p.) with lipopolysaccharide (LPS; 20 μg/g body weight) or phosphate-buffered saline (PBS). For virus injections, 1 × 109 plaque-forming units per recombinant virus were administered by way of tail vein injection. In each experiment seven animals received identical treatments. Mice were sacrificed 5 days after adenovirus injection in the fasted state 8 hours after LPS administration. Organs including liver, epididymal fat pads, kidneys, and gastrocnemius muscles were collected after the corresponding time periods, weighed, snap-frozen, and used for further mRNA, protein, or metabolic analysis. All animal procedures were approved by local authorities and are in accordance with National Institutes of Health (NIH) guidelines.
Blood Metabolites.
Serum levels of glucose, ketone bodies, and free fatty acids were determined using an automatic glucose monitor (One Touch, Lifescan) or commercial kits (Sigma, Munich, Germany; Randox, Crumlin, Northern Ireland; Wako, Neuss, Germany, respectively). Serum cytokine levels were determined by Multiplex enzyme-linked immunosorbent assay (ELISA; Millipore, Billerica, MA).
Quantitative Taqman Reverse-Transcription Polymerase Chain Reaction (RT-PCR).
Total RNA was extracted from homogenized mouse liver or primary hepatocytes using the Qiazol reagent (Qiagen, Hilden, Germany) kit. Complementary DNA (cDNA) was prepared by reverse transcription using the oligo dT primer (Fermentas, St. Leon-Rot, Germany). cDNAs were amplified using assay-on-demand kits and an ABI Prism 7700 Sequence detector (Applied Biosystems, Darmstadt, Germany). RNA expression data was normalized to levels of TATA-box binding protein RNA.
RNA Interference.
Oligonucleotides targeting mouse PGC-1α (5′-GGTGGATTGAAGTGGTGTAGA-3′) were annealed and cloned into pENTR RNAi vector (Invitrogen, Karlsruhe, Germany). Nonspecific oligonucleotides (5′-GATCTGATCGACACTGTAATG-3′) with no significant homology to any mammalian gene sequence were used as nonsilencing controls in all experiments as described.24
Primary Cell Culture.
Primary mouse hepatocytes were isolated and cultured as described.25 Cells were treated with dexamethasone (DEX; 10 nM) or cAMP agonist forskolin (Fsk) (10 μM), or both, for 24 hours and harvested for mRNA expression analysis. In additional experiments, cells were treated with DEX and Fsk as above and conditioned RAW264.7 macrophage medium collected from LPS-primed cell cultures for 8 or 24 hours.
Protein Analysis.
Protein was extracted from frozen liver samples, loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto nitrocellulose membrane. Western blot assays were performed as described12 using antibodies specific for PGC-1α or valosin-containing protein (VCP) (Abcam, Cambridge, UK).
Plasmids, Cell Culture, and Transfections.
Promoter luciferase constructs containing the PEPCK or PGC-1α 5′-flanking regions or an isolated p65/RelA response element, −490 PEPCK, PGC-1αLuc, and NFk-Luc have been described.12, 26
Murine RAW264.7 macrophages were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and stimulated with LPS (100 ng/mL) for 6 hours before collecting the supernatant. Cytokine enrichment of the supernatant was subsequently confirmed by Multiplex ELISA (Millipore). Rat H4IIE hepatocytes were transfected using the calcium phosphate precipitation method as described.12 Cells were left untreated or treated with DEX (10 nM), Fsk (10 μM) (Sigma), and/or conditioned RAW264.7 supernatant for 24 hours and were harvested for luciferase assays 48 hours after transfection. The luciferase activity was normalized to beta galactosidase activity.
Statistical Analysis.
Statistical analyses were performed using a 2-way analysis of variance (ANOVA) with Bonferroni-adjusted posttests, or t test in one-factorial designs, respectively. The significance level was P = 0.05.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
The importance of PEPCK for hepatic gluconeogenesis and systemic glycemia prompted us to develop an in vivo monitoring system for PEPCK promoter activity in mice. To this end we employed an adenoviral backbone vector carrying distinct sequences of the PEPCK 5′-flanking region, thereby controlling expression of a luciferase reporter gene (Fig. 1A). Adenovirus was produced according to standard procedures, purified by cesium chloride purification, and used to infect H4IIE hepatocytes to initially verify the functional integrity of this reporter system in a cell-autonomous manner. As shown in Fig. 1B, a viral construct carrying 1330 bp of the PEPCK 5′-flanking region (−1330 PEPCK) produced a basal luciferase signal as compared to the absence of luciferase activity in cells infected with an adenoviral construct lacking any PEPCK promoter fragments (Fig. 1B). In addition, treatment of the cells with the glucocorticoid analog DEX or the cAMP agonist Fsk stimulated −1330 PEPCK activity by 5-fold (Fig. 1B), but had no effect on a control vector lacking PEPCK promoter fragments (Supporting Fig. S1A). Consistent with previous reports,9 cotreatment of the cells with both DEX and Fsk led to a synergistic 16-fold activation of −1330 PEPCK activity (Fig. 1B), demonstrating the principle functional integrity of critical hormone-responsive elements in the adenoviral background. To test this in a relevant in vivo setting, we delivered PEPCK reporter adenoviruses into C57Bl6 mice by way of tail vein injection. At day 5 after injection, all PEPCK reporter viruses produced a robust luciferase activity above background levels in whole-liver lysates as compared to control adenovirus lacking PEPCK regulatory elements (data not shown). Importantly, luciferase activity of constructs carrying either 1330 (−1330 PEPCK) or 490 (−490 PEPCK) bp of the PEPCK 5′-flanking region was induced 3-7-fold by fasting as compared to refed animals (Fig. 1C), thereby recapitulating the fasting induction