text:
Hi! My name is Sandy Musk, and I am the music teacher at Timberwilde. This is my 37th year of teaching music in Northside ISD. I have been at Timberwilde since it opened in 1980. It has been a joy for me to teach the children of this neighborhood. My first 2 years of teaching music began at Esparza Elementary.
I am a local girl. I went to Holmes High School, Incarnate Word College, and graduated from The University of Texas in San Antonio, with a Music Education Degree and an Early Childhood Endorsement. My Masters of Music Degree is from Sam Houston State University with a Kodaly Certification. My two children have grown up with band, dancing, sports, and have become like many kids, computer savvy.
My "specialty," is playing the organ for weddings. Would you believe that I started playing for weddings when I was only 13 years old? My introduction into the music world began when I started taking dancing lessons at age 4. It followed with piano and organ lessons in 3rd and 6th grade. Besides teaching, I have a church music job, where I am the organist, conduct a children's chorus, and direct a hand chime group. This is just another venue to expand my musical learning.
In 2008, I was honored to be "The Spurs Teacher of the Game." It was a very fun night. I have also been selected "Teacher of the Year."
At Timberwilde, the music program tries to emphasize many elements of music. There is a strong emphasis on Kodaly and Orff methods of teaching. We sing, play instruments, use rhythmic and solfege notation, listen to music, and play lots of music games. We also incorporate folk dancing and square dancing throughout the year. In fourth and fifth grade, the students learn how to play the soprano recorder.
The featured group of the school is the Timberwilde Choir. It is an auditioned group of fourth and fifth graders, (with a few selected third graders), that meet before school 3 days a week at 7:20 A.M. The choir has proudly sung at Fiesta Texas, Trinity University, the State Principal's Convention at the downtown Mariotte Hotel, St. Anthony Hotel, and the Hill Country Hyaat Hotel. The choir has also performed at Firstmark's Credit Union Shareholder Banquet, as well as grand openings, Los Leones Art Show, District Choir Festivals, H.E.B., nursing homes, malls, community parties and patriotic events. We have a well known group that just loves to sing.
In April 2008, the choir was highlighted at Sea World's 20th Anniversary Celebration, that was live, nationally web casted. A lot of fun and educational perks came along with it. A big thrill was when the Timberwilde Jubilant Singers made their debue on TV, singing for a holiday special on KABB/FOX TV in 2007. We returned in 2009 and 2011.
My planning/conference time is from 11:25 A.M.-12:10 P.M. Feel free to email me at firstname.lastname@example.com, or call 3970400 ext3151 if you have any questions.
My personal goal is to spread the joy of music to the children of Timberwilde.


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Carlos Martinez (martica04)
From BR Bullpen
Carlos Ernesto Martinez
known as Carlos Matias prior to 2011
- Bats Right, Throws Right
- Height 6' 0", Weight 185 lb.
- Born September 21, 1991 in Colinas del Sur, Puerto Plata, D.R.
[edit] Biographical Information
Carlos Martinez is a top pitching prospect who made his debut with the St. Louis Cardinals in 2013. He was previously known as Carlos Matias. He was originally signed by the Boston Red Sox under that name in 2009, but the contract was voided by Major League Baseball before he could play for the Red Sox, because of concerns over his true identity and birth documents. He was suspended for a year and when the delay ended, signed with the Cardinals in April of 2010. The source of the problem was that he was born in a rural area of Puerto Plata province in the Dominican Republic, where record-keeping is poor; his mother died when he was young and he was brought up by an uncle whose last name was Matias, hence the confusion. His place of birth, Colinas del Sur, simply refers to a district in his native province.
Carlos made his professional debut at age 18, pitching for the DSL Cardinals in 2010. He went 3-2 in 12 starts, dominating opponents with an 0.76 ERA and 78 strikeouts in 59 innings. He led the Dominican Summer League in ERA (.10 ahead of runner-up Henry Perez), lowest opponent average (.144), WHIP (.71, tied with Jayson Aquino) and strikeouts per 9 innings. Baseball America listed him as the Cards' #3 prospect. That earned him a ticket to the United States, and he started the 2011 season in full-season A ball, playing for the Quad Cities River Bandits of the Midwest League; this was already a huge leap for a young pitcher from the Dominican Republic, as the Rookie-level Appalachian League would have been a more common next destination. Carlos did extremely well against Midwest League batters though, putting up a 2.33 ERA over his first 8 starts, with 50 strikeouts over 38 2/3 innings, to earn a promotion to the Palm Beach Cardinals of the Florida State League. He vaulted so far up the prospect rankings that he was selected as one of two Cardinal minor leaguers to play in the 2011 Futures Game. He pitched the 4th inning for the World team. He struck out Paul Goldschmidt to open the inning, hit Devin Mesoraco with a pitch, and then induced Will Middlebrooks to hit into a double play grounder to end the frame. He hit 98 mph on the radar gun during the outing, showing the sort of stuff that makes scouts salivate. He was working on developing a change-up to complement his outstanding fastball. Martinez did not pitch as well after the Futures Game, finishing at 3-3, 5.78 for Palm Beach, for a combined pitching line of 6-5, 3.93 in 18 starts. Baseball America rated him as having the top fastball in both the FSL and MWL. They listed him as the 6th-best prospect in the FSL, after Shelby Miller, Matt Harvey, Hak-ju Lee, Trevor May and Alex Colome, as well as the 27th-best prospect in baseball, second to Miller in the Cards chain.
In 2012, Martinez was back at Palm Beach to start the year, and put up an ERA of 3.00 with a 2-2 record in 7 starts. He was then promoted to the Springfield Cardinals of the Texas League, where he was 4-3, 2.90 in 15 games. Overall, he pitched 22 games, with a record of 6-5, 2.93, to confirm his status as a top prospect. After the season, Baseball America listed him at #38 on its top 100 prospects list and third among Cardinals minor leaguers after Oscar Taveras and Miller. They ranked his fastball as the best in the FSL and put him down as the circuit's 6th-best prospect, behind position players Jurickson Profar, Taveras, Wil Myers, Mike Olt and Jonathan Singleton.
He was back at Springfield to begin the 2013 season and picked up a win in his first three starts, during which his ERA was 2.31 and he had a 9/1 K/W ratio in 11 2/3 innings. He had missed all of spring training because of trouble in obtaining a visa, which explains why he had pitched so little when called up to St. Louis on May 3rd to replace the struggling Mitchell Boggs. Although he had been a starter throughout his minor league career, the Cardinals were looking to him as a reliever and it is in that role that he made his debut on the day of his call-up, pitching a scoreless 7th inning in relief of another top prospect, Miller, with the Cardinals holding a 6-1 lead over the Milwaukee Brewers. He allowed a single to the first batter he faced, Yuniesky Betancourt, but the induced pinch-hitter Logan Schafer to hit into a double play and Norichika Aoki to ground out to end the inning before being replaced by pinch-hitter Ty Wigginton.

subset: commoncrawl


text: Shapeways: Bring your creativity to life in 3D http://www.shapeways.com/forum/index.php Shapeways: Bring your creativity to life in 3D nice place you guys have here http://www.shapeways.com/forum/index.php?t=rview&goto=31874&th=5979#msg_31874 I have upload a couple simple items to see how things work around here, so far so good. . did a test video also. let me know what you think.]]> mrgm 2011-07-31T00:11:21-00:00 Re: nice place you guys have here http://www.shapeways.com/forum/index.php?t=rview&goto=32024&th=5979#msg_32024 Very nice start. Have you been through the material specifications to make sure your items are printable? Some of them look pretty small. http://www.shapeways.com/materials/ Hope to see some prints in the "It Arrived" section soon!]]> Youknowwho4eva 2011-08-03T17:14:19-00:00 Re: nice place you guys have here http://www.shapeways.com/forum/index.php?t=rview&goto=32056&th=5979#msg_32056 . I took some pictures and will post in the appropriate section. i will try to keep things thicker than a 1/16" if in the tuff stuff and thick for the lighter materials. ]]> mrgm 2011-08-04T14:41:12-00:00


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Works great for our iguana
posted 7 years ago
I'm surprised by the other reviews. Our vet (who is an iguana vet) recommended we get a heat rock with an auto shut-off to help with digestion and keeping him warm. Our rock shuts-off every so often on its own. We've had no indication of burning and our iguana does every BUT sit on it all day.