of PEPCK mRNA levels as well as correlating with fasting hypoglycemia in the same animals (Fig. 1D, Supporting Fig. S1B). Importantly, elimination of the GRU (−355 PEPCK) completely abolished fasting-induced luciferase activity, whereas the regulation of endogenous PEPCK mRNA by food deprivation was still detectable in these animals (Fig. 1C,D). These data indicated that adenoviral transfer of PEPCK promoter deletion fragments can be used to monitor the transcriptional response of individual cis-regulatory elements to endogenous signaling cues in vivo, thereby allowing dissection of the contribution of individual transcriptional complexes to gluconeogenic PEPCK activity under the given metabolic conditions. Indeed, consistent with the importance of both the GR and CREB for basal and fasting-induced gluconeogenesis,10 point mutation of either the GR or CREB binding site within the PEPCK promoter (GREmut and CREmut) was found to decrease basal refed luciferase activity and to significantly impair the fasting-dependent induction of PEPCK promoter activity (Fig. 1C), further substantiating the functional integrity and specificity of the in vivo reporter system. To this end, no luciferase activity could be detected in tissue lysates of adipose tissue and skeletal muscle (data not shown), validating the previously reported liver-specificity of the adenoviral gene delivery technology.12
Despite improved clinical protocols, the 40%-60% mortality rates of septic patients have not been substantially improved over the past 20 years,27 and hypoglycemia still represents a major predisposing factor for the poor prognosis of critically ill patients under septic conditions.28 Impairment of gluconeogenesis and particularly PEPCK gene expression has been known to be critically involved in endotoxic hypoglycemia16, 17; however, the molecular mechanisms of in vivo PEPCK inhibition under these conditions are still unknown.
To address hepatic gene-regulatory events responsible for endotoxic hypoglycemia, we employed the PEPCK in vivo promoter system as described above to map critical cis-regulatory elements in a standard mouse model for sepsis. To this end, C57Bl6 mice were fasted overnight, injected with a sublethal dose of bacterial LPS or vehicle control (PBS), and analyzed 8 hours after LPS administration. Consistent with previous reports,16, 17 LPS triggered a significant decrease in blood glucose levels as compared to controls (Fig. 2A), correlating with substantially increased levels of circulating cytokines, including interferon γ, interleukin-1β, interleukin-6, and tumor necrosis factor α (TNF-α) (Supporting Fig. S1C). Noteworthy, these proinflammatory conditions also decreased total serum ketone bodies (Supporting Fig. S1D), indicative of decreased hepatic β-oxidation. Consistent with endotoxic hypoglycemia, mRNA levels of gluconeogenic key enzymes, PEPCK, and G6Pase were down-regulated 5-10-fold in livers of LPS-treated animals (Fig. 2B), thereby underscoring the importance of improper gluconeogenic control as a hallmark of septic hypoglycemia. We next sought to confirm the direct impact of the hormonal-cytokine antagonism on the hepatocyte by mimicking the in vivo septic milieu in cell culture. Indeed, in primary mouse hepatocytes PEPCK and G6Pase mRNA expression were synergistically enhanced by combined Fsk and DEX exposure of the cells, and this induction was decreased by 90% upon costimulation of the cells with a cytokine-enriched supernatant from LPS-conditioned RAW264.7 macrophages (Fig. 2C,D), demonstrating the cell autonomy of the observed inhibitory effects of sepsis on gluconeogenic gene expression in hepatocytes. Interestingly, treatment of these primary cells with LPS had no influence on PEPCK gene expression, whereas G6Pase was significantly inhibited by LPS stimulation (Fig. 2C,D), indicating that the LPS effects in endotoxemic mice are most likely mediated by both direct LPS/Toll-like receptor 4 (TLR4) signaling and indirect elevation of cytokine levels as shown before (Supporting Fig. S1C). Consistent with this assumption, inhibition of PEPCK and G6Pase gene expression by conditioned macrophage medium was slightly impaired in primary hepatocytes from TLR4-deficient animals as compared to wildtype controls (data not shown).
To map sepsis-responsive cis-regulatory elements within the PEPCK gene promoter, C57Bl6 mice were injected with adenoviral luciferase constructs carrying distinct PEPCK promoter mutants, fasted overnight, and rendered septic by sublethal LPS administration. As shown in Fig. 3, activity of −1330 PEPCK and −490 PEPCK was decreased 5-fold in septic animals as compared to control PBS-injected littermates (Fig. 3A), thereby closely matching LPS-induced hypoglycemia and decreased PEPCK mRNA levels in the same animals (Supporting Fig. S2A). Point mutation of the GR binding site within the PEPCK −490 bp 5′-flanking region had no effect on relative PEPCK LPS responsiveness (Fig. 3B), suggesting that the GR per se does not represent the target of proinflammatory signaling on the PEPCK gene during endotoxemia. In contrast, point mutation of the CRE (Fig. 3C) or the deletion of the entire GRU (Fig. 3D) completely abolished the inhibitory effect of septic conditions on PEPCK promoter activity, whereas repression of endogenous PEPCK mRNA levels as well as hypoglycemia (Supporting Fig. S2B-D) was still evident in these mice. Importantly, the GRU-deficient reporter construct could be still repressed by coadministration of a dominant-negative CREB (ACREB) (Fig. 3D), verifying the specificity of the LPS insensitivity of this promoter fragment in mice.
These data indicate that the inhibition of gluconeogenic PEPCK expression during sepsis requires two distinct cis-regulatory sites within the PEPCK 5′-flanking region in vivo, namely, the CRE as well as the GRU.
To explore the mechanistic basis for the observed dual track inhibition of PEPCK expression during endotoxic conditions, we subsequently profiled the expression of various critical transcriptional regulators of PEPCK GRU activity in septic and control animals. As shown in Fig. 4A, LPS treatment had no effect on the hepatic expression of GR, steroid receptor coactivators (SRC)1 and 2, CREB-binding protein CBP, p300, hepatocyte nuclear factor (HNF)-4α, CREB, and CREB regulated transcription coactivators 1 and 2 (Fig. 4A). In contrast, mRNA of Foxo1 and PGC-1α were significantly reduced. Whereas Foxo1 mRNA was mildly reduced, mRNA as well as protein levels of nuclear receptor coactivator PGC-1α were found to be strongly down-regulated upon LPS treatment as compared to control littermates (Fig. 4A and inset). Indeed, PGC-1α levels were also decreased in primary mouse hepatocytes exposed to cytokine-enriched macrophage supernatant (data not shown), demonstrating the cell autonomy of this effect. Consistent with an impairment of PGC-1α gene transcription by cytokine signaling, in transient transfection assays of H4IIE rat hepatocytes PGC-1α promoter activity was significantly inhibited by treatment of the cells with conditioned, cytokine-enriched macrophage medium (Supporting Fig. S3A), whereas a reporter gene carrying an isolated binding site for the proinflammatory transcription factor RelA/p65 was induced by 2.5-fold under the same conditions (Supporting Fig. S3A), demonstrating the specificity of the effects.
The cAMP-dependent induction of PGC-1α expression and its subsequent coactivator function for GRU-bound transcriptional regulators, including HNF-4α, Foxo1, and the GR, had been previously identified by us and others as the molecular mechanism underlying the synergistic action of cAMP and glucocorticoid pathways in the induction of PEPCK and hepatic gluconeogenesis.12, 19 The specific decrease of PGC-1α expression in response to LPS therefore favored the hypothesis that the inhibition of PGC-1α coactivator function represents a critical step in endotoxic hypoglycemia.
Therefore, we next sought to functionally link PGC-1α activity to inhibition of PEPCK gene expression under proinflammatory conditions. To experimentally mimic the down-regulation of PGC-1α under septic conditions, we employed PGC-1α-specific short hairpin RNA (shRNA) constructs in transient transfection assays using the PEPCK −490 bp promoter vector in cultured H4IIE hepatocytes. Consistent with our previous reports,12 PGC-1α shRNA significantly impaired both DEX- and Fsk-stimulated PEPCK promoter activity, and also blocked synergistic DEX/Fsk action by 4-fold (Fig. 4B), verifying the integrity of the shRNA system. Treatment of the cells with conditioned macrophage medium further blocked −490 PEPCK activity 3-4-fold in the presence of endogenous PGC-1α, but had only minor or even no significant effects upon PGC-1α shRNA treatment (Fig. 4B), suggesting the requirement of functional PGC-1α as a target of proinflammatory inhibitory signaling.
To finally test this assumption independently, we overexpressed PGC-1α in primary mouse hepatocytes treated with conditioned macrophage or control medium by way of adenoviral PGC-1α cDNA delivery. Treatment of the cells with cytokine-enriched macrophage medium significantly decreased endogenous PEPCK mRNA levels as compared to control cells (Fig. 4C), and PGC-1α adenovirus infection fully rescued PEPCK mRNA to control levels under these conditions (Fig. 4C). Interestingly, whereas PGC-1α overexpression rendered PEPCK expression insensitive to treatment with cytokine-enriched macrophage supernatant in the absence of hormonal stimulation, the macrophage supernatant was still able to repress PGC-1α-driven PEPCK mRNA expression in the presence of additional Fsk/DEX treatment (Fig. 4C). Taken together, the fact that (a) shRNA-mediated PGC-1α suppression was sufficient to down-regulate PEPCK expression and to impair cytokine-dependent PEPCK inhibition, and (b) PGC-1α restoration under cytokine stimulation led to PEPCK expression levels well above control levels further substantiated the notion that the loss of PGC-1α upon proinflammatory, septic conditions represents a key step in the repression of PEPCK and presumably hepatic gluconeogenesis. The down-regulation of PGC-1α during sepsis may, thereby, contribute to the pathogenesis of endotoxic hypoglycemia in critically ill patients, and may further aggravate the risk for fatal complications under these conditions.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Detailed molecular knowledge about the pathogenesis of clinically severe hypoglycemia in critically ill, septic patients is still rare. By employing a novel in vivo promoter mapping system, we identified the repression of nuclear receptor coactivator PGC-1α through proinflammatory cytokine signaling as a critical event in the suppression of PEPCK expression, a key step in hepatic gluconeogenesis during sepsis.