Pros: This works great for him
Cons: None
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posted 6 years ago
As a leopard gecko owner, I strongly dislike this product. The burn rate for leo's is pretty high. I recommend a heat pad over a heat rock any day.
Pros: does provide heat
Cons: can cause bad burns
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Results 11 to 20 of 38
Style: MMA--
Yeah I have to figure out how to get my hands on that. Normally they drop off the reports a week or two later at the bar for our TPW charges, but he got hit with a few more charges that I am not sure we will see.
My witness statement doesn't include the ninja mask and the stars, that was what the Sheriffs told me after the fact. But that should be in the police report I believe, he had some sort of outstanding warrant on him too, and I think got some sort of charge for the weapons (being concealed and illegal, butterfly knife is over 3 1/2" which is what you can carry in in FL).
No telling what I can get a copy of, but I know I have the witness statement up at the bar, I may be able to get a report when the DA calls us in a month or so. We pressed charges on him for battery, simply because we wanted to go to court with this lol as it was too damn funny.
Chances are he'll plead out though and we won't be able to tell a judge the hilarity that ensued. I will keep you guys posted though.
Be careful of them ninjas!
Light Heavyweight
- Join Date
- Jan 2006
- Location
- Orlando, Florida
- Posts
- 3,378
Posted On:
1/26/2007 3:08pm
Style: BJJ/Pekiti Tersia/Hsing-I--
This story's amazing. +Rep for the retard of ninja.
The insane's facination with the martial arts is always hilarious to me.
Two of my girlfriends uncles are pretty much wacked out of their skull. They're also both convinced that they're martial arts masters (One is convinved he's half elf, but thats another matter entirely).
The other one is a schitzo and he was living in a house with his friends. Apparently he got into a argument with someone near by and they ended up calling the cops on him. My girlfriends father arrived there because he was bringing his brother-in-law some dinner when he asked the cops what was going on. The cops were hesitating going in and arresting him because the uncle yelled from inside that he "knew kung fu" and would "take them all out" if they came in there. Her father started laughing and said, "Thats just Tim... he's crazy.. he doesn't know ****. Let me talk to him." About 2 minutes later he brought him outside. The cops that were there recognised him (apparently they had to deal with him before) and went... oh it's Tim." Talked to him for a minute and let him go back in.
Last edited by Bustardo; 1/26/2007 6:16pm at .
- Join Date
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Posted On:
1/26/2007 9:21pm
Style: CMA,Muay Thai ,Yudo,TKD--Originally Posted by GoldenJonas
Style: Rehab--
Senior Member
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Posted On:
1/26/2007 9:41pm


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Stocks log best month since January on Fed pledgeBy STEVE ROTHWELL
The Associated Press | August 01,2013AP PHOTO
A television screen on the floor of the New York Stock Exchange shows the rate decision by the Federal Reserve, as specialist Jeffrey Berger works at his post, Wednesday.NEW YORK (AP) After a day of stalled rallies, the stock market closed out July with its best monthly gain since January.
The Standard & Poors 500 index ended the month 4.95 percent higher. Thats the biggest increase since January, when it rose 5.04 percent. The Dow Jones industrial average also had its best month since January.
Markets surged in July after Fed Chairman Ben Bernanke assured investors that the central bank wouldnt pull back on its stimulus program until the economy was strong enough. The central bank is buying $85 billion of bonds a month to keep down interest rates to encourage borrowing and hiring.
On Wednesday, the Fed reaffirmed its commitment to support the economy in a statement released after the end of a two-day meeting. The central bank dropped hints that it might need to maintain its stimulus, and slightly downgraded its assessment of U.S. economic growth from moderate to modest.
That initially gave stocks a boost, pushing the S&P 500 within two points of breaching the 1,700 level for the first time in afternoon trading. But the rally faded in the final hour, leaving the S&P flat at the end of the day.
Given the markets big gains in July, stocks may struggle to climb further in the coming months, said Phil Orlando, chief equity market strategist at Federated Investors.
I would not be the least bit surprised to see some modest consolidation, said Orlando.
Stocks started higher Wednesday after the government said that the economy grew at a faster pace in the second quarter than economists had forecast.
The gain was mostly gone by the time the Fed statement came out at 2 p.m. Eastern Daylight Time. The market staged another rally for about an hour after the Feds announcement, then ended little changed.
The U.S. grew at an annual rate of 1.7 percent from April through June as businesses spent more and the federal government cut less spending, the Commerce Department said Wednesday. Economists had expected growth of 1 percent, according to the data provider FactSet.
There was also an encouraging report on hiring ahead of the governments monthly jobs survey due out Friday.
U.S. businesses created a healthy 200,000 jobs this month, payroll company ADP said, as companies hired at the fastest pace since December. ADP also raised its estimate of the number of jobs the private sector created in June.
The Standard & Poors 500 index ended little changed at 1,685.73. The Dow Jones industrial average ended the day 21 points lower, or 0.1 percent, at 15,499.54.
The Nasdaq composite gained 9.90 points, or 0.7 percent, to 3,626.37. The index fell just five times during the month and is at its highest level in more than a decade.
On the bond market, investors anticipated that the Feds slightly weaker assessment of the economy would imply a longer period of bond purchases. Bond yields fell as demand increased for U.S. government debt. The yield on the 10-year Treasury note fell to 2.58 percent from 2.66 percent just before the announcement.
Investors were also tracking company earnings Wednesday.
Comcast rose $2.37, or 5.5 percent, to $45.08 after the parent company of the NBC network and Universal Studios reported earnings and revenue that exceed analysts expectations in the second quarter.
Software company Symantec, which makes the Norton antivirus software, surged after the company reported earnings and revenue that beat analysts forecasts. The stock rose $2.33, or 9.6 percent, to $26.68.
Analysts forecast that second-quarter earnings rose an average of 4.75 percent for S&P 500 companies, according to S&P Capital IQ. That would be the slowest rate of growth in three quarters.
In commodities trading, the price of oil rose $1.95, or 1.9 percent, to $105.03 a barrel. Gold dropped $11.80, or 1 percent, to $1,313 an ounce.MORE IN National / World BusinessWASHINGTON An emotional Amtrak CEO pledged to lawmakers Tuesday that safety technology that... Full StoryWASHINGTON U.S. Full StoryWASHINGTON Al and Saundra Karp have found an unconventional way to raise money and help save... Full Story
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Shannon Says:
July 28th, 2011 at 7:41 AM
Ah, incest fanfiction. I'm all too familiar with it, mostly in the Harry Potter & LOTR fanfiction world. In LOTR, it's usually between Elrond's sons/Arwen's brothers Elladan and Elrohir; in HP I've seen it in many different forms, from the popular Fred/George to Twins/Ron, Ron/Ginny, Bellatrix/Narcissa, Bellatrix/Sirius, etc, etc, etc. An ex-girlfriend of mine was pretty into Elladan/Elrohir fanfic, actually. It doesn't faze me anymore, but I still find it a little strange. I get that it's always "between consenting adults" and all, but it's still just a little weird.