PGC-1α was originally cloned as a coactivator for nuclear receptor PPARγ in brown adipocytes, responsible for the tissue-specific induction of thermogenesis in response to cold-exposure and β-adrenergic signaling.29
During fasting, hepatic PGC-1α is activated in response to catecholamine and glucagon stimulation by way of a direct cAMP/cAMP responsive element binding protein (CREB)-mediated effect on its gene promoter.12 PGC-1α then, in turn, mediates activation of the gluconeogenic program including PEPCK in response to glucocorticoid signals through direct interactions with the GRU-binding transcription factors, GR and HNF-4α.12, 19, 30 Apart from the induction of PGC-1α, CREB mediates cAMP-dependent as well as basal PEPCK promoter activity by way of a consensus cAMP-responsive element (CRE).2, 4–6 Importantly, this CRE site is required for the full glucocorticoid response of the PEPCK promoter and has been described as an essential accessory site for GR action.31 Consequently, the impairment of PGC-1α expression and the subsequent disruption of hormonal synergism by proinflammatory signals provide an effective mechanism for the repression of gluconeogenic PEPCK gene expression during sepsis as identified by our current study. In this regard, preliminary data indicate that proinflammatory signaling in addition to interfering with PGC-1α gene expression (Fig. 4A) may also directly affect PGC-1α protein function under these conditions (unpublished data), which might further add to the impairment of PGC-1α-dependent PEPCK transcription during sepsis. Intriguingly, whereas PEPCK expression became insensitive to cytokine inhibition when driven by exogenous PGC-1α only, proinflammatory mediators were still able to repress PEPCK mRNA levels when activated by both PGC-1α and additional Fsk/DEX treatment (Fig. 4C). These findings are consistent with previous reports demonstrating the importance of the CRE for TNFα-mediated repression of PEPCK promoter activity in vitro,32 and might argue for a primary effect of LPS on suppressing cAMP signaling on both the PEPCK as well as the PGC-1α promoter, resulting in a secondary PGC-1α-dependent amplification of gluconeogenic gene suppression under proinflammatory conditions and explaining the important contribution of the GRU in this context in vivo (Fig. 3).
PGC-1α has been shown to suppress the production of proinflammatory mediators in muscle cells,33 and the reduction of PGC-1α mRNA levels in skeletal muscle of type II diabetic patients34 is thought to be tightly associated with the chronic inflammatory status of these subjects.35 By acting as a negative target of inflammatory cues in the liver, the down-regulation of PGC-1α during septic conditions may thereby reflect the "glycemic" aspect of a broader involvement of this coactivator in (anti-)inflammatory programs at a systemic level. In this respect, our preliminary studies do favor a direct impact of LPS/TLR4 signaling on the PGC-1α/PEPCK axis (Fig. 2 and data not shown), and additionally suggest the involvement of proinflammatory, macrophage-derived cytokines in gluconeogenic gene repression. Indeed, previous work has shown that neutralizing anti-TNFα antibodies prevent endotoxic repression of PEPCK gene expression in LPS-challenged mice,17 and TNFα treatment has been successfully used to inhibit the activity of PEPCK promoter constructs in in vitro transfection assays,18, 32 mediated at least in part by way of the CRE.32 Consistently, the NFkappaB/RelA/p65 as a major transcriptional mediator of proinflammatory signaling has been implicated in the control of PGC-1α expression in human cardiac myocytes.36 In addition, RelA has been demonstrated to inhibit PEPCK promoter activity independently of a putative RelA binding site within the PEPCK 5′-flanking region.18 Interestingly, expression of the PC gene was not impaired under proinflammatory conditions in mice (Fig. 2B) and primary hepatocytes (data not shown). Indeed, whereas the liver-specific promoter regulation of the PC gene remains largely unexplored,37 PC gene expression was found to be unresponsive to glucocorticoid stimulation,38 arguing for a generally differential mode of transcriptional control as compared to PEPCK and G6Pase.
Taken together, our results are consistent with a model in which the elevation of proinflammatory cytokines, such as TNFα, during sepsis promotes the inhibition of PGC-1α gene expression, which in turn abrogates the GRU/CRE-mediated hormonal synergism required to maintain hepatic PEPCK levels. As PGC-1α is also known to control G6Pase promoter activity,30 the inhibitory impact of endotoxic mediators on the PGC-1α coactivator provides an amplification mechanism to efficiently shut down hepatic gluconeogenesis and to promote endotoxic hypoglycemia. Maintenance of hepatic PGC-1α activity might thereby represent an attractive therapeutic defense in the antihypoglycemic treatment of critically ill patients.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
We thank members of our labs for experimental advice and technical support, and M. Montminy (La Jolla, CA) for providing reagents.
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
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Supporting Information
- Top of page
- Abstract
- Materials and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Additional Supporting Information may be found in the online version of this article.
Filename | Format | Size | Description |
HEP_23194_sm_SupFig1.tif | 907K | Supporting Figure 1. A, Relative luciferase activity in H4IIE hepatocytes infected with an adenovirus lacking all PEPCK 5′-flanking regions. Cells were treated with forskolin (FSK, 10 μM) and/or dexamethasone (DEX, 10 nM) as indicated for 24 h before harvesting the cells (means ± SEM, n=9). B, Blood glucose levels of C57Bl6 mice infected with adenoviruses carrying distinct PEPCK promoter luciferase fragments at day 5 after injection as indicated. Mice were either fasted for 12 h or fasted for 12 h and refed for 6 h (means ± SEM, n=9). C, Serum levels of interferon (Ifn)γ, interleukin (IL)1β, IL6, and tumor necrosis factor (TNF)α in C57Bl6 mice treated with lipopolysaccharide (LPS; 20 μg/g body weight) for 8 h (means ± SEM, n=9). D, Serum total ketone body levels in same animals as in C (means ± SEM, n=9). *, p < 0.05. | |
HEP_23194_sm_SupFig2.tif | 958K | Supporting Figure 2. A-D, Quantitative PCR analysis of hepatic PEPCK mRNA (left panels) and blood glucose (right panels) levels in C57Bl6 mice infected with adenoviruses carrying 1330 (A), 490 (B-D) or 355 (D) bp of wild-type PEPCK promoter luciferase fragments or 490 bp PEPCK promoter fragments harboring point mutations within the PEPCK GRE (B) or CRE (C) at day 5 after injection. Mice were fasted overnight and injected with LPS (20 μg/g body weight) for 8 h (means ± SEM, n=9). *, p ≤ 0.05. | |
HEP_23194_sm_SupFig3.tif | 218K | Supporting Figure 3. A, Transient transfection assay of H4IIE hepatocytes transfected with luciferase reporter constructs carrying 1000 bp of the PGC-1α 5′-flanking region (PGC1αLuc) or 3 copies of an isolated nuclear factor κ B (NFκB)/RelA/p65 response element (3xNFκB). Cells were treated with LPS-conditioned macrophage medium (RAW) for 24 h as indicated (means ± SEM, n=9). *, p ≤ 0.05. |
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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Dworkin Construction Corporation
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nancy m
Half poet, half geek with a heavy dose of techno joy.
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Cut that mike up for me bass
Let's do this thang boy,
Deadly sounds right back at ya
Can't stop none of this?
Playa, check this out
They don't want this chorus all they want is Murder
They don't feel my pain but they gone feel my presence
And I've been stressin' since a adolescent
They don't want this chorus all they want is Murder
They don't feel my pain but they gone feel my presence
Now I ain't never robbed rally's
But it was close, I've done stick ups for Bally's
We love to smoke as I recall I heard you mention my name
What you love to see me stressin'?
This ain't part of the game you must be high
'Cause you rockin' like a Bass Head
You hear me, I'm grindin', workin' hard for mine ya feel me
Four hundred years of pain and now this
It's like them cockroaches got you trained, just like a bitch
You see they smile in yo face now what they after
And now they wanna take my place
Them back stabba's, blucka blucka, I'mma get you sucka
We roll tinted windows on the black Humma Trucka
And after it rain he might be dead like Jesse James
Trained for pain even when I lose everything I gain
It's simple mathematics when you go ballistic
Statistics show we breed soldiers in my district
It ain't like Mr. Rodgers I learned the game
But it wasn't from the Dodgers
Pissed from pain, and Welfair wasn't a question
Just a decision, we embraced it as a blessin'
'Cause food was missin' and my tattoos tell a story
I'm Bossainie and Kevin died in his glory
I know he see me, I'm runnin' but I ain't movin'
It's like a dream, 'cause all they want is Murder
That's what it seem
They don't want this chorus, all they want is Murder
They don't feel my pain but they gone feel my presence
And I've been stressin' since a adolescent
They don't want this chorus all they want is Murder
They don't feel my pain but they gone feel my presence
And I've been stressin' since a adolescent
Come meet me in the projecets 'cause it's a set up
And the only way to stop, watch him get wet up
Ain't no since in you fakin' with me
I'm just a pebble and the window you got is bigger than me
Watch me shadow while blood splattered and hit the pave
I'm puttin' fresh flowers on my empty grave
Tryna sell a million so I can provide for my lil' one
Shit, they want little mess like the rest of the ghetto children
It's confusin', I'm losin' pieces to a broken puzzle
Russian Roulette to my head it's down bubble muzzle
I'm Kamakazi, you try me put yourself right beside me
I'm a wanted man call Baby Mamma so she can hide me
The only victim is all the brotha locked up in prison
Gettin' letters and pictures, I know they people miss em'
Take a ride to Texas in a stolen Lexus
Three mack 11's is ready, that's me only protection
"Who do I believe in really?", I put my faith in God
I was dealt some bad cards, he's fightin' in school yards
And fightin' at night behind bars the devil hit me wit a murder charge
All they want is Murder boy
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C-Murder - Miscellaneous Album Lyrics | |
1. | Ain't Nuttin' Personal |
2. | Won't Let Me Out |
3. | My Life |
4. | I Heard U Was Lookin' 4 Me |
5. | Yall Heard of Me |
6. | Stressin |
7. | Mamma How You Figure |
8. | Back Up |
9. | Betta Watch Me |
10. | Camouflage & Murder |
11. | Hustlin' (w/ Master P) |
12. | Murder & Daz |
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1 | I Won't Stop |
2 | Don't Make Me |
3 | Can't Take (feat. Shop Boyz) |
4 | Part (feat. Shop Boyz |
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homemade rear suspension
I'm starting a 47 chevy sedan delivery.Thinking of using dodge ramcharger front springs for rear suspension.Tried this on a 39 buick 4 door traded it on this,didn't seem to come out to bad,as far as stance never got it finished.These springs have one reverse eye(pointed down)and one up,same design as CE and others.Ones I used 3leaves,1 1/2 arch,1400 spring capacity.Others listed,5 leaves,1 1/4 arch,1550 spring capacity: another5 leaves,1 5/8 arch.1550 spring capacity.All these are 2 1/2 wide,48inch eye centers.Then insead of shackles using leaf spring sliders.The reason I'm wanting to try this you can buy these springs for around $150.a pair.I can make my own brackets,I'm thinking the 3 leaves 1400lbs,would be about right although CE are 5 leaves I think around
1 1/2 arch.I don't know why this won't work,be a very reasonably price rear suspension. Any thoughts?
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"jalopy",thanks for the reply,would you know or someone,on figuring leaf spring rate I looked in knowledge base,on being to stiff or to soft of springs,giving this some thought or go ahead and buy complete rear suspension.just trying to stretch the $$$.
Recent Suspension - Brakes - Steering posts with photos |
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Diesel fuel spills after semi and truck crash
Roughly 180 gallons of diesel fuel spilled Friday morning when a Chevrolet truck and a semitrailer crashed along Highway 371, south of Brainerd near Fort Ripley.
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Tribology Letters
Tribology Letters is devoted to the development of the science of tribology and its applications, particularly focusing on publishing high-quality papers at the forefront of tribological science and that address the fundamentals of friction, lubrication, wear, or adhesion. The journal facilitates communication and exchange of seminal ideas among thousands of practitioners who are engaged worldwide in the pursuit of tribology-based science and technology.
- 57 Volumes
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Takahiro Nihira, Kazuyoshi Manabe, Chiharu Tadokoro, Shingo Ozaki… (January 2015)
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Here we go Cam is the example you come up with.