Wincest is just… no. And canon? I don't buy it at all. Now, Cas/Dean, on the other hand, that I might buy as canon.
Paula Says:
July 28th, 2011 at 1:42 PM
Lol Yeah even I can see how someone would see Dean/Cas from canon! I think that the writers like to have a little fun with it. Thanks for commenting!
Dayle Fraschilla Says:
December 18th, 2011 at 6:43 AM
Um, ohh kay.
I've only seen one episode of Supernatural (I'm still working out how to start watching it from the beginning), but I can understand your disgust.
I've never been into fanfiction. I tried with Buffy, but I just get annoyed, so I stick with the "actual" story lines.
About incest writing in general, have you ever read Flowers in the Attic and the sequels? I read them in middle/high school and was highly perturbed at the time!
Dayle Fraschilla recently posted..Not-a-diet week something-or-other: Making it Happen
Paula Reply:
December 18th, 2011 at 3:04 PM
I really think you would like Supernatural! It's one of those shows that got better as time went on, with the 4th and 5th seasons being the best.
The only fanfiction I really enjoyed reading was Harry Potter. It helped make the long stretches between books bearable.
I never read Flowers in the Attic, but I remember being around ten and my older cousin was reading it. She told me about it, and I was just totally creeped out. At ten, I had never heard of incest, so it was like the first corruption of my brain. lol And I've just never had any desire to read those books, because if it.
Wincest fan Says:
November 19th, 2014 at 6:39 PM
Okay so…I understand that you may not like wincest, and I am a fan, but most cultures it is not abnormal for incestuous relationships. For example, there's Cleopatra, and in the middle east, cousins marry another. Its a social norm. As for not reading the fanfics, well, I'm not quite sure why you would write about something you do not have much knowledge on. Know that I am referring back to you in saying this, not trying to demean you. If you disagree with incest, I'd much rather you explain why incest is wrong, rather than wincest. As for why I read it, it is because after watching about all the seasons, I believe that Sam and Dean are meant to be. If a human and angel can be together (Dean and Cas) why not siblings? Two consenting adults should be able to express their love for one another despite the fact that they are related. Not all teenage girls write fanfic because its easier, most write it because they too believe that Sam and Dean would make a good couple. To me, it is canon because despite everything they have been through, at the end of the day its Sam and Dean. I would recommend that you read fanfics without a sexual focus, those can explain better why wincest is a legitimate ship. I am not trying to be rude, I am merely expressing my opinion, which happens to differ from your own.
Lealdanan Reply:
March 11th, 2015 at 7:54 AM
@Wincest fan, Hi. I have to say, I am not against incest. In fact, I ship myself some incestuous relationship, like Lucrezia and Cesare from The Borgias (the Showtime version). So, I am not allergic to the idea at all. And I would have been open to the idea of a relationship between Dean and Sam, IF, and only IF there really was something to go on with. But there isn't. There is no sexual tension, no inappropriate touching, no romantic chemistry. In other sibling incest situation, you can usually feel the tension building between the two, or just by the way they look at each other, like Cas and Dean. These two are brothers (that love each other very much), but just brothers. If you really take off your fangirl glasses, you would know that yes, it's all about Sam and Dean, but in no way these two would have anything else than this very brotherly bond that they share, bickering and fighting and saving each other and forgiving each other, like only siblings do.
Why Wincest Is Wrong
You may be asking yourself, "What in the world is Wincest?" Well, unfortunately for you, I am about to tell you. Wincest is Supernatural fanfiction that features a sexual relationship between brothers Sam and Dean. (Or one of them and their Dad.) I know! I felt like gouging my eyes out the first time I read about it too!
Okay, I have to admit that I have never actually read any Supernatural fanfiction. I only know that Wincest exists because I lurk at the TWOP forums and it is talked about a lot there. I use to read Harry Potter fanfiction all the time though and it has it's own eye-gouging trends. (Twincest for one. Yes, as in Fred and George! ICK!)
Anyway, I don't have to actually read Wincest to know that it is wrong. It is about incest, for pete's sake! Incest is illegal (in most places and cultures), abnormal and gross! Why would you want to write about it, at least in a positive light, let alone read it? I just don't understand!
I have read articles that say most fanfiction of this nature is written by teenage girls who have a crush on one of the male characters. Apparently it is easier for them to share their crush with male characters than another female character. I have no idea if this is true or not, it's kind of silly if it is, and makes me feel a little bit sorry for these girls.
Honestly, the thing that bothers me the most about Wincest is the people that insist that it is canon. WTF? Where are they getting this?
Of course, Sam and Dean love each other. They are brothers, who have literally been to hell and back for each other and they have a strong family bond, but that does not equal sexual attraction. There are many different types of love. Can't people today tell the difference? (I know the show sometimes makes allusions to Wincest but always with the idea that it is ridiculous. To quote Dean: They know we're brothers, right?)
You are probably wondering why I even care when these are just fictional characters that I am talking about. Well, these characters are near and dear to my heart and I hate to see them put through something so… gross!
So, I have just one thing to say those of you writing fanfiction, I say, "Grow up and create a Mary Sue to fulfill your sexual fantasies with your favorite Winchester brother and leave the Wincest alone!"
Are you a fan of Supernatural? If so, what are your views on Wincest?
Let's be clear that I am not talking about Slash fanfiction. It's not my thing, but as long as the characters aren't related, I say go with whatever floats your boat.


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Eating Lunch at Your Desk? It's Worse for You Than You Think
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Judge Bars Cracker Barrel from Grocery Stores: Supermarket Savvy
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Rabbi Speaks at Glenn Beck Gathering
Tells us Bible Belt is 'Judaism's safety belt'
Glenn Beck's "Restoring Honor" mega-rally Saturday, billed as a Christian religious revival, drew a crowd of between 50,000 and 600,000 (depending on who is counting). On Friday, however, the Fox News host gathered closer to 5000 of his closest friends at the Kennedy Center for a quieter event, "America's Divine Destiny," which featured all-stars like the Rev. John Hagee, Chuck Norris, and … Orthodox Rabbi Daniel Lapin.
Lapin, who has notoriously been tied to Jack Abramoff, echoed the larger themes of the night: "When you sever a flower from its roots, it dies," he reportedly told the crowd. "I think what is happening in America is we're being severed from our Biblical roots." According to one of the event's hosts, he urged the crowd to "study the Bible, make more money and say extra prayers for America."
Of course, it was Shabbat, and so a microphone was a no-no. The audience, however, could apparently hear him fine, and even greeted him with a "Shabbat Shalom."
The rally, Lapin told Tablet Magazine yesterday, demonstrated that "America is a country that is rooted in Christianity, and that this is one of the factors that have made America one of the most tranquil and prosperous homes that Jews have enjoyed for 2000 years." He added, "I think of the Bible Belt as Judaism's safety belt."
At Lincoln Memorial, a Call for Religious Rebirth [NYT]
Glenn Beck Goes Messianic at America's Divine Destiny Event Before 2500 Screaming Fans [Alternet]
Glenn Beck's 'Divine Destiny' Event Focuses on Faith [Daily Caller]
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The signal transducer and activator of transcription 3 (STAT3) mediates gene expression in response to numerous growth factors and cytokines, playing an important role in many cellular processes. To better understand the molecular mechanisms by which Stat3 influences gene expression in the lung, the effect of pulmonary epithelial cell specific deletion of Stat3 on genome wide mRNA expression profiling was assessed. Differentially expressed genes were identified from Affymetrix Murine GeneChips analysis and subjected to gene ontology classification, promoter analysis, pathway mapping and literature mining.