Dude i've ask you this before ( which you've avoided the question) but can I get 1 or 2 "WR" as the example of this? Since we are talking about WR can I get a Wr as the example. What WR this raw this unpolished with this little of college production and experience was a top 20 pick?
Find me a QB with less than 300 passing attempts and one year of starting and coming out of a gimmicky spread offense coming out of college and being drafted 1st overall.
Go ahead, I'll wait.


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Microsoft Office 2001 is a great suite that allows you to perform a variety of tasks, such as sending email, creating newsletters or other documents, creating charts and graphs, and putting together presentations. Mark Maggiacoma guides you through this Virtual Training Company tutorial by introducing you to the basics common to all the different features, like creating new documents and saving. He then covers the Project Gallery, where you can set up menus, shopping lists, workout logs, and much more. You will be led from the basics, through editing, layout, special items, and finish with how to print in Word 2001, Excel 2001, and PowerPoint 2001. You will also learn how to use Entourage, including how to set up multiple email accounts. To get started, simply click one of the topics below.
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DictionaryMy•raPronunciation: (mī'ru), Strict Standards: Non-static method FenSites::linkTo() should not be called statically in /site/html/dictionary/myra.html on line 73 [key] —n. 1. an ancient city in SW Asia Minor, in Lycia. 2. a female given name: from a Latin word meaning "extraordinary." Random House Unabridged Dictionary, Copyright © 1997, by Random House, Inc., on Infoplease.
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Cirque du Soleil's Varekai: An Enchanted Forest of Acrobatic Wonders
* Additional fees apply. No coupon or promo codes necessary to enjoy the displayed discount price.
The last date listed for Cirque du Soleil: Varekai was Sunday August 31, 2014 / 5:00pm.
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Under the Big Apple Circus Big Top, you're never more than 50 feet from the oohs, ahhs and wows! … More
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Featured review from Ursula
view more less of this reviewNice place, clean bathrooms. Very good show. A lot of great gymnastics, beautiful costumes and good comedians.
star this review starred report as inappropriateThe set up is really cool. The music was awesome, but the story was a little jumbled and the acts seemed to be very short. It is a good Cirque experience, but if you are a regular, it will be a little sub-par.
star this review starred report as inappropriateGreat theater as it was small and every seat was a good seat. Not my favorite Cirque show, very slow start, a little too long on the "fluff" skits in between the acts. I would rather see dancing, hear music but the slapstick humor was long and...continued
star this review starred report as inappropriateOne of the best "small " C d S show!


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Sino ang mas EVIL?
Total Members Voted: 40
Voting closed: August 22, 2012, 10:18:32 PM
It is no secret (here at the Candy HQ, at least) that I am crazy about One Direction cutie, Harry Styles. ICYDK who he is�and I'm really hoping that's not the case because if it...


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Mouse offspring conceived by in vitro fertilization are metabolically different from naturally conceived mice.
Mouse offspring conceived by in vitro fertilization are metabolically different from naturally conceived mice.
Engineered proteins can tether multiple fluorescent molecules to give a brighter signal—and that's not all.
Embryonic stem cells likely mucked up the cultures used in the debunked "stimulus-triggered acquisition of pluripotency" studies.
The National Institutes of Health shutters its initiative to track the health of 100,000 children through adulthood.
Scientists make hydrogel coats for individual cells that can be tailored to specific research questions.
Bacteria in the gut of a pregnant mouse strengthen the blood-brain barrier of her developing fetus.
The right mix of mouse embryonic stem cells in a dish will start forming early embryonic patterns, according to two studies.
Some institutions are changing their protocols for rodent euthanasia, as research finds there may be more humane methods.
Scientists create a photosensitive pharmaceutical to target a glutamate receptor.
A paper published last month claims that electromagnetic exposure facilitates cell reprogramming, but some scientists question the evidence.


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Olympians and Champions Gather During Second Day of California Dressage Society Championships
Championship dressage continued Friday at the Murieta Equestrian Center in Rancho Murieta, CA for the 44th Annual California Dressage Society Championship Show presented by HorseShow.com, in conjunction with the CDS Young Horse Futurity (including the Cal-Bred Futurity), the GAIG/USDF Region 7 Dressage Championships, and the GAIG/USDF Breeders Championships West Coast Series Final. During the day's competition, three-time Olympian Guenter Seidel was on hand to sign autographs and present awards; during evening hospitality, Seidel was interviewed by announcer Brian O'Connor, fielding questions from fans in the audience and via Facebook.
Five rings of competition featured horses and riders from across the region vying for top honors in several CDS Horse of the Year divisions. Emerging the victor from a large CDS Horse of the Year Open Prix St. Georges class to claim the Bent Roswall Memorial Trophy was Rebecca Rigdon of Encinitas, CA, riding her Holsteiner mare "Solei" to a final score of 70.395%. In the Adult Amateur division, the Global Imaging Perpetual Trophy and a score of 63.553% was earned by "Souvenir", a 12-year-old Trakehner gelding owned and ridden by Jill Thornton from Ramona, CA. At the Grand Prix level, Leslie Webb of Bakersfield, CA rode Harmony Sporthorses' Oldenburg gelding "Harmony's Sandro" to the blue ribbon and the Sherry de Leon Memorial Trophy with 68.511%. Continuing their previous day's success, Kimberly Noon-Fishel from Walnut Creek, CA and her gray Oldenburg gelding "Casino Royale" once again danced victory in the Fourth Level freestyle, taking home the Ran Averett Memorial Perpetual Trophy with a score of 67.333%.