Total of 791 mRNAs were significantly increased and 314 mRNAs were decreased in response to the deletion of Stat3Δ/Δ in the lung. STAT is the most enriched cis-elements in the promoter regions of those differentially expressed genes. Deletion of Stat3 induced genes influencing protein metabolism, transport, chemotaxis and apoptosis and decreased the expression of genes mediating lipid synthesis and metabolism. Expression of Srebf1 and 2, genes encoding key regulators of fatty acid and steroid biosynthesis, was decreased in type II cells from the Stat3Δ/Δ mice, consistent with the observation that lung surfactant phospholipids content was decreased. Stat3 influenced both pro- and anti-apoptotic pathways that determine cell death or survival. Akt, a potential transcriptional target of Stat3, was identified as an important participant in Stat3 mediated pathways including Jak-Stat signaling, apoptosis, Mapk signaling, cholesterol and fatty acid biosynthesis.
Deletion of Stat3 from type II epithelial cells altered the expression of genes regulating diverse cellular processes, including cell growth, apoptosis and lipid metabolism. Pathway analysis indicates that STAT3 regulates cellular homeostasis through a complex regulatory network that likely enhances alveolar epithelial cell survival and surfactant/lipid synthesis, necessary for the protection of the lung during injury.
The signal transducers and activators of transcription (STATs) were initially identified as a family of latent cytoplasmic transcription factors that are activated by various cytokines, growth factors and other stimuli, and phosphorylated by many protein kinases [1-4]. In response to various stimuli, STAT family members are phosphorylated by receptor associated kinases, form homo- or heterodimers and are translocated to the cell nucleus where they activate transcription [5]. Recent studies also support the role of unphosphorylated STAT3 that accumulates in nucleus and activates transcription by binding to NFkappaB [6]. STAT3 regulates a variety of biological processes, functioning at both transcriptional and non-transcriptional levels to influence cell growth, survival and metabolism. Its capacity to induce cell transformation and tumorigenesis makes it a potential therapeutic target for various cancers [7,8].
Systemic deletion of Stat3 is embryonic lethal in the mouse, indicating its important role in embryogenesis [9]. Biological roles of STAT3 in various organs and cells have been studied in vitro as well as cell specific deletion in the mouse in vivo. The biological consequences of Stat3 deletion are surprisingly diverse and sometimes contradictory. For example, cardiomyocyte-specific STAT3 deficiency caused cardiac fibrosis and heart dysfunction with advanced age [10]. Hepatic cell specific deletion of Stat3 caused insulin resistance associated with increased expression of gluconeogenic genes [11]. Conditional ablation of Stat3 in respiratory epithelium of the mouse (Stat3Δ/Δ mice) did not alter lung morphogenesis or function but enhanced susceptibility to hyperoxia and adenoviral induced lung injury whereas overexpression of Stat3C (a constitutive active form of STAT3) in pulmonary epithelium protects against hyperoxic lung injury, suggesting that STAT3 is required for the maintenance of surfactant homeostasis and lung function during injury [12-14]. While STAT3 has been proposed as an anti-apoptotic protein through the induction of survival genes such as Bcl2-like 1(Bcl-X) and B-cell leukemia/lymphoma 2 (Bcl-2), STAT3 also exerts pro-apoptotic effect through the regulation of insulin-like growth factor binding protein 5 (IGFBP-5) to modulate mammary epithelial apoptosis [4,15]. STAT3 is abundantly and ubiquitously expressed in many tissues and distributed between the cell cytosol and nucleus. A direct effect of non-phosphorylated, cytoplasmic STAT3 on cell motility was reported recently through direct protein-protein interactions [16], indicating a non-transcriptional function of STAT3.
The functions of STAT3 vary in different cellular and physiologic contexts, influencing diverse gene targets by interaction with other proteins and genes. The diversity of STAT3 functions indicate that STAT3 is involved in complex genetic networks to maintain cellular homeostasis rather serving a singular role in acute phase responses as initially defined. In the present study, we sought to systemically study the role of STAT3 in pulmonary epithelial cell homeostasis. Using knowledge based gene expression profiling approaches and a conditional system that selectively deleted Stat3 in the respiratory epithelium; we identified a large STAT3-dependent network that influences a wide variety of biological processes in type II alveolar cells in the lung.
Results and Discussion
Identification of differentially expressed genes in alveolar type II epithelial cells from Stat3Δ/Δ mice
In previous studies Stat3 mRNA and protein expression were markedly reduced in Type II cells isolated from Stat3Δ/Δ mice, being less than 10% of control levels [12]. To identify the RNAs influenced by the conditional deletion of Stat3Δ/Δ, RNAs isolated from alveolar epithelial type II cells of control and Stat3Δ/Δ mice were compared using Affymetrix murine genome MOE430 gene chips. The complete dataset can be found at Gene Expression Omnibus (GEO) [17]; Accession no. GSE6846. Total of 1105 genes were identified as significantly altered using the criteria described in Method. Among them, 791 mRNAs were increased and 314 mRNAs were decreased in response to the deletion of Stat3Δ/Δ in the lung (Additional file 1). Changes in mRNA expression of a subset of genes including Malt1, Rnt4, Reg3g, Bcl2l1, Cds2, Cdipt, Fasn, Acox2, Akt2, Gpam, Foxj1, Abca3, Srebf1, Srebf2 and Scap were validated by real-time RT-PCR. Genes cross validated by RT-PCR are listed in Table 1 and indicated by asterisks (*) in Additional file 1.
Additional file 1. Genes Differentially Expressed in Stat3Δ/Δ Mice. Additional file descriptions text (including details of how to view the file, if it is in a non-standard format).
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Table 1. Comparison of mRNAs by RT-PCR and RNA microarray
Functional classification of differentially expressed genes revealed the dysregulation of various biological processes in type II epithelial cells from Stat3Δ/Δ mice
Differentially expressed genes were classified according to Gene Ontology (GO) classification on Biological Process. The Fisher Exact Test was used to calculate the probability of each category that was overrepresented in the selected list using the entire MOE430 mouse genome as reference dataset. Deletion of Stat3 from type II cells significantly induced the genes involved in protein metabolism, protein transport, chemotaxis and apoptosis and decreased the expression of genes in lipid synthesis and metabolism (Table 2).
Table 2. Functional Classification of Genes Differentially Expressed in Stat3Δ/Δ mice
Promoter analysis identified putative common regulators of the differentially expressed genes
To identify putative common transcription factors regulating the type II cell responses to Stat3 deletion, promoter region (-2kb to exon1) of differentially expressed genes were searched for overrepresented cis-elements using MatInspector (Genomatix) vertebrate matrix library. In compare with the sequence of random chosen gene promoters, the cis-elements significantly enriched in the promoter region of differentially expressed genes were selected based on a binomial probability calculation and their percentage frequency in our selected gene list (Adjusted p Value < 0.001 and frequency > 50 %). STAT, EGRF, AHRR, SP1F, ZF5F, E2FF, HIFF, SREB and AP2F were the most overrepresented cis-elements and may therefore mediate changes in gene expression in cells from Stat3Δ/Δ mice (Table 3). The finding that STAT was the most enriched cis-elements indicated the identification of a sub-group of potential direct transcriptional targets of Stat3 in lung epithelial cells. Sterol regulatory element binding factors (Srebf1 and 2, known as important transcriptional regulators of fatty acid and steroid biosynthesis), were significantly decreased in Stat3Δ/Δ mice. The SREB binding site was overrepresented in differentially expressed genes indicating that it is a potential regulator of the lipid metabolism pathways altered in Stat3Δ/Δ mice. Other significantly enriched TFBS include SP1F and HIFF. STAT3 and SP1 function cooperatively to activate the C/EBP promoter, the SP1 site being required for IL-6 induction and transactivation by STAT3 [18]. HIF1A, SP1, SMAD3 AND SMAD4 can form multifactor complex, regulating VEGF and erythropoietin gene transcription through functional cooperation and association [19-21]. Consistent with the promoter analyses and literature findings, the mRNA expression of Klf5 (a member of the SP1 family) and Hif1a, Smad3 and Smad4 was simultaneously increased by Stat3 deletion, indicating the potential transcriptional complex formation among the corresponding transcription factors.