The CDS Four-Year-Old Futurity concluded Friday, with the California-bred "Caliente DG" (00 Seven x Satina by Contango, bred by DG Bar Ranch) impressing judges Kristi Wysocki and Paula Lacy to claim the title with a Training Level Test 2 score of 72.600%, which combined with the previous day's test for an overall winning score of 70.854%. This bay Dutch Warmblood mare, owned by Brenda Linman, was ridden to a decisive victory by Craig Stanley of Madera, CA. In the Adult Amateur division of the Four-Year-Old Futurity, Melissa Mulchahey of Livermore, CA turned heads with her winning mount, "Outrageous". Despite his small size, this chestnut Westfalen pony gelding (Out-Break x Diddi by Donnerhall Sir, bred in Germany) is big on character and movement, scoring 68.400% today for an overall average of 68.575%. "These German ponies have all of the ability the big horses have, and I think ponies are an ideal mount for adult amateur riders like me," said Mulchahey. "This CDS futurity is a great opportunity for amateurs with young horses to compete against our peers."
GAIG/USDF Regional Championship action continued today with several highly-competitive classes. In the Open Intermediaire II division, a strong field of competitors resulted in a highly unusual tie for the top placing, which was unable to be broken. As a result, Tracey Lert of Soquel, CA riding her own Dutch Warmblood gelding "Udo" shared the championship honors with Heidi Gaian of Hollister, CA and her Holsteiner "Chicago", both scoring 66.053% from judges Lorraine Macdonald and Michael Osinski. Akiko Yamazaki of Los Altos Hills, CA, owner of U.S. Team horse "Ravel", Yamazaki enjoyed her own time in the saddle today, guiding her black Hanoverian mare "De La Noche" to a winning score of 66.711% in the Intermediaire II Adult Amateur division.
In the Open Training Level division, Bethany Nehse of Livermore, CA rode her own ISR mare "Winkayla" to emerge victorious with a score of 73.400%, while for Adult Amateurs, Nancy Szakacs from Hollister, CA earned a score of 74.400% and the win with her Westfalen gelding "Rudi Regali". In the Open Second Level championship, Leslie Webb continued her day's winning ways, partnering with Harmony Sporthorses' Dutch Warmblood mare "Harmony's Armani" for 70.714%. Lisa Johnston (Woodland, CA) rode her own Oldenburg gelding "Venzerro" to a decisive win at Second Level for Adult Amateurs with a score of 68.214%. Rounding out the day's Regional Championship classes were the Open Intermediaire I and Grand Prix Freestyles, where Charlotte Jorst of Reno, NV claimed the championship sash in both divisions with Skagen Holdings' "Tristan" and "Asterios", scoring 71.563% and 66.000%, respectively.
Championship competition continues Saturday, including the five- and six-year-old divisions of the CDS Young Horse Futurity, the GAIG/USDF Breeders Championships West Coast Series Final, and a full roster of evening entertainment. Daily results and complete information about the 44th Annual California Dressage Society Championship Show presented by HorseShow.com is available on the CDS website, www.california-dressage.org.
Para Dressage
Friday, January 16, 2015 - 05:20
Kjell Myhre talks to riders at the 2015 USEF High Performance Southeast Para Equestrian Dressage Symposium in Wellington, FL, January 12-13. (Photo: (C) Lindsay Y. McCall)
Wellington, FL - Following the 2015 Adequan Global Dressage Festival CPEDI3* select U.S. Para-Dressage athletes and coaches had an opportunity to attend the USEF High Performance Southeast Para-Equestrian Dressage Symposium. This symposium took place January 12-13, 2014, at the Global Dressage Festival show grounds in Wellington, Florida. Over two days U.S. High Performance and developing Para-Dressage coaches and riders were immersed in the international equestrian discipline of Para-Dressage. Topics included Para-Dressage theory, scales of training, para-dressage test riding, quality of horse, freestyles, preparation...
Tuesday, January 13, 2015 - 04:46
USA Team (Left to right): Sydney Collier, Annie Peavy, Rebecca Hart, U.S. Chef D' Equipe Kai Handt, and Roxanne Trunnell (Photo: (C) Lindsay Y. McCall)
Wellington, FL - Thirteen Para-Dressage horse and rider combinations made their way to Wellington, Florida, this January for the first CPEDI3* of the 2015 show season. Para-Equestrians from Canada and the United States in Grades Ia, Ib, II, III, and IV, competed at the spectacular Global Dressage venue in the Van Kampen Arena. FEI Ground Jury members Mr. Freddy Leyman (BEL), Mr. Kjell Myhre (NOR), and Ms. Kristi Wysocki (USA) were on hand to offer their expertise for each Para-Dressage duo. This CPEDI3* is one of the qualifiers for the 2015 USEF Para-Equestrian Dressage National Championships, 2016 USEF Para-Equestrian Dressage National C...
Monday, January 12, 2015 - 09:29
Robyn Andrews and Fancianna (Photo: SusanJStickle.com)
Ottawa, ON. - On the final day of competition at CPEDI3* Global Dressage Festival I presented by Adequan, Robyn Andrews won her test for the second day in a row for Canada. The competition was held at The Stadium at Palm Beach International Equestrian Center in Wellington, Fla., Jan. 9-11, 2015. Andrews of St. Johns, N.L. secured the victory in the Grade Ia Freestyle test riding her 2014 World Equestrian Games mare 14-year-old Fancianna, a Friesian-Andalusian cross, to a score of 67.917%. "I was pleased with Fancy today," said Andrews. "I was a bit tired, but we pulled it off. Many thanks must go to the Global Dressage Festival for putting on a great show. Special thanks to my coach, Amanda Kalvoda; chef d'équipe, Elizabeth Quigg; Pa...