Table 3. Enriched TFBS In Genes Differentially Expressed in Stat3Δ/Δ mice
Pathway analysis revealed known and novel functions of STAT3 in the lung
Pathway enrichment test is an unbiased way to answer the question, ''Are the differentially expressed genes enriched in certain pathways?'' To address this issue, we compared the overlap of differentially expressed genes in Stat3Δ/Δ mice with the known biological pathways in KEGG (Kyoto Encyclopedia of Genes and Genomes) using 1) the mouse genome and 2) a list of genes shown to be least changed in response to the Stat3 deletion in the gene array as background. Results from both analyses were consistent. Jak-Stat Signaling Pathway, Apoptosis, Cytokine-Cytokine Receptor Interaction, Insulin Signaling Pathway, Mapk Signaling Pathway, Focal Adhesion, and Wnt Signaling Pathway were among the most enriched pathways identified from the analysis of differentially expressed genes (Table 4). mRNAs mediating steroid biosynthesis and fatty acid metabolism were mostly decreased in Stat3Δ/Δ mice. The most overrepresented pathways and the differentially expressed genes associated with those pathways are illustrated in Additional files 2. The finding that known Stat3 functions, including the Jak-Stat Signaling Pathway were identified from microarray analysis of Stat3Δ/Δ mice type II cells using knowledge integration approaches provides support for the utility of the analysis to detect novel pathways regulated by Stat3.
Additional file 2. Differentially Expressed Genes In Jak-Stat, Insulin and Wnt Signaling Pathway. Pathways were downloaded from KEGG website [82]. Each rectangle represents one or multiple gene products. Genes up-regulated in Stat3Δ/Δ mice are highlighted in red. Genes down-regulated in Stat3Δ/Δ mice are highlighted in blue. Rectangle contains more than one gene products and changes expression in opposite directions are highlighted in yellow.
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Table 4. Enriched Pathways In Genes Differentially Expressed in Stat3Δ/Δ mice
Stat3 influences protein metabolism in lung type II cells
''Protein metabolism'' was the most enriched biological process (5.83E-10), accounting for 20% of the induced genes caused by the deletion of Stat3. More than 40 genes encoding proteins involved in protein ubiquitination/ubiquitin cycle were present (Table 5). Of interest, Casitas B-lineage lymphoma b (Cblb) was increased 7.3 fold. CBLB is a member of Cbl ubiquitin ligases (E3) protein family that are tyrosine-phosphorylated in response to a wide variety of receptor mediated stimuli, including epidermal growth factor receptors, cytokine receptors such as colony stimulating factor family receptors (increased 2–3 fold in the Stat3Δ/Δ cells) and immune complex receptors such as Fcgr2b (increased 2.7 fold). CBLB functions as an adaptor protein that regulates many signal-transduction pathways including JAK-STAT signaling pathways through its ability to interact with critical signaling molecules [22]. The expression of genes encoding known CBLB binding partners was significantly increased in type II cells from the Stat3Δ/Δ mice, including SYK kinase (increased 4 fold), JAK2 (increased 2.1 fold), PKC (increased 2.6 fold), phosphatidylinositol 3-kinase (Pik3r1 increased 1.8 fold, Pik3c2a increased 3 fold), adenylate cyclase-associated protein 1 (Cap1 increased 5 fold) and Crk (increased 2.5 fold). These signaling molecules can in turn modulate various down stream signaling cascades including those mediated by small G-proteins (multiple members of RAS oncogene family were induced 2–3 fold), as well as PI3K/AKT mediated signaling and Nfat induction (increased 4.1 fold) [23-27]. CBLB physically interacts with another ubiquitin-protein ligase, NEDD4 (increased 3.8 fold), an interaction that reverses CBLB effects by targeting CBLB for ubiquitination and proteasomal degradation [28]. SLIM is a known E3 ubiquitin ligase specifically interacts with activated STAT1 and STAT4, inducing their ubiquitination and degradation [29]. We speculate that CBLB may act in a similar manner to regulate STAT3 signaling. In addition, SUMO1 and SUMO1 specific peptidase 2 were both increased more than 2 fold in Stat3Δ/Δ mice. SUMO1 can covalently modify many targets including STAT1 and glucocorticoid receptor NR3C1 (increased 5 fold) and regulate protein stability and transcriptional activity [30]. Taken together, deletion of STAT3 activates a number of molecules regulating protein metabolism, stability and routing, indicating the potential role of ubiquitination and sumoylation in cytokine signaling and STAT3 activation that influences cellular adaptation.
Table 5. Genes In Ubiquitin Cycle Were Largely Induced In Stat3Δ/Δ Mice
STAT3 influences phosphate metabolism and protein kinase cascade in lung type II cells
Expression of genes regulating ''Phosphate metabolism'' and ''protein kinase cascade'' were significantly increased in Stat3Δ/Δ mice (3.1E-07 and 1.38E-06, respectively), accounting for 7.5% of total induced genes (Table 6). Expression of genes encoding a number of kinase that phosphorylate STAT3 in vivo or in vitro were increased [31-35], including Janus kinase 1 and 2 (increased 3.1 and 2.6 fold, respectively), ribosomal protein S6 kinase polypeptide 3 (Rps6ka3, increased 3.1 fold), met proto-oncogene (Met, increased 4.2 fold), mitogen activated protein kinase 8 (Mapk8, increased 4.7 fold) and Dual-specificity tyrosine-phosphorylation regulated kinase 1a (Dyrk1a, increased 2.6 fold). The increased expression of these genes indicates a potential compensatory mechanism related to the lack of activation of STAT3 or its targets.
Table 6. Genes In Phosphate Metabolism Were Largely Induced In Stat3Δ/Δ Mice
STAT3 influences lipid homeostasis in lung type II cells
Expression of genes encoding sterol regulatory element binding factor 1 and 2 (Srebf1 and Srebf2), their cleavage activating protein (Scap) and multiple SREBP target genes involved in lipid metabolism were decreased in Stat3Δ/Δ mice type II cells. As depicted in Figure 1, genes dedicated to the biosynthesis of fatty acid, phospholipid and cholesterol were down regulated, with the exception of 3-hydroxy-3-methylglutaryl-Coenzyme A synthase (Hmgcs), which was increased. HMGCS participates in other metabolic pathways, including valine, leucine and isoleucine degradation (KEGG 00280). This analysis indicates that metabolic pathways regulating fatty acid, phospholipid, and cholesterol biosynthesis were coordinately decreased after deletion of Stat3 in type II cells, supporting an important role of STAT3 in regulating lipids biosynthesis in the lung. LDL receptor (Ldlr), mediating cholesterol uptake, and ATP-binding cassette A3 (Abca3), important for phospholipid transport, lamellar body formation and pulmonary surfactant secretion in alveolar type II cells were decreased in the Stat3Δ/Δ mice cells [36-38]. Recent in vitro studies from our group confirmed the direct binding of SREBP1c to the Abca3 promoter (Besnard et.al, submitted for publication). On the other hand, the increased expression of Abca1, a key facilitator of cellular cholesterol and phospholipid export [39,40] and high density lipoprotein binding protein (Hdlbp), which may function in the removal of excess cellular cholesterol, suggests that cholesterol and phospholipid clearance were induced after deletion of Stat3. Adenosine kinase (Adk), was increased 8.2 fold. Cellular role of ADK in lipid metabolism is somewhat controversial [41], Adk deficient mice developed neonatal hepatic steatosis and die within 14 days with fatty liver [42]. Increased expression of lipid export genes and decreased expression of genes mediating lipid biosynthesis likely cause a reduction of total lipids level in the type II cells of Stat3Δ/Δ mice.
Figure 1. Genes involved in lipids biosynthesis and clearance pathways were altered in type II cells from Stat3Δ/Δ mice. A. Srebf1 and Srebf2 regulate genes encoding the major metabolic enzymes in the fatty acid, cholesterol and phospholipids biosynthesis. The arrow (↑) indicates that the mRNA level was induced in Stat3Δ/Δ mice; (↓) indicates the mRNA level was reduced in Stat3Δ/Δ mice. Gene symbols, descriptions and the expression changes are listed in panel B.
Previous studies demonstrated the susceptibility of Stat3Δ/Δ mice to lung injury and death related to surfactant dysfunction [12]. Consistent with our prediction from the present mRNA microarray analysis, the saturated phosphatidycholine (SatPC) content in bronchoalveolar lavage fluid was significantly decreased in Stat3Δ/Δ mice (Figure 2). Significantly decreased SatPC synthesis and abnormalities in lamellar body numbers and morphology were also observed in Stat3Δ/Δ mice (data not shown). Taken together, a number of genes regulating surfactant lipid homeostasis were altered in type II cells isolated from the Stat3Δ/Δ mice, consistent with biochemical, functional, and morphologic changes in the surfactant system that is exacerbated by oxidant stress [12] or expose to pathogens [13].
Figure 2. Surfactant saturated phosphatidylcholine (SatPC) was significantly decreased in bronchoalveolar lavage fluid from the Stat3Δ/Δ mice, n = 8 per group. Aliquots of BALF were extracted with chloroform/methanol (2:1) and SatPC was isolated with osmium tetroxide followed by measurement of phosphorus as described previously [12]. Statistical differences were analyzed by Student t-test.
Role of Akt in Stat3 regulated lipid metabolism
Our observations support the view that the decrease in SREBP, at least in part, results in decreased expression of genes regulating lipid biosynthesis and metabolism in type II cells from Stat3Δ/Δ mice. SREBPs are master regulators of lipid metabolism. The transcriptional targets, and the pathways mediated by SREBP in liver have been well studied [43]. SREBPs are expressed in the developing lung, SREB1c increases in the developing lung concomitantly with the perinatal increase in surfactant and lipid synthesis, surfactant protein and Abca3 expression, genes critical for surfactant function.
However, the role of Stat3 in regulating SREBP and associated lipid metabolism in the lung is largely unknown. In the present analysis, we sought to identify mechanisms by which Stat3 regulates SREBPs and associated lipid biosynthesis pathways in alveolar type II cells in the lung. The regulation of SREBPs occurs at both transcriptional and post-transcriptional levels. The post-transcriptional regulation requires SCAP. Cre-mediated disruption of Scap significantly reduced Srebf1 and 2 levels as well as SREBP target gene expression in liver [44]. There are multiple potential STAT sites on the Scap promoter. Thus, Stat3 may influence lipid biosynthesis through the transcriptional regulation of Scap. At transcriptional level, nuclear hormone receptors (Nr1h2 and Nr1h3) and Pgc1-alpha and beta are known to regulate SREBPs expression in liver. Increased expression of Srebf1 and Fasn was associated with increased hepatic triglyceride content in Stat3 deficient mice [10,45]. In the present study, expression of SREBP and their down stream targets were decreased in type II cells from Stat3Δ/Δ mice (a finding that contrast with the findings in the liver) without changes in other known regulators such as Nr1h2, Nr1h3, Pgc1-alpha or Pgc1-beta, indicating the likely presence of alternative regulatory mechanisms in lung cells.
We hypothesize that AKT plays an important role in Stat3 regulated SREBP expression and associated lipogenesis in lung based on the following observations: 1) Protein and mRNA levels of Akt were increased in cells constitutively expressing active Stat3 and were reduced after Stat3 depletion [46]. STAT3 binds to Akt1 promoter was confirmed by ChIP assay [47], suggesting that A kt maybe a direct transcriptional target of Stat3. AKT, on the other hand, inhibits Stat3 transcriptional activity and phosphorylation [48]. Decreased Akt gene expression seen after deletion of Stat3 may represent a direct effect of Stat3 deficiency or to a negative regulatory response to STAT3 deficiency, 2) AKT activation induces both Srebf1 and Srebf2 mRNAs and proteins as well as key enzymes in the cholesterol, fatty acid and membrane lipid biosynthesis pathways [49], 3) Multiple lines of evidence suggest that PI3K influences Stat3 activation. STAT3 binds directly to the PI3K regulatory subunits [50-52]. The expression of genes encoding for several PI3K subunits were altered after deletion of Stat3 (see Additional file 1), supporting the involvement of PI3K/Akt signaling in Stat3 regulated bioprocesses in lung, and 4) Since AKT physically interacts with FOXA2 and regulates FOXA2-dependent transcriptional activity [53], the effects of AKT may be mediated, in part, via Foxa2. Foxa2 expression was reduced more than 2 fold in cells from the Stat3Δ/Δ mice. FOXA2 regulates lipid metabolism in both lung and liver [54,55]. Deletion of either Stat3 or Foxa2 resulting in decreased expression of a number of the overlapping genes that play important roles in surfactant homeostasis including Abca3 [55]. Thus, interactions between AKT and FOXA2 represent another potential mechanism by which lipid metabolism is influenced in Stat3Δ/Δ mice.
Stat3 influences expression of genes mediating apoptosis and cell survival
Many genes modulating apoptosis/cell survival were altered in response to the deletion of Stat3 from type II cells, including multiple Bcl-2 family members (Bcl2, Bcl2l1, Mcl1, Bcl2l11 and Bad), caspase 3 (Casp3), FADD-like apoptosis regulator (Cflar, also known as Flip), mucosa associated lymphoid tissue lymphoma translocation gene 1 (Malt1), prostaglandin-endoperoxide synthase 2 (Ptgs2) and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor, Nr3c1). In depth literature mining identified more apoptosis related genes than did analysis by Gene Ontology annotation. As depicted in Figure 3, our study indicates that STAT3 regulates apoptosis in a complex manner via processes that occur in multiple intracellular locations. Thus, STAT3 appears to serve as key regulator of apoptosis in alveolar type II cells. Among those apoptosis related genes, Malt1, Ptgs2 and Nr3c1 were strongly induced (> 5 fold in compare with control). MALT1 interacts with BCL10 (increased 2 fold in Stat3Δ/Δ mice). The formation of this complex is essential for NF-kappaB activation that, in turn, play a role in cell survival. IKKB phosphorylates BCL10 in its MALT1 interaction domain, causing BCL10 and MALT1 to disassociate, resulting in attenuation of NFKB signaling and cytokine production [56,57]. PTGS2 also known as COX-2, a key enzyme in prostaglandin biosynthesis, that is highly expressed in alveolar type II cells. The expression of Ptgs2 is increased in epithelial tumors, including non-small cell lung and prostate cancers via activation of the IL-6/GP130/STAT3 signaling pathway [58]. This pathway could contribute to tumor formation by promotion of tumor cell resistance to apoptosis via inhibitor of apoptosis (IAP)-dependent mechanism [58,59]. Consistent with these observations, Ptgs2, Il6st and two of the IAP family members (Birc4 and Birc6) were correspondingly induced in Stat3Δ/Δ cells (5.7, 1.7, 3.2 and 2.3 fold respectively). Nr3c1 (increased 5.1 fold) encodes a receptor for glucocorticoids that can act as both a transcription factor and as a regulator of other transcription factors. STAT3 and NR3C1 physically interact to mediate effect of glucocorticoid on the IL-6-mediated inflammatory response [60-62]. NR3C1 also interacts with stress-responsive transcription factors (Hif1a, increased 4.7 fold), mitogen activated protein kinase 8 (Mapk8 or Jnk, increased 4.7 fold) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (Ywhal, increased 3.3 fold), a 14-3-3 family of proteins implicated in the pathogenesis of small cell lung cancer [63,64].