Friday, January 9, 2015 - 05:31
U.S. Chef D' Equipe Kai Handt jogs Rubicon 75, ridden by Mary Jordan, owned by Vinceremos Therapeutic Riding Center (Photo (c) Lindsay Y. McCall)
Wellington, FL - Para-Equestrian Dressage athletes from around the North American continent have landed in Wellington, Florida, for the 2015 Adequan Global Dressage Festival CPEDI3*. This is the first CPEDI3* event for the 2015 calendar year. Thirteen horse and rider combinations presented their mounts in front of FEI Ground Jury members Mr. Freddy Leyman (BEL), Mr. Kjell Myhre (NOR), and Ms. Kristi Wysocki (USA). Para-Equestrians from Canada and the United States will compete January 9-11, in The Van Kampen covered arena at the Global Dressage Festival. Friday, January 9, is the FEI Para-Equestrian Dressage Team Test, followed by Saturday's FE...
Thursday, January 8, 2015 - 07:52
Kai Handt and Wes Dunham showcase collaborative coaching at the Northeast Symposium in 2013. Rider Sydney Collier aboard her own Wentworth works on gaits. (Photo (C) Lindsay Y. McCall)
Wellington, FL - The United States Equestrian Federation, Inc. (USEF), is excited to announce a two-day Para Equestrian Dressage Symposium to be held at Global Dressage, Wellington, Florida, January 12 & 13, 2015, 8 AM-4 PM. Select U.S. High Performance and developing Para- Equestrian Dressage riders and coaches will be attending the symposium. Auditors are welcome to attend for $75. Pre-registering as an auditor is not required. Please contact Laureen Johnson at USEF at email@example.com if you are interested in audit...
Wednesday, December 24, 2014 - 05:00
Nora Grassmyer, Mibis, Barbara Grassmyer, and Bob Grassmyer. (Photo: (c) Lindsay Y. McCall)
El Dorado Hills, CA - On December 19, 2014, the Dressage Community lost friend, volunteer, and parent Nora Grassmyer after a three year battle with bladder cancer. Nora was heavily involved with the Dressage and Para-Dressage disciplines both internationally and at the national level. She was also a longtime volunteer at the California Dressage shows managed by Connie Davenport. Nora was a driving force behind her daughter and Paralympian Barbara Grassmyer. She was there at every moment for Barbara including when Barbara served as an alternate for the U.S. Paralympic Team in 2000, competing in Sydney, Australia; in 2001 and 2002 when Barbara was the USEF Para-Equestrian Dressage National Champion;...
Thursday, December 18, 2014 - 06:30
Rebecca Hart and Schroeters Romani rode to the high score of 78.382% during the Polar Express Show at White Fences.
Wellington, FL - Rebecca Hart and Schroeters Romani shined Saturday afternoon during the Polar Express show held at the White Fences Equestrian Center in Loxahatchee, Florida. The talented duo rode to the score of 78.382 percent for the FEI Para Team Grade II test during the national show, kick starting their season as they prepare for the Adequan Global Dressage Festival in CPEDI3* in January 2015.Hart traveled to Florida in early December to work with Wellington-based international dressage trainer Todd Flettrich out of Cherry Knoll Farm, which is also a partial owner of Romani. Hart and Flettrich have worked consistently on warm-up and preparation with Romani after takin...
Sunday, December 14, 2014 - 17:41
Sydney Collier riding Willi Wesley (Photo: SusanjStickle.com)
Lexington, Ky. - The United States Equestrian Federation (USEF) is pleased to announce Sydney Collier as the 2014 Junior Equestrian of the Year. Seventeen-year-old Collier is a talented Para-Equestrian who competed at the international level in 2014, including the Alltech FEI World Equestrian Games. A native of Ann Arbor, Mich., she was diagnosed at the age of seven with a rare congenital birth defect called Wyburn-Mason Syndrome. In spite of this challenge, Collier pursued her love of riding and has become a top equestrian athlete. A prime example of courage, sportsmanship, and dedication, Collier will be awarded the Ruth O'Keefe Meredith Memorial Trophy as the 2014 Junior Equestrian of the Year. Having her biggest year to da...
Sunday, November 30, 2014 - 06:53
Trainer Wes Dunham and WEG athletes Sydney Collier's mount Willi Wesley at the 2014 Adequan Global Dressage Festival CPEDI3* horse inspection . (Photo: Lindsay McCall)
Wellington, Florida - November 26, 2014 - Entries are now being accepted at www.equestrianentries.com through December 22, 2014, for the 2015 Adequan Global Dressage Festival CPEDI3*. Para-Equestrian Dressage athletes kick-off the show season with their their first international show of the year January 7-11, 2015, in Wellington, FL, in the Van Kampen covered arena. This show will include FEI Para-Equestrian Dressage athletes in Grades Ia, Ib, II, III, and IV. Thursday, January 8, is the Horse inspection at approximately 5:30 PM. Friday, January 9, is the FEI Para-Equestrian Dressage Team Test, followed by Saturday's FEI P...
Tuesday, November 11, 2014 - 06:18
We salute our veterans this Veterans Day. Thank you for protecting our freedoms. On this day we ask you to take a moment to think about those that have served our country. We would like to salute our disabled veterans who are part of not only the equestrian sport but all Paralympic sports. Thank you, - USPEA. We too, at DressageDaily, would like to take a moment to thank all veterans who have served our country to protect our liberties and rights. We truly appreciate all past and current members of our military for all that you do! History of Veterans Day - From the U.S. Department of Veterans Affairs: World War I - known at the time as "The Great War" - officially ended when the Treaty of Versailles was signed on June 28, 1919, in the Palace of Versailles outside the town of Versaille...