Figure 3. Genes involved in apoptotic pathways were altered in type II cells isolated from Stat3Δ/Δ mice. Genes associated with apoptosis were identified via literature mining using Pathway Studio 4.0 (Ariadne Genomics, Inc.). STAT3 regulates apoptosis positively and negatively, via processes occurring in multiple intracellular compartments. Genes up-regulated in Stat3Δ/Δ mice are framed in red. Genes down-regulated in Stat3Δ/Δ mice are framed in green. Each dashed line demarcated with a larger dot linking "apoptosis" and each gene node indicates a regulatory relationship based on the literature references. Regulatory relationships are denoted by line colors (green, positive regulation; red, negative regulation; and gray, regulation direction is unknown).
STAT3 is likely to regulate apoptosis by multiple mechanisms including gene transcription. Bcl-xL is the direct transcription target of STAT3[65]. STAT3 can serve as an anti-apoptotic factor by transcriptional up-regulating the expression of Bcl-xL [66]. The decrease of Bcl-xL may represent a direct response to Stat3 deletion. The fact that expression of Bcl-xL blocked the apoptotic effects of the adenovirus in lung injury suggested that Bcl-xL may mediate the role of STAT3 in the regulation and survival of the respiratory epithelium [13]. The Pi3k-Akt pathway represents a second mechanism by which STAT3 influences cytoprotection. Pi3k-Akt signaling mediates a wide range of down stream targets to regulate apoptosis [67]. For example, AKT phosphorylates multiple Bcl-2 family members, including BAD and Bcl-xL [68], inhibits caspase 3 activation [66] and blocks cytochrome C release from mitochondria [69]. Another mechanism by which STAT3 modulates apoptosis is through protein-protein interactions. Bcl-xL is a direct transcriptional target of STAT3; Bcl-xL interacts with VDAC1 to regulate the outer mitochondrial membrane channel induce apoptosis [70,71]. CASP3 can interact with multiple apoptosis proteins including CFLAR, BIRC4 and 6, BCL2 and APP [72-75]. The expression of Casp3 as well as its interaction partners was induced in Stat3Δ/Δ mice. NR3C1 has both pro- and anti-apoptotic effects. NR3C1 physically interact with STAT3, HIF1A, MAPK8, YWHAL [63,64,76]; these stress-responsive transcription factors and signaling molecules were largely induced in the present array from Stat3Δ/Δ type II alveolar epithelial cells. The close transcriptional communication and physical interactions among these transcriptional regulators likely play a critical role in regulating the balance of apoptosis and cell survival. In the present study, effects of STAT3 deletion were assessed in type II epithelial cells purified from the adult mouse lung. mRNA was isolated immediately after isolation to avoid cell culture dependent alteration in gene expression. It is possible that the cells have undergone added cellular stress during protease treatment, isolation and purification, which in turn may influence the expression of genes. Our results support the view that STAT3 regulates the balance between a subset of pro- and anti-apoptotic genes, determining the cell death or survival through multiple mechanisms. Consistent with the present microarray prediction, cleaved caspase-3 and TUNEL positive cells were significantly increased in Stat3Δ/Δ mice following adenoviral infection and the apoptosis can be blocked by expression of Bcl-xL [13].
Our previous studies demonstrated that Stat3 plays critical role in cyto-protection during lung injury [12,13]. Present data support the role of Stat3 in enhancing epithelial cell survival and surfactant lipid synthesis that contribute to the maintenance of lung function. Deletion of Stat3 from type II alveolar epithelial cells induced the expression of the genes regulating protein metabolism, protein transport, chemotaxis and apoptosis while decreasing the expression of genes regulating lipid synthesis and metabolism. Critical to pulmonary function during injury, Stat3 influences the expression of genes regulating surfactant lipid synthesis and surfactant homeostasis including Abca3. As illustrated in Figure 4, the present study identified a complex regulatory network by which Stat3 regulates gene expression in type II alveolar cells that is required for cellular homeostasis following injury. STAT3 likely interacts with AKT/FOXA2 in the regulation a number of biological processes in alveolar type II cells, including cell survival/apoptosis, cholesterol and fatty acid biosynthesis required for surfactant homeostasis and lung function.
Figure 4. Schematic representation of the proposed role of Stat3 in cytoprotection of the lung in normal (A) versus Stat3Δ/Δ mice (B). Stat3 plays critical role in cyto-protection during lung injury [12, 13]. Present data support the role of Stat3 in enhancing epithelial cell survival and surfactant lipid synthesis that contribute to the maintenance of lung function. For simplicity, only representative genes were included.
Transgenic Mice
SP-C-rtTA/(tetO)7CMV-Cre/Stat3flx/flx triple-transgenic mice were generated as described previously [12]. Stat3flx/flx mice were a kind gift of Dr. Takeda (Hyogo College of Medicine, Japan) [8]. In the presence of doxycycline, exon 21 of the Stat3 gene is permanently deleted from respiratory epithelial cells prior to birth (Stat3Δ/Δ mice) [12]. Stat3 deleted transgenic (Stat3Δ/Δ) and non-deleted littermates (double transgenic,SP-C-rtTA, or tetO7CMV-Cre and Stat3flx/flx) were used for the experiments. Doxycycline was administered to the dams in the food at a concentration of 625 mg/kg (Harlan Teklad, Madison, WI) from embryonic day 0 (E0) to postnatal day 25 (P25), resulting in extensive deletion of Stat3 in respiratory epithelial cells [12]. As previously described, deletion of Stat3 did not alter lung size, morphology or survival under non-stressed condition [13].
RNA Extraction
Alveolar type II cells were isolated from 8 weeks old, sex and age matched littermate control and Stat3Δ/Δ mice using collagenase and differential plating as described by Rice et al.[77]. Type II cells from 3 mice were pooled to obtain one cell pellet. Three independent pools were generated from control and Stat3Δ/Δ mice separately for purification of RNA and microarray hybridization. Type II cells were homogenized with TRIzol reagent (Invitrogen, Carlsbad, CA). RNA concentration was measured by spectrophotometer and normalized prior to cDNA synthesis. These cell isolates consist of more than 90% alveolar type II cells with residual alveolar macrophages as the major contaminating cell. Purity was assessed by modified Papanicolaou stain. Purity and number of type II cells isolated from Stat3Δ/Δ mice were not different from controls.
RNA Microarray Analysis
mRNA was extracted from three independent pools of isolated type II cells from adult Stat3Δ/Δ and control mice. The cRNA was then hybridized to the murine genome MOE430 (consists of ≈ 45000 gene entries) chips (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol. The RNA quality and quantity assessment, probe preparation, labeling, hybridization and image scan were carried out in the CCHMC Affymetrix Core using standard procedure. RNA quality and quantity were analyzed by spectrophotometer. The A260/A280 ratio was used to determine RNA purity with the acceptable region of 1.9–2.1. Affymetrix Microarray Suite 5.0 was used to scan and quantitate the gene chips under default scan settings. Normalization was performed using the Robust Multichip Average model [78,79]. Data were further analyzed using affylmGUI from R/Bioconductor package [80]. Differentially expressed genes were selected with the threshold of T-Test P-value ≤ 0.05, False Discovery Rate (FDR) ≤ 10% and fold change ≥ 1.5. We prioritized the mRNAs whose abundance consistently changed in multiple probe sets by selecting them without the FDR consideration. Unknown cDNA clones/ESTs and duplicated gene entries were removed from further functional analysis.
Gene Ontology Analysis
Gene Ontology Analysis was performed using public available web-based tool David (database for annotation, visualization, and integrated discovery) [81]. Overrepresented biological processes were selected at the threshold of Fisher Exact Test P-Value ≤ 0.05 and minimum gene counts belonging to an annotation term ≥ 2%.
Promoter Analysis: Transcription Factor Binding Sites Search
Promoter sequences of all differentially expressed genes (-2000 bp upstream from the tentative TSS) were retrieved from our database (originally downloaded from UCSC Genome browser) and searched for over-represented TFBS (Transcription Factor Binding Site) in these sequences using MatInspector (Genomatix) using the complete Vertebrate Matrix Library 6.2. The P value is calculated using binomial distribution probability by comparing the matrix match of the promoter regions of differentially expressed genes with the promoters from random mouse genes sets (-2kb for all the promoters). The single-step Bonferroni adjustment is used to control for the multiple comparison effect (i.e., multiplication of p-value by the number of TFBS in Genomatix Vertebrate Matrix Library).
Pathway Enrichment Test
Overrepresented pathways were identified by comparison the overlap of differentially expressed genes and all genes in MOE430 mouse genome (reference) with the known KEGG pathways. A Fisher's exact test for 2·2 contingency table was used to calculate the statistical significance. A pathway is considered to be over-represented when a probability P value = 0.01 and gene frequency (genes in the pathway/total number of differentially expressed genes) ≥ 2%.
Literature Mining
Potential protein-protein or protein-DNA interactions were identified using Pathway Studio (Ariadne Genomics) that contains MedScan, an automated text-mining tool to search the entire PubMed and other public sources (The current version database contains more than 15 million Medline abstracts/full text). Gene expression profiling results was imported into the Studio and used to interpret pathways, gene regulation networks, and protein interaction maps.
Validation of mRNAs
Real-time RT-PCR was used to cross validate changes in a subset of genes from microarray selection. mRNA was extracted from alveolar type II epithelial cells isolated from Stat3Δ/Δ and control mice using RNeasy Protect mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. RNA concentration was measured by spectrophotometer. cDNA was made with SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Malt1, Rtn4, Reg3g, Bcl2l1, Abca3, Scap, Fasn, Srebf1 and Srebf2 were detected using primers listed Changes in mRNA were determined in type II cells isolated from Stat3Δ/Δ and controls (n = 3–4/group). The following primers were used: Malt1: forward, TAT CCA GGA GGA CCC CAT GT and reverse, TCT GAT CAA AGC CAG TTA GCA TCAT; Rtn4: forward, AAG TGG AAG GAG TTT GAG AGA GCA and reverse, CTG TCT CAA AGC AGA TGT GAA AGC; Reg3g: forward, TGC CAA AAG AGC CCT CAG GA and reverse, TGC CTG AGG AAG AGG AAG GAT TCG; Bcl2l1: forward, TCT CTC TCC TCT GTC CAC CCT TG and reverse, TGC CCC TCA GAA GCC AGA AC; Abca3: forward, GCA TTG CCC TCA TTG GAG AGC CTG and reverse, TCC GGC CAT CCT CAG TGG TGG G; Scap: forward, TGA CCA CAA ACA AGG AGA GC and reverse, CAG GAA CAC CAA ACA GCA AG; Srebf1: forward, AAG CCG GGT GGG CGC CGG CGC CAT and reverse, GTC GTT CAA AAC CGC TGT GTC CAG; Srebf2: forward, CAT CCA GCA GCC TTT GAT ATA CCA G and reverse, AGG ACC GGG ACC TGC TGC ACC TGT G. Fasn: forward, GGA CAT GGT CAC AGA CGA TGA C and reverse, GTC GAA CTT GGA CAG ATC CTT CA. The PCR product was separated by the 1% agarose gel electrophoresis. β-actin was used as the internal control. Taqman Gene Expression Assay were used to confirm the expression of Akt2, Cdipt, Acox2, Cds2 and Gpam using Applied Biosystems 7300 Real-Time PCR System and company designed probes (Applied Biosystems). Statistical differences were determined using unpaired Student's t-tests.
We thank Ann Maher for secretarial assistance, Valerie Besnard for scientific discussion. We also thank Dr. K. Takeda (Hyogo College of Medicine) for the gift of Stat3flx/flx mice.
This work was supported by National Institutes of Health Grant HL61646 to M. Ikegami, Y. Xu and J.A. Whitsett.
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Tablet. Hotels for Global Nomads
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6526 Yount Street, Yountville, CA, USA
Check availability as rates may vary for dates. Best rate guaranteed.
A decade or two ago, Napa Valley was the place to go for a little slice of Renaissance Tuscany. It made sense at the time, with the California wine industry really hitting its stride, for the architecture to aspire toward a classic European sort of gravity. These days, however, Napa is ground zero for a new green building movement, and its hotels are leading the way — no doubt something greener will come along, but at the time of its opening, Bardessono was pretty much as sustainable as it gets.
The construction, which looks like sandstone or blond concrete, is actually rammed earth — for several slightly boring reasons it's greener than all the alternatives, and anyway it lends a distinctive look. You'll see a lot of wood as well — when sourced properly it's the other green building material, and most of it is reclaimed or recycled. And it's not just the building that's green, but the operation — great unglamorous pains are taken to reduce paper or water waste, to automatically shade the rooms from the midday sun, that sort of thing.
At the same time, Bardessono is pretty close to the state of the art in luxury, and all the concern about sustainability doesn't change that. The suites are essentially high-end luxury apartments, complete with gas fireplaces and lavish living spaces, and the bathrooms are designed to function as private spas — literally, as in, that's where the therapist takes you when it's time for your in-room treatment.
Add a massive wine collection (some from the on-site organic vineyard) and an upscale contemporary Californian restaurant (serving produce from the hotel's own farm) and you've got a pretty complete package, quintessentially Napa. With several restaurants by Thomas Keller, among others, Yountville isn't just a wine country destination, but arguably one of the capitals of the US culinary world as well.
How to get there:
Bardessono is approximately a 1 1/2 hour drive from San Francisco International Airport and a 1 hour drive from both Oakland International Airport (OAK) and Sacramento International Airport (SMF).
- 62 Rooms
- Style: Modern Design
- Atmosphere: Quiet
- 22 Guest Reviews
- Bardessono
- 6526 Yount Street
- Yountville, CA, USA
Hotel amenities and services
- 100% non-smoking hotel
- 24 hour concierge
- 24 hour room service
- Babysitting (on request)
- Bar
- Central air conditioning
- Free parking
- Free wi-fi
- Garden
- Golf nearby
- Lounge
- Multi-lingual staff
- Offsite gym facilities
- Outdoor swimming pool
- Pets allowed (charges apply)
- Restaurant
- Sauna
- Spa
- Spa treatments (on request)
- Turkish bath
- Wake-up service
- Yoga
Other Napa / Sonoma Valleys hotels
22 Guest Reviews
All reviews are from Tablet guests with verified stays.
- Chandra
- Stayed July 2013
- Rating: Excellent
What I liked:
The perfection
What the hotel could do better:
Nothing, had a wonderful stay!
- Yvonne
- Stayed December 2012
- Rating: Good
What I liked:
Large room, fireplace & patio
What the hotel could do better:
Not much
- Louis
- Stayed August 2012
- Rating: Excellent
What I liked:
This is my second time staying at this hotel and I have nothing but wonderful things to say. Staff is great, the rooms are fabulous and it is just a great place for a weekend get away. The outdoor shower was incredible. All of this made leaving the room difficult.
What the hotel could do better:
Nothing, had a wonderful stay